Actipix D100

Transcript

For more information about Paraytec Ltd, please visit our web site at http://www.paraytec.com or contact: Paraytec Ltd 1a St Georges Place Tadcaster Road York, YO24 1GN Tel: (44) (0)1904 526270 Fax: (44) (0) 1904 652101 Email: [email protected] Web: www.paraytec.com Part Number 110-2100 Revised: 05.06.2009 Published by Paraytec Ltd, York, UK Copyright © 2009 Paraytec Ltd. All rights reserved. Printed in the UK. ActiPix D100 Table of Contents ActiPix D100.............................................................................................................................1 Application software......................................................................................................1 How the detector works..........................................................................................................2 What is a pixel?............................................................................................................2 Active pixel sensor........................................................................................................2 Signal and reference pixels..........................................................................................2 Process of collecting a single image.............................................................................3 Converting an image into absorbance values...............................................................3 Processing multiple images into a single absorbance peak.........................................3 Principles of absorbance........................................................................................................4 The Beer-Lambert law..................................................................................................4 Interpreting data......................................................................................................................5 Modes of operation..................................................................................................................6 Capillary based experiments (CE, LC, Sizing)......................................................................7 Working with capillaries..........................................................................................................8 Focusing from capillary.................................................................................................8 Multiple windows...........................................................................................................8 Running in capillary mode......................................................................................................9 Online capillary mode...........................................................................................................10 Method........................................................................................................................10 Running a capillary experiment..................................................................................10 Capillary location........................................................................................................10 Detector calibration.....................................................................................................11 Data collection............................................................................................................12 Trace view controls...............................................................................................................13 Rolling trace view........................................................................................................13 Full trace view.............................................................................................................13 LC system setup....................................................................................................................15 Flow rate.....................................................................................................................15 Flow direction..............................................................................................................15 CE system setup....................................................................................................................16 Length to window........................................................................................................16 Separation start..........................................................................................................16 Flow direction..............................................................................................................17 Imaging mode........................................................................................................................18 Online imaging mode............................................................................................................19 Method........................................................................................................................19 Data............................................................................................................................20 i ActiPix D100 Table of Contents Dissolution imaging..............................................................................................................21 Principle of operation..................................................................................................21 Creating a dissolution method.............................................................................................22 Dissolution data capture.......................................................................................................23 Dissolution data processing.................................................................................................25 Converting a dissolution image to absorbance image................................................25 Generating profiles................................................................................................................28 Cartridge Editor.....................................................................................................................31 Basic cartridge editor..................................................................................................31 Advanced cartridge editor...........................................................................................31 Cartridge files.........................................................................................................................32 Opening cartridge files..........................................................................................................33 Creating a new cartridge file.................................................................................................34 Cartridge type selection..............................................................................................35 Editing channel parameters........................................................................................35 Basic cartridge editor............................................................................................................37 Advanced cartridge editor....................................................................................................38 Method Editor.........................................................................................................................40 Capillary methods.......................................................................................................40 Imaging methods........................................................................................................40 Method files............................................................................................................................41 Opening method files............................................................................................................42 Creating a new method.........................................................................................................43 Capillary specific settings (CE, LC and Sizing)..................................................................44 Method Details............................................................................................................44 Timing Details.............................................................................................................44 Output Data................................................................................................................44 Experimental Conditions.............................................................................................45 Settings.......................................................................................................................45 Imaging method settings......................................................................................................47 Method Details............................................................................................................47 Timing Details.............................................................................................................47 Output Data................................................................................................................47 Lamp...........................................................................................................................47 Imager.........................................................................................................................48 ii ActiPix D100 Table of Contents Folder settings.......................................................................................................................49 Online Mode...........................................................................................................................50 Detector status window........................................................................................................51 Checking detector status......................................................................................................52 Detector controls...................................................................................................................53 Previewing data.....................................................................................................................54 Collecting data.......................................................................................................................55 Extending runtime.................................................................................................................56 Runtime clock.............................................................................................................56 Trace view controls...............................................................................................................57 Rolling trace view........................................................................................................57 Full trace view.............................................................................................................57 Plot controls...........................................................................................................................59 Zoom in.......................................................................................................................59 Zoom out.....................................................................................................................60 Panning.......................................................................................................................60 Channel selection.......................................................................................................61 Settings...................................................................................................................................62 Folders........................................................................................................................62 Connection..................................................................................................................63 Upgrade......................................................................................................................64 Diagnostic Log............................................................................................................65 Offline Mode...........................................................................................................................66 Default data folder.................................................................................................................67 Viewing traces........................................................................................................................68 Add trace....................................................................................................................69 Remove trace.............................................................................................................69 Clear all.......................................................................................................................69 Plot controls...........................................................................................................................70 Zoom in.......................................................................................................................70 Zoom out.....................................................................................................................71 Panning.......................................................................................................................71 Channel selection.......................................................................................................72 Offline image view.................................................................................................................73 Image window.............................................................................................................73 Playback.....................................................................................................................73 Frame Selection..........................................................................................................74 Cursor.........................................................................................................................74 Display........................................................................................................................74 iii ActiPix D100 Table of Contents Offline image view Time Marker................................................................................................................75 Timing.........................................................................................................................75 Region Selection.........................................................................................................75 Profile..........................................................................................................................75 Absorbance.................................................................................................................75 Correlate.....................................................................................................................76 2D image controls..................................................................................................................77 Zoom in.......................................................................................................................77 Zoom out.....................................................................................................................77 Pan.............................................................................................................................77 Region selection.........................................................................................................78 Marker placement.......................................................................................................78 3D image controls..................................................................................................................79 Zoom in.......................................................................................................................79 Zoom out.....................................................................................................................79 Rotate.........................................................................................................................80 Tilt...............................................................................................................................80 Pan.............................................................................................................................80 Reviewing analysis data.......................................................................................................81 Sizing analysis............................................................................................................81 Adjusting parameters..................................................................................................82 Peak fitting..................................................................................................................82 Printed report..............................................................................................................83 Viewing frames......................................................................................................................84 Data reprocessing.................................................................................................................85 File reprocessing........................................................................................................85 Image to trace conversion....................................................................................................86 Loading an image.......................................................................................................86 Setting up a method....................................................................................................86 Generating trace data.................................................................................................87 Processing frames.................................................................................................................88 Loading a frame..........................................................................................................88 Generating trace data.................................................................................................88 Parameter optimisation...............................................................................................88 Offline sizing analysis...........................................................................................................89 Loading traces............................................................................................................89 Adjusting analysis parameters....................................................................................89 Selecting the data.......................................................................................................89 Reviewing the analysis...............................................................................................91 Folder settings.......................................................................................................................92 File types................................................................................................................................93 Configuration..............................................................................................................93 Data capture...............................................................................................................93 iv ActiPix D100 Table of Contents File types Post processing..........................................................................................................93 Additional....................................................................................................................93 Analysis files..........................................................................................................................94 Cartridge files.........................................................................................................................95 Frame files..............................................................................................................................96 Image files..............................................................................................................................97 Method files............................................................................................................................98 Trace files...............................................................................................................................99 Calculator tools....................................................................................................................100 Absorbance conversion......................................................................................................101 Extinction coefficient..........................................................................................................102 Flow rate conversion...........................................................................................................103 Hydrodynamic radius..........................................................................................................104 Advanced operation............................................................................................................105 Updating the D100 firmware...............................................................................................106 Incompatible firmware message...............................................................................106 Updating the firmware...............................................................................................106 Configuring the ethernet connection.................................................................................109 Default configuration.................................................................................................109 Network settings.......................................................................................................109 Modifying the settings...............................................................................................110 Diagnostic logging..............................................................................................................111 Setting log level........................................................................................................111 Locating the log file...................................................................................................111 Removing the log file................................................................................................112 v ActiPix D100 The ActiPix ® D100 UV area imaging detector has been designed for use in a variety of applications in research, development and quality control. It provides quantitative imaging in the ultraviolet region of the electromagnetic spectrum. Application software The ActiPix software consists of a suite of four applications: ActiPix D100 Cartridge Editor is used to create/modify cartridge definition files. Cartridge files contain the physical parameters that relate to actual ActiPix cartridges, e.g. an LC cartridge file could contain the number capillaries in the cartridge and dimensions of each capillary. ActiPix D100 Method Editor is used to create/modify experimental method files. Method files are created as LC, CE, sizing or imaging, and contain the parameters required to run a particular experiment. ActiPix D100 Online Mode is used for running LC, CE, Sizing or Imaging experiments and capturing data in the form of traces, frames or images. When operating in online mode, method files provide the settings for the experiment. ActiPix D100 Offline Mode is used reviewing previously captured data, e.g. traces files from an LC experiment. This mode also allows the reprocessing of captured image data. 1 How the detector works What is a pixel? A pixel is a common term made popular with digital cameras referring to how many millions of pixels they contain. A basic description of a pixel is a funnel that produces a current of electricity when a photon of light of suitable wavelength contacts the inside surface of the funnel. At the bottom point, or base, of the funnel are the internal workings of any computer microchip: resistors, capacitors, transistors and diodes. The pixel and associated electronics occupy an area typically less than 10 µm x 10 µm. The efficiency of converting photons into electric current is directly related to parameters such as Gain and Offset. When properly set these values will allow several thousand photons to not only be turned into electric current but to actually amplify the photocurrent, making the pixel even more sensitive. Continuing with the assumption that a pixel is shaped like a funnel, one can visualize that the funnel can accept only so much material (photons) before it overflows (saturates). Once a pixel has saturated it can accept no more photons and is essentially unusable until it is emptied. Thus controlling how many photons reach each pixel per image is important, as are the readout and reset times. Active pixel sensor An active pixel sensor typically comprises 1 million or more pixels. This area of pixels is considered the viewing area. The overall dimension of each pixel is the limitation for resolution. In the case of the ActiPix, the array detector measures 1280 columns by 1024 rows, given a total area of (1280 x 1024) = 1310720 pixels or 1.3 Mpixels. Each pixel is 7 µm W x 7 µm D for a total area of 49 µm2. This gives a maximum total viewing area of 63 mm2 (9 mm W x 7 mm D). Two important unique capabilities of an area detector are: 1. The ability to bin pixels. Binning of pixels is the addition of data from a group of pixels in one or more dimensions to give an overall higher value but with a loss of resolution. The ActiPix D100 uses a binning of 10 in the direction of the capillary axis, the pixel rows, providing for an increase of size to give effective pixels of 70 µm in this dimension, with photocurrent a factor of nearly 10 greater relative to a single pixel. 2. Because of the electronics built into each pixel of an active pixel sensor, Paraytec special software can address a selected region of the image, turning off pixel rows not required in the analysis. Signal and reference pixels The ActiPix assigns signal and reference pixels in the selected region. To obtain the photocurrent, every single effective pixel is read out in complete order: Nominally each pixel in the array detector will be read at approximately 10 MHz, providing for very high imaging rates. Once each pixel is read it is empty and available for refilling. Inherent to all the electronics connected to each pixel is a more or less constant level of background electronic signal, the dark current. Although quite small, this needs to be measured periodically and subtracted from the intensity value of each effective pixel immediately after it is read out. 2 Dark current measurements are made at the beginning of a run. This phase lasts for about 10 seconds, and to establish dark current values for all pixels the ActiPix turns the lamp off. The associated counterpart to this measurement is the baseline light intensity, Io, the intensity with just solvent in the capillary at the beginning of the run. After the dark current has been measured, the solvent inside the capillary is used as the absorbance baseline and the ActiPix determines Io and the baseline zero value for the remainder of the run. Process of collecting a single image To get accurate intensity values using an array detector it is important to control the intensity of light reaching the pixels to avoid saturation. This is done by timing very carefully the flash from the xenon arc lamp and the reading out of all the pixels. An image is collected by reading out the value at each pixel selected in the viewing area and then subtracting its dark current value. This takes a few hundred microseconds and then the sensor is ready to be read again. The signal is sent to the lamp to provide the next flash and the process starts again. Converting an image into absorbance values The process of calculating an absorbance value from an array of pixels begins by assigning certain pixels as signal and reference and turning others off. Once this pixel map is created (i.e. cartridge file), this template is applied to each image collected. On applying the map, those pixels assigned as signal (sig) and reference (ref) are averaged together respectively within a column of effective pixels. The final result for each sample channel is a single signal and reference value per column, which are put into the following formula to return a single absorbance value for that column. A = log {(Io / Isig) / (Io / Iref)} With a binning factor of 10, there will be 102 (1024/10) columns and 102 absorbance values per image. Processing multiple images into a single absorbance peak Because the analyte is continuously moving past the sensor, several images will be taken of the same analyte band. Knowing the exact linear velocity is critical to assigning a single absorbance value. In LC and nano-sizing applications, where the flow rate is constant, the flow rate (µL/min) is input by the user to allow the ActiPix to determine the linear velocity (mm/s) and thus the appropriate spatial displacement between successive images (snapshots) to bring peaks from a single analyte band to a common centre. In this process of addition of time-displaced snapshots, the signal accumulates in proportion to the number of snapshots, n, whilst the noise grows only as the square root of this number of snaphots, thus providing an improvement in signal to noise of n1/2 in comparison to a single snapshot. In capillary electrophoresis, the linear velocity is analyte-dependent and readily calculated by the ActiPix knowing the length of the capillary to the detector and the time of arrival of the analyte peak. It is important to note that no spatial resolution is sacrificed in the process of procsssing of multiple images in this way, and whilst the total length of the imager is 9 mm the spatial resolution is determined by the effective pixel size of 70 µm. 3 Principles of absorbance Absorbance measures the proportion of light absorbed by molecules present in a cell positioned between the light source and the detector. Typical absorbance measurements in a fluid flowing system are taken such that the absorbance of the solvent, commonly referred to as baseline, is by convention set to zero. This is helpful, since what is of interest is the absorbance difference between the sample, present as a dilute solution in the solvent, and the solvent. Changes to the solvent may result in a shift, either up or down, of the baseline. As well as baseline shifts due to changes in absorbance of the solvent at the wavelength of interest, shifts will also arise if there are any changes of refractive index (RI) of the solvent. This is because the proportion of light lost by reflection at the wall-solvent interface decreases as the difference between refractive index of solvent and wall material decreases. It should be noted that changes in baseline due to RI changes are typically expected to be of magnitude milli absorbance units (mAU). All molecules absorb light. Most molecules associated with biological applications and many small molecules absorb in the ultraviolet (190-400 nm). The ActiPix is capable of measuring light in the UV and the visible regions of the spectrum. Wavelengths in this range have a frequency measured in nm (nanometres). Each compound will absorb light over a range of nm, with the maximum being taken as the characteristic wavelength associated with that compound. Additionally, as each molecule typically has many different types of chromophores, responsible for the electronic transitions which lead to absorption in the UV-vis region, every compound absorbs light with a different efficiency. The Beer-Lambert law The Beer-Lambert law links absorbance, A, to concentration, c, via the relationship A = εbc where ε is the absorption coefficient at the wavelength of measurement and b the cell pathlength. Normally concentrations are expressed in molar units (moles per litre) and pathlengths in centimetres. Thus ε is the absorbance at unit concentration (1 mol per litre) and unit pathlength (1 cm). The key thing is that absorbance is directly proportional to concentration, and once this molar absorption coefficient is known, the exact concentration of the compound (c) can be calculated from the absorbance read by the detector, dividing absorbance A by ε and b. c = A /εb In cases where the molar absorption coefficient is not known, the response factor, R, linking absorbance to concentration (R = εb) can be determined by measuring absorbance as a function of concentration, typically using a dilution calibration series of the compound in question in the solvent, and taking the slope of the graph. 4 Interpreting data All UV detectors used with flow cells in separation science, e.g. liquid chromatography (LC) and capillary electrophoresis (CE), output data for graphs of absorbance as a function of time, t, with A displayed in the y dimension and t in the x dimension. Diode array detectors typically also provide information on absorbance as a function of time at a set of wavelengths. A fluidic separations system entails an injection stage, in which a plug of analyte solution is placed into the flow stream. The concentration of the analyte as a function of distance (and time) in the flow stream is initially a rectangular function, with concentration equal to zero outside the injection zone and the injection concentration inside the injection zone. However, when this zone passes the detector the plotted response is typically a rise and fall in absorbance units that resembles a Gaussian peak. Obviously, conditions and reactions prior to the detector can affect the peak shape: the further the detector from the injector, and the smaller the injection volume, the more likely is the peak to be Gaussian. Special caution should be used with regard to air bubbles. Unlike analyte zones, bubbles do not spead out with time. Any bubble of gas moving along the fluid system will cause an apparent large absorbance signal. The D100 is unique in providing information unobtainable from other standard LC detectors. Because of the large detection area, multiple capillary windows can be placed over the sensor and monitored for absorbance simultaneously. These windows can be from separate capillaries or from a single capillary looped back over the detector. Because of this and many other unique features, the D100 can provide absorbance readings that will allow researchers to influence the analyte zone in a controlled way and be able to observe in real-time from the peak shape what the effect this influence has on the sample. This simple principle can be used in many ways to provide valuable insights that were previously unobtainable for microscale processes and reactions. 5 Modes of operation The ActiPix D100 has two main modes of operation: • Capillary Mode - multiplexed UV absorbance measurement (up to eight capillaries). • Imaging Mode - UV wide area and dissolution imaging. Choosing a mode depends on the type of application and to some extent the type of cartridge being used. All cartridges or applications using capillaries should generally provide absorbance data plots rather than movies. All other types of applications are collected as an image and have far fewer method parameters but offer more possibilities for data measurements. See also:Capillary method settings 6 Capillary based experiments (CE, LC, Sizing) Capillary mode is used to collect and display absorbance data from up to eight capillaries in realtime. Absorbance measurements are saved directly to disk, and can exported in .csv and .NetCDF formats. Advanced algorithms are used for automated capillary location, simplifying the setup and running of experiments. Automated analysis provides instant access to results after each run completes. Captured traces can be viewed, overlaid and post processed. Capillary absorbance measurement and post processing 7 Working with capillaries The spatial resolution of the ActiPix D100 is determined by the effective pixel size of 70 µm in the capillary dimension. This allows for a high resolution between peaks in highly complex samples, such as those in protein digests. Greater resolution between peaks is possible than with other absorbance detectors for use with capillaries in nanoLC applications, e.g. those with Z-cell design, which have sample volume at the detector much greater than the effective pixel volume in the ActiPix. Another benefit with the ActiPix is that, because the sensor window width is significantly larger than with normal single-point absorbance detectors, the time-at-detector (TAD) allows the analyte to spend more time over the sensor. This is of particular relevance when making comparison with single point detectors for capillary electrophoresis. Focusing from capillary The role of the capillary is also an important contributor to the whole sensor system. The natural benefits of fused silica capillary is that it acts like a cylindrical lens, focusing light passing through the solution containing the analyte onto a few pixels while diverting light passing through the outer walls away onto other regions of the imager. This puts the entire signal onto just a few pixels, which is enough to monitor the whole analyte band. At this point, it should seem obvious that any movement by the capillary will in fact then move the signal onto different pixels and cause an erroneous signal. Special combs are used to tightly hold the capillary of the cartridges used in the D100 and prevent movement. Instructions are given in the Cartridge Manual for correct mounting and assembly of capillaries. Multiple windows A single capillary can have multiple windows, thus allowing detection at many points along the fluid path such as before and after a reaction zone. Current absorbance detectors normally measure only the absorbance from one point, whereas the ActiPix D100 allows researchers to measure absorbance at multiple points and provide greater insight into a reaction or processes. 8 Running in capillary mode The following steps are required for running an experiment with capillaries: 1. Create a new cartridge file using the Cartridge Editor, or identify a preconfigured cartridge suitable for the experiment. 2. Create a CE, LC or Sizing method file using the Method Editor, or modify a default method file. Load the cartridge file into the method, update the settings as required and save. 3. Load the method file into the Online Mode application, start the detector and run the experiment. 4. Review/process the saved absorbance data using Offline Mode. See also: Cartridge Editor, Creating cartridge files 9 Online capillary mode ActiPix D100 Online Capillary Mode is used for running capillary based experiments (LC, CE or Sizing) and recording absorbance trace data. A method file provides settings for the experiment and must be loaded prior to operation of the detector. Method The Method tab contains the settings specified in the LC, CE or Sizing method file (as produced by the Method Editor). Various settings in the method tab can be modified on a run by run basis, however these settings are not saved back to the original method file. Main Online Window (Capillary mode - Method) Running a capillary experiment When running a capillary based experiment, the following steps are performed automatically when the Start button is pressed: 1. Transfer of the method settings to detector. 2. Cartridge location and validation. 3. Detector calibration. 4. Data collection and display. 5. Post-run analysis (Sizing only). Pressing the Preview button also starts the capillary location, calibration and data collection process, but no data is saved to disk. Capillary location Prior to data collection and display, the ActiPix analyses the cartridge to accurately locate the capillaries. The signal and reference zones, as defined in the cartridge file, are used to identify the number and position of each capillary channel. 10 The following capillary location steps are performed automatically at the start of each capillary mode run: 1. Collect preview images and determine the imaging area required for the capillaries selected in the cartridge file. 2. Zoom in to locate the capillaries and reference area. 3. Locate the signal zones inside the capillary based on cartridge file parameters. If capillary location is successful the signal zones are coloured to match the data traces (blue, green, etc.). If capillaries cannot be located an error message Cartridge Image analysis failed is shown. Cartridge image analysis failed Detector calibration Data collection to begins as soon as the capillary location has completed, or when an input trigger signal is received. Prior to data collection, calibration data is recorded: 1. The lamp is turned off for 10 seconds to collect the dark current information. The dark current is subtracted from each image frame during data collection. 2. The lamp is turned back on and the Io is collected for each signal and reference area. This baseline value is used to calculate each true absorbance value for each data point. If any analyte or different buffer is present in the capillary the true baseline will be incorrect or change once the capillary volume is changed over to fresh buffer. INFORMATION: It is important to have only buffer in the capillary during the detector calibration. If analyte is present during calibration, sample peaks that follow may not show true absorbance values. 11 Data collection Following the calibration steps, data is collected at the sample rate selected in the method. Absorbance traces for each channel are displayed on the Data tab. Absorbance trace Note: These steps are repeated each time the Preview or Start button is pressed. This is done because it is not known if the cartridge or capillary has been changed or replaced between runs. Furthermore, for the best results the determination of the optimum signal location should be done for each run. See also: Offline Mode 12 Trace view controls When running a capillary based experiment, the measured absorbance of each channel is plotted on onscreen in real-time. As the run length increases, the trace window will scale automatically to accommodate the data. Rolling trace view If the Trace view is set to Rolling, only the last section of captured data is displayed. The Max width parameter sets the length (in minutes) of the data to be shown. During long runs, it is sometimes useful to set the Trace view to Rolling as it is more efficient than the Full display and requires less time to update. Rolling trace view controls Rolling absorbance trace view Full trace view On setting the Trace view to Full, the whole of the trace data is displayed during acquisition. The The Max width parameter is disabled. Full trace view controls 13 Full absorbance trace view See also: Plot controls 14 LC system setup To run the ActiPix D100 with a CE method, it is necessary to know two important pieces of information: • Flow rate past the detector • Flow direction past the detector This enables images from the detector to be combined correctly to form trace data. Flow rate and Flow direction values should be entered into the LC method. Flow rate Flow rate of the sample past the detector window. Flow direction Flow direction of the sample past the detector window for each channel (Left to right, or Right to left). The flow direction can be set independently for each channel. Typical liquid chromatography (LC) setup 15 CE system setup To run the ActiPix D100 with a CE method, it is necessary to know three important pieces of information: • Length to detector window • Time to separation start • Flow direction past the detector This enables images from the detector to be combined correctly to form trace data. Length to detector, Separation start and Flow direction values should be entered into the CE method. Length to window The length to the detector window from the capillary inlet. This is necessary to allow correction for analytes that travel slower towards the end of the separation. Typical capillary electrophoresis (CE) setup Separation start Time in seconds from when the detector is triggered and the separation voltage is applied. CE separation start time 16 Flow direction Flow direction of the sample past the detector window for each channel (Left to right, or Right to left). The flow direction can be set independently for each channel. 17 Imaging mode As an area imager, the ActiPix D100 can monitor an area of 7 mm x 9 mm, providing quantitative measusements of UV absorbance at tens of thousands of pixels in real time. Using a dissolution cell, it is possible to post process image data to generate wide area UV absorbance measurements. Plugins are available to enable the importing of image data into the NIH ImageJ application, so further measurements can be performed. It is also possible to capture image sequences of capillaries, and using a suitable method/cartridge file, reprocess the image data to generate absorbance traces. Imaging data recorded as a movie can require a large ammount of disk space (several hundred Megabytes). Imaging files can be replayed and post processed in Offline Mode. All of these components are powerful tools for the researcher to obtain data never before achievable in the ultraviolet. Image Capture and Post Processing The following steps are required for running an imaging experiment: 1. Create an imaging method file using the Method Editor, or modify a default method file. Update the settings as required and save. 2. Load the method file into the Online Mode application, start the detector and run the experiment. 3. Review/process the saved image data using Offline Mode. 18 Online imaging mode ActiPix D100 Online Imaging Mode is used for collecting image data, and is is activated by loading an imaging method file. The display consists of two tabs, the Method settings and a Data view. Method The Method tab contains the settings specified in the imaging method file (as produced by the Method Editor). Various settings in the method tab can be modified on a run by run basis, however these settings are not saved back to the original method file. Main Online Window (Imaging mode - Method) 19 Data The Data tab displays a preview of the image data, as well as settings and controls to allow measurements from the image display. Main Online Window (Imaging mode - Data) See also: Imaging method settings 20 Dissolution imaging When used with a dissolution cartridge, the ActiPix becomes a flexible, multi-purpose tool for formulation development research. It can provide a mechanistic insight into the release active pharmaceutical ingredients from a tablet surface, allow rates of diffusion of species through a membrane to be monitored, and provide information of amorphous to crystalline transformations. Principle of operation During operation, the sample is placed inside the dissolution cartridge. The cartridge is then mounted into the ActiPix Dissolution Imager. Side clamps fix the imaging cartridge in place. Using an external pump, liquid flows through the imager cell, and the resultant dissolution is recorded. Image data can be reviewed to enable time dependent profiling or to identify significant events, e.g. the break away from the surface and subsequent dissolution of a particle of the active pharmaceutical ingredient. The following steps are required for running and processing data from a dissolution imaging experiment: 1. Create a new image method using the Method Editor, or modify a default method file. 2. Load the method file into the Online Mode application, start the detector and collect dissolution image data. 3. Review/process the saved image data using Offline Mode. See also: Creating a dissolution method 21 Creating a dissolution method 1. Start the ActiPix D100 Method Editor. 2. Create a new image method by selecting File→New→ImageMethod. Creating a image new method 3. Modify the methods parameters as desired (adjust the horizontal/vertical pixel binning to the desired image resolution, and select the window start/end lines). Dissolution image method 4. Save the method file. INFORMATION: The sample rate and integration time are automatically adjusted depending the horizontal/vertical pixel binning and the imager window start/end line settings. See also: Dissolution data capture 22 Dissolution data capture To enable the conversion of raw images into absorbance images, it is necessary collect reference dark and Io frames at the beginning of a run. Initially, the cell should only contain the dissolution solution and no tablet. After capturing reference data, the Pause button can be used to suspend data transfer while a tablet is inserted into the cell. Data collection can then resume. The image can then be reprocessed in Offline Mode to generate absorbance data. The following steps are required to capture dissolution image data: 1. Load the Dissolution Cartridge into the ActiPix Sensor Head. 2. Start the ActiPix D100 Online Application. 3. Open the newly created dissolution imaging method using File→Open. Opening dissolution imaging method 4. Press the Preview button and check that the window start and end lines are correct for the area to be imaged. 5. With the Dissolution cell filled with solution, but no tablet, press the Start button. Data will be saved to the location designated in the Output Data group controls. 6. The detector starts by collecting dark and Io frames. These are used later in the offline processing to calculate the absorbance. Data frames 23 7. Wait until the data frames are collected and press the Pause button to stop further data being written to the output image file. Data collection suspended message is displayed in the Messages window. Data collection suspended The detector Status reports that data collection is paused and the Elapsed time resets to zero and flashes. Paused status 8. Remove the Dissolution cartridge and empty the solution from the cell. 9. Place the sample tablet into the cell and load the Dissolution cartridge into the ActiPix Sensor Head. 10. Restart data collection by pressing the Pause button again. The Messages window indicates that data collection has resumed and the Elapsed time couunter restarts. Data collection resumed 11. Flow solution into the cell and capture the image data. Data collection can be stopped at any time by pressing the Stop button. See also: Dissolution data processing 24 Dissolution data processing Using the Offline Mode, it is possible to view, convert, measure and export image data. Converting a dissolution image to absorbance image 1. Start the ActiPix D100 Offline Application. 2. Open the newly captured image file using File→Open making sure to select the d100_image file type. Opening dissolution image file 3. The offline image processing window is displayed. 25 Offline image processing 4. To convert the image to absorbance data, select the Absorbance tab of the Process controls and press the Absorbance button. Choose a filename and press Save. Absorbance conversion 5. Open the newly created absorbance file by selecting File→Open. 6. The dark and Io frames are not modified during the conversion process, however images in the data frames will now be in terms of absorbance values. 26 See also: Generating profiles 27 Generating profiles Offline image processing mode provides a Profile tool for detailed analysis and export of dissolution data over a specific time frame. 1. After loading the absorbance image, use the frame slider (located under the image) to move to a data frame where the profile is to start. 2. Press the Set button next to the Start frame spin box to set the processing start frame. 3. Move the slider to select the stop frame and press the Set button next to the Stop frame spin box. Frame selection 4. The display settings can be adjusted to improve the contrast in the image. 5. Select the desired dimensions (pixels, um or mm) and intensity units (ADC counts or absorbance mAU). For absorbance profiles, select Absorbance mAU. Display settings 6. Holding the SHIFT key and the LEFT mouse button, drag an area on the image to create a red Profile box. 28 Profile box 7. Select the Profile tab from the Process group controls. To output time and position labels select the check boxes. Press the Profile button and select a filename with which to save the profile. Press Save to generate the profile. Profiles are output as comma delimited CSV files, and can be viewed and plotted in MS Excel. 29 Profile output 30 Cartridge Editor ActiPix D100 Cartridge Editor is used to create/modify cartridge definition files. Cartridge files contain the physical parameters that relate to actual ActiPix cartridges, e.g. an LC cartridge file could contain the number capillaries in the cartridge and dimensions of each capillary. The Cartridge Editor can operate in two modes, Basic and Advanced. Basic cartridge editor The basic cartridge editor provides access to a set of preconfigured cartridge settings that can be used for the most experiments. It also provides options for changing settings on a per capillary basis. By default the Cartridge Editor will start in basic mode. Advanced cartridge editor The advanced cartridge editor provides access to all of the cartridge parameters and is only recommended for the expert user. See also: Basic cartridge editor, Advanced cartridge editor, Method Editor 31 Cartridge files ActiPix D100 cartridge files are used in capillary mode. They describe the physical setup of a cartridge, including signal and reference zone locations and capillary dimensions. The software comes pre-loaded with a library of cartridge files for all types and numbers of capillaries. If you a capillary size is not found, it is not a size recommended by Paraytec for use in the ActiPix D100. However, this does not prohibit the user from making a cartridge file to fit their specific research needs. The default cartridge library can be found in C:\Paraytec\ActiPix D100\Cartridge. They are organized by ID and OD size and number of capillaries. See also: Method Files 32 Opening cartridge files In the cartridge folder you will find cartridge files for almost every size capillary. These files have been specifically optimized for best universal performance for many applications. To open an existing cartridge, open the Cartridge Editor and select File→Open... from the menu. Opening a Cartridge File Navigate to C:\Paraytec\ActiPix D100\Cartridge to see the list of default cartridge files. Select a cartridge from the list and select Open. Default Cartridge File List 33 Creating a new cartridge file To create a new cartridge, open the Cartridge Editor and select File→New Capillary Cartridge, a new screen will appear, allowing the new cartridge to be configured. Creating a New Capillary Cartridge A description of the cartridge can be entered, and the cartridge type selected using the drop down tab listing. Cartridge Editor 34 Cartridge type selection Select the required cartridge type from the list of the standard ActiPix cartridges. The cartridge type number should match the number on the front of the actual cartridge. On selecting a cartridge type, the corresponding channel parameters are display. Cartridge Type Selection Editing channel parameters Once the capillary sizes have been selected on the next screen you will be able to edit the sizes of the capillary if necessary. The channel parameters can be adjusted according to the actual physical configuration of the cartridge. Editing channel parameters 35 For each cartridge type common ID/OD settings are shown. However, it is possible to fine tune the channel parameters using the Custom... setting. Custom capillary settings After configuring the new cartridge it can be saved, ready for use in a method. 36 Basic cartridge editor The basic cartridge editor provides access to a set of preconfigured cartridge settings that can be used for the most experiments. It also provides options for changing settings on a per capillary basis. By default the Cartridge Editor starts in basic editing mode. See also: Advanced cartridge editor 37 Advanced cartridge editor The advanced cartridge editor provides access to all of the cartridge parameters and is only recommended for the expert user. Advanced Cartridge Editor 38 CAUTION: Changing the advanced parameters can cause a cartridge file to fail. Please contact your Distributor or Paraytec for further advice. See also: Basic cartridge editor 39 Method Editor ActiPix D100 Method Editor is used to create/modify experimental method files. Method files are created as LC, CE, sizing or imaging, and contain the parameters required to run a particular experiment. The method files been have named and created to allow operation in many standard experiments. Other experiments can be performed under a particular method, and so the method type refers to the physical design of the cartridge and not to the experiments capable of being run. Capillary methods LC method - methods utilizing any pumping system that has a constant velocity regardless of volumetric flow rate. Can use one or more windows or capillaries. CE method - methods utilizing voltage for analyte movement and thus a non constant velocity. Can use one or more windows or capillaries. Sizing method - specific method for pumping through a single capillary with two windows that automatically calculates the hydrodynamic radius of a single analyte injected. Imaging methods Image method - method allowing capture of images to create a movie. Dependent upon total viewing area and not velocity. Images can be further processed offline for dissolution profiling and absorbance conversion. See also: Cartridge Editor 40 Method files ActiPix D100 method files determine the mode of operation of the ActiPix, and enable the configuration of experimental parameters. Method files are used in both imaging and capillary mode. There are four different types of method file used by the ActiPix D100: • LC method used for running liquid chromatography experiments. • CE method used for running capillary electrophoresis experiments. • Sizing method used for running protien sizing experiments. • Image method used for dissolution experiments. The first three methods are capillary based, and contain both experiment and cartridge settings (loaded from a cartridge file). An image method is a special case used for capturing image data directly from the sensor. The method folder comes with a standard method library. These methods are identified by the type and number of capillaries. The standard method library can be found in C:\Paraytec\ActiPix D100\Method. Capillary method file structure See also: Cartridge files 41 Opening method files To open an existing method, start the Method Editor and select File→Open from the menu. Opening a Method File Navigate to C:\Paraytec\ActiPix D100\Method to see the list of default method files. Select a method from the list and select Open. Default Method File List The method is loaded into the Method Editor where the parameters can be modified. The method can then be saved using a different file name. 42 Creating a new method Start the Method Editor and from the menu select File→New, and choose the method type to create. Creating a new method Modify the method parameters, load a cartridge file (if creating a capillary based method) and save. The method can then be loaded into the ActiPix D100 Online Mode application and used for data collection. New method 43 Capillary specific settings (CE, LC and Sizing) Described below are descriptions of each parameter field to be used as a guide for creating or modifying your own capillary methods. Method Details Parameter Type Description Location Created by Date created Description Method type, e.g. CE, LC, Sizing or Imaging (non-editable) A user supplied description of the method Location where the method file was originally saved Name of the method author/creator Date the method was created (non-editable) Timing Details Parameter Runtime Trigger in Trigger out Trigger out time Trigger out duration Sample rate Filter type Time constant Description total run time for data collection Wait for external rising/falling edge trigger signal to start data collection (input 5V pk) Set the trigger out pulse type: none, positive or negative Trigger out time in seconds Trigger out pulse duration in seconds Output absorbance data sample rate (Note: this cannot be higher than the data collection frame rate) Smoothing filter selection: none, exponential or moving average Filter time constant selection, larger values provides more smoothing Output Data Parameter Create new folder Auto index filename Auto restart detector Folder Filename File type Description Create a new data folder automatically for each run Auto index the filename by appending the run number Automatically restart the detector after a run completes to allow multiple runs Select the folder to save the output data Set the output file name • Trace and Frame files - ActiPix D100 proprietary format. • NetCDF (network Common Data Form) machine independent data format accepted for import by most instrument and chromatography software. 44 • csv (comma delimited) - compatible with Microsoft Excel. Experimental Conditions Parameter Sample Instrument Filter Type Buffer (CE, Sizing) Injection time (CE) Injection pressure (CE) Injection voltage (CE) Buffer A (LC) Buffer B (LC) Injection volume (LC, Description a description about the sample information about instrument used (serial number, model, etc.) name of the method used by the instrument for future reference selected filter wavelength information about the buffer record the injection time pressure and time used for hydrodynamic injections voltage and time used for injections information about the buffer A information about the buffer B injection volume and time Gradient rate (LC) Column name (LC) Notes rate of gradient and conditions the name of the column for data reporting additional notes about experiment or sample Instrument method Sizing) Settings Parameter Cartridge Lamp stablisation Flow direction Separation start (CE) Length to window (CE) Flow rate (LC) Description A cartridge file MUST be chosen. Files are typically stored in C:\Paraytec\ActiPix D100\Cartridge. This file provides the parameters necessary to determine how many and location of signal areas Set the lamp stablisation On/Off. Lamp stablisation can reduce the baseline drift as the sensor head warms up. Select the flow direction of the sample across the detector. Time in seconds from when the detector is triggered, and the voltage is applied to start the separation See also: CE system setup Analytes travel slower towards the end of the separation. The exact length of the capillary from inlet to 1st detection window must be input so that data frames are collected slower as the run progresses to accommodate for the slower moving analytes. The Run Time is also related to this factor. See also: CE system setup Analytes travel at a constant velocity, the exact flow rate past the detection window is required to determine how many data frames belong to each data point. This value is independent of the Run Time. 45 Capillary ID (Sizing) Capillary OD (Sizing) Analogue scale Analogue offset Channels Value set from the cartridge file. Value set from the cartridge file. Full scale analogue output in terms of mAU/V. For a setting of 1000 mAU/V and an offset of 0 mAU the analogue output would be 0 V for 0 mAU, and 1 V for 1000mAU. There is a 5 s delay between data collection and analogue output. Analogue output offset measured in mAU. For the above scale, an offset of 500mAU would give an output of 0 V for -500 mAU, 0.5 V for 0 mAU, and 1 V for 500 mAU. More than one channel, or signal, can be collected simultaneously. Even though multiple capillaries are loaded into the cartridge, data does not have to be collected from each one but only the ones chosen here. Directions match flow direction as facing the sensor. Unique to sizing, the flow direction past the second detection window is opposite the first window in a sizing cartridge only. Other cartridges provide different options. • Channel - Channel index. • Enable - Enables a particular channel. • Flow direction - Select the flow direction of the sample across the detector for a particular channel. • Length to window (CE) - The length of the capillary from inlet to 1st detection window. • Analogue - Select the channel to link to the analogue output 46 Imaging method settings Described below are descriptions of each parameter field to be used as a guide for creating or modifying your own imaging methods. Method Details Parameter Type Description Location Created by Date created Description Method type, e.g. CE, LC, Sizing or Imaging (non-editable) A user supplied description of the method Location where the method file was originally saved Name of the method author/creator Date the method was created (non-editable) Timing Details Parameter Runtime Trigger in Trigger out Trigger out time Trigger out duration Description total run time for data collection Wait for external rising/falling edge trigger signal to start data collection (input 5V pk) Set the trigger out pulse type: none, positive or negatve Trigger out time in seconds Trigger out pulse duration in seconds Output Data Parameter Create new folder Auto index filename Auto restart detector Folder Filename Estimated file size Description Create a new data folder automatically for each run Auto index the filename by appending the run number Automatically restart the detector after a run completes to allow multiple runs Select the folder to save the output data Set the output file name An estimate of the file size in MB based on the imager settings and the runtime Lamp Parameter Lamp intensity Power Description Manual/auto adjustment of the lamp power level Manually set the lamp power level 47 Imager Parameter Dimensions Pixel time Top row Bottom row Height Integration time Image subsample Image fps Frame time Description Sets the dimensions of the imager controls Sets the pixel size for image collection Sets the top row of the imager readout Sets the bottom row of the imager readout Displays the total height of the imager readout Sets the imager integration time Sets the imager subsample interval saving data to disk Displays the frames per second of the saved data Displays the time between frames 48 Folder settings The Method Editor settings can be accessed from Tools→Settings menu. Select the preferred folders for opening and saving Method and Cartridge files. Method editor folders settings See also: Default data folder 49 Online Mode ActiPix D100 Online Mode is used for running LC, CE, sizing or imaging experiments and capturing data in the form of traces, frames or images. When operating in online mode, method files provide the settings for the experiment. See also: Offline Mode 50 Detector status window The detector status window provides feedback from the detector whilst operating. • Messages window displays status updates from the detector. • Image window shows the capillary image location and zone assignment. • Hardware displays detector hardware status indicators, and is useful for diagnosing faults with the system. • Information window shows parameters specific to the current run, such as lamp power, pixel value, data buffer, frame rate and sensor head temperature. • Status field indicates the overall state of the detector and can be Ready, Busy or Error. It also displays the elapsed and remaining time of the current run. Detector Status (Error) Detector Status (Ready) INFORMATION: The detector will not collect data until all Hardware status indicators are healthy (green ticks), when this occurs the hardware indicators are replaced by the Information window. See also: Detector controls 51 Checking detector status Make sure the Sensor cable and USB are connected to the ActiPix D100 Control Box, and a cartridge is loaded into the sensor head. Switch on the ActiPix D100 Control Box and start the Online Mode application. When the application loads, check that the Detector Status window shows that the ActiPix is in a Ready state. This may take several seconds. Detector Status (Ready) If the sensor head is not connected to the control box, or another error condition occurs, the status indicator will show an Error. Data cannot be collected from the detector if it is in an error state. See also: Detector controls 52 Detector controls The detector can be started/stopped using the control buttons on the toolbar. The detector controls consist of Preview, Start, Pause, Stop and Extend runtime. Detector controls Preview - starts the detector, but does not save the captured data. Start - starts the detector and saves captured data. Pause - temporarily stops writing data to file and resets the elapsed time counter. Stop - stops the detector. Extend runtime - increases the experiment runtime. See also: Previewing data, Collecting data 53 Previewing data Before beginning with a run, careful planning of the experiment, set up and parameters must be undertaken. The ActiPix provides a useful diagnostic and time saving tool with the Preview mode. Quick experiments or condition changes can be monitored without having to record a data file. Care should be taken however, to ensure that the Preview mode is not accidentally used during a valuable data file collection. To begin a run, choose either the Preview or Start button from the tool bar. With either mode the system will go through a series of calibration routines prior to data plotting. This applies only during absorbance collection and not during image mode. The Preview button is used to view the data from the detector without saving it to file. This is useful when setting up and testing the detector prior to an experiment or run. Previewing the detector output Press the Preview button to activate the detector. The image from the detector is shown in the Data tab. During data collection, the detector produces an audible click each time the lamp flashes, this is normal. Status messages are displayed while the detector is running and the detector Status shows the blue Busy indicator. Please note, the image may vary depending on the cartridge type loaded into the Sensor Head. The detector will operate for the time specified in the Runtime setting in the method, and stop automatically when this has elapsed. It is also possible to stop the detector manually by pressing the Stop button. Manually stopping the detector See also: Offline mode 54 Collecting data Whilst operating in Preview mode, no data is saved to file. To save data to file, it is necessary to start the detector using the Start button. Start the detector and save data Data is stored from the detector in real-time. Therefore, it is necessary to select the folder and filename to which the output data is stored prior to starting the detector. This information can be specified in the Output Data section of the Method tab. Click the Browse... button to chose a location and type a filename in the Filename field. For convenience, a data directory is automatically setup when the ActiPix software is installed C:\Paraytec\ActiPix D100\Data\. Output data location Having selected a filename and folder for the data to be saved, press the Start button to initialise the detector and collect image data. CAUTION: Image data files can take up a large amount of disk space. It is recommended that when operating in Imaging Mode, the runtime is kept relatively short. See also: Default data folder 55 Extending runtime During a recorded run it is sometimes necessary to extend the run time. This is easily done by pressing the button and entering the additional time desired. Extending the runtime Runtime clock The runtime clock is shown as both Remaining and Elapsed time. Extending the runtime will adjust both of these clocks. The Runtime clock begins counting when the detector initiates its calibration routine at the start of a run, and the first dark images are collected. In capillary mode, there is a 30 s delay before valid trace data is displayed, while the detector is being calibrated. During this period a flat line is shown on the display. Runtime clock See also: Offline mode 56 Trace view controls When running a capillary based experiment, the measured absorbance of each channel is plotted on onscreen in real-time. As the run length increases, the trace window will scale automatically to accommodate the data. Rolling trace view If the Trace view is set to Rolling, only the last section of captured data is displayed. The Max width parameter sets the length (in minutes) of the data to be shown. During long runs, it is sometimes useful to set the Trace view to Rolling as it is more efficient than the Full display and requires less time to update. Rolling trace view controls Rolling absorbance trace view Full trace view On setting the Trace view to Full, the whole of the trace data is displayed during acquisition. The The Max width parameter is disabled. Full trace view controls 57 Full absorbance trace view See also: Plot controls 58 Plot controls Pan, zoom and channel selection features are available on absorbance plots in both Online and Offline modes. Pan and zoom mouse controls Zoom in Using the Left mouse button drag a box around the area of interest. The plot will automatically zoom to this area. Multiple zooms can be applied. Absorbance plot zoom selection 59 Zoom out To zoom out click once with the Right mouse button. Each right click will zoom out one level. Absorbance plot zoomed in Panning To pan the trace, press the Centre wheel button and drag the mouse to move the plot in any direction. Releasing the button resets the time and absorbance axes. Absorbance plot panning 60 Channel selection By clicking on a channel number located to the right of the absorbance plot, any channel of choice can be turned off to show only select channels. To turn back on, click the channel button again. Absorbance plot channel selection 61 Settings The Online Mode settings can be accessed from Tools→Settings menu. Folders Select the preferred folders for saving Data and opening Method files. Online mode folders settings See also: Default data folder 62 Connection Configure the D100 Control Box to use a USB or Ethernet connection. Connection settings See also: Configuring the ethernet connection 63 Upgrade Update the D100 Control Box firmware. Firmware upgrade settings See also: Updating the D100 firmware 64 Diagnostic Log Set the diagnostic logging level. Diagnostic log settings See also: Diagnostic logging 65 Offline Mode ActiPix D100 Offline Mode is used reviewing previously captured data, e.g. traces files from an LC experiment. This mode also allows the reprocessing of captured image data. The Online Mode application generates four data file types that can be viewed and reprocessed in Offline Mode: • Trace files - can be loaded into the offline trace view, exported as csv/NetCDF or reprocessed to generate a sizing analysis. • Analysis files - can be viewed offline, adjusted and formatted into printed reports. • Image files - can be viewed and further processed to generate profiles or absorbance trace data. • Frame files - can be viewed and processed to optimize experimental parameters and generate trace data. Double clicking the icon any of the above file types will launch a new instance of the Offline Mode application. INFORMATION: File icon types match the colour of the mode they are used with. All data files (.d100_trace; .d100_analysis; .d100_image; .d100_frame) appear with the green ActiPix icon and can be opened in Offline Mode. See also: Online Mode 66 Default data folder For convenience, the ActiPix software is installed with a predefined data directory. By default, this is located in the C:\Paraytec\ActiPix D100\Data\ folder. Unless a new data folder has been selected, any data collected in Online Mode will be placed in this folder. INFORMATION: If a particular file cannot be located, a search can initiated from the Windows Start Menu using the *.d100_trace, *.d100_analysis, *.d100_image or *.d100_frame search terms. 67 Viewing traces In Offline Mode, one or more absorbance traces can be loaded into a plot window. To open a trace, select File→Open... from the menu and navigate to location of the trace file. In the Open File... dialog, make sure Trace-Files type is selected. Click the trace file and press the Open button. Opening a trace file The absorbance trace is loaded, along with its method data that was saved when it was recorded. Viewing a trace 68 Add trace Further traces can be added/removed from the plot window using the Add Trace, Remove Trace and Clear All buttons. Add/remove/clear trace controls Pressing the Add Trace button, launches a Open File... dialog. One or more trace files can be selected and loaded into the plot window. Adding a trace to a plot Remove trace Removes a selected trace from the plot window. The trace to be removed is chosen from a drop down list that is presented when the Remove Trace button is pressed. Clear all Clears all the traces from the plot window. See also: Plot controls 69 Plot controls Pan, zoom and channel selection features are available on absorbance plots in both Online and Offline modes. Pan and zoom mouse controls Zoom in Using the Left mouse button drag a box around the area of interest. The plot will automatically zoom to this area. Multiple zooms can be applied. Absorbance plot zoom selection 70 Zoom out To zoom out click once with the Right mouse button. Each right click will zoom out one level. Absorbance plot zoomed in Panning To pan the trace, press the Centre wheel button and drag the mouse to move the plot in any direction. Releasing the button resets the time and absorbance axes. Absorbance plot panning 71 Channel selection By clicking on a channel number located to the right of the absorbance plot, any channel of choice can be turned off to show only select channels. To turn back on, click the channel button again. Absorbance plot channel selection 72 Offline image view In Offline Mode, raw and absorbance images can be loaded. To open an image, select File→Open... from the menu and navigate to location of the image file. In the Open File... dialog, make sure Image-Files type is selected. Click the image file and press the Open button. Image window Offline image window Parameter 2D tab 3D tab Pixel intensity/Absorbance scale Frame slider Frame index Frame time Description Selects the 2D (flat) view. Selects the 3D view. Colour bar intensity scale for the image. Adjusts the displayed image frame. Index of the current image frame. Time of the current image frame. Playback The Playback controls allows a sequence of captured images to be displayed. Parameter Play Description Starts playback of the image movie. Playback starts from the position indicated by Start frame in the Frame Selection controls. Playback automatically stops when the Stop frame is reached. 73 Loop Stop Speed Continuously loops through the image sequence between the Start frame and the Stop frame of the Frame Selection controls. Stops playback of the image movie. Adjusts the speed of the playback. Frame Selection Frame start/stop playback range selection and image capture. Parameter Start frame Stop frame Set Frame block Capture Description Start frame for playback and profile export functions. Stop frame for playback and profile export functions. Sets the start/stop frames for playback and profile export functions. Number of frames to average when using the profile export functions. Captures a copy of the current image frame and allows the image to be saved in PNG format. Cursor Cursor coordinates and measurements. Parameter Cursor (x,y) Pixel intensity Marker (x,y) Delta (x,y) Distance Description Cursor x,y position. Units are set by Dimensions setting in the Display controls. Pixel intensity of the cursor position. Units are set by Intensity units setting in the Display controls. Marker cross x,y position. The marker position is set by a single LEFT mouse click in the image display. Cursor x,y position relative to the marker position. Distance from the marker position to the cursor. Display Parameter Black White Color map Dimensions Origin Intensity units Description Adjusts the image contrast. Adjusts the image brightness. Selects the image colour map overlay. Selects the image display/cursor dimensions (pixels, µm, mm). Sets the origin relative to the current image window or the imager chip. All measurements are made relative to the origin. Sets the intensity scale units, ADC counts for raw images and mAU for converted absorbance images. 74 Time Marker The time marker can be used to time stamp specific events in the image sequence. Time relative to the marker is displayed. Parameter Marker time Relative time Set Marker Goto Marker Description Frame time stamp stored when the Set Marker button is pressed. Current frame time relative to the stored Marker time. Sets the Marker time. Displays the image frame at the Marker time. Timing Parameter Dark frames Io frames Data frames Frame rate Description Indicator is active when the current image frame is a dark frame. Indicator is active when the current image frame is an Io frame. Indicator is active when the current image frame is data frame. Frame rate of the image sequence. Region Selection Parameter Top row Bottom row Left column Right column Selection height Selection width Description Top limit of the selected ROI. Bottom limit of the selected ROI. Left limit of the selected ROI. Right limit of the selected ROI. Selected region height. Selected region width. Profile Parameter Time label Position label Profile Mean Description Adds time labels to the export data. Adds position labels to the export data. Output profile data. Output mean data. Absorbance Parameter Output data Absorbance Description Selects absorbance, offset, normalise or linearise data for export. Output absorbance data. 75 Correlate Parameter Interpolation Search range Output data Correlate Description Adjusts the interpolation factor. Adjusts the search range in pixels. Selects output data type, position, profile or correlation. Output correlation data. See also: Dissolution data processing, Generating profiles 76 2D image controls Pan and zoom features are available on images in both Online and Offline modes. Offline mode also provides region selection and marker placement features. 2D image pan and zoom mouse controls Zoom in Using the Left mouse button drag a box around the area of interest. The image will automatically zoom to this area. Multiple zooms can be applied. 2D image zoom region selection Zoom out To zoom out click once with the Right mouse button. Each right click will zoom out one level. Pan To pan the image, press the Centre wheel button and drag the mouse to move the image in any direction. Panning is only available after a zoom has been applied. 77 Region selection A region of interest (ROI) can be selected by holding the SHIFT key and Left mouse button down whilst dragging. The region selection is shown as a Red rectangle. Initially, the whole image is placed within the selection region. 2D image region selection Marker placement A reference marker can be placed by a single Left mouse click in the image. The reference marker is a Red cross. 2D image marker placement See also: 3D image controls 78 3D image controls Pan, zoom, rotate and tilt features are available on 3D images in Offline mode. The 3D image represents the Red region selection area of the 2D image. 3D image mouse controls Zoom in Pushing the Centre mouse wheel forwards causes the image to zoom in. 3D image zoom in Zoom out Pushing the Centre mouse wheel backwards causes the image to zoom in. 3D image zoom out 79 Rotate Pressing the Left mouse button whilst dragging in any direction causes the image to rotate. Tilt Holding the SHIFT key and Left mouse button whilst dragging tilts the image. Pan Holding the CTRL key and Left mouse button whilst dragging pans the image. See also: 2D image controls 80 Reviewing analysis data Analysis files will show the all the results and parameters (i.e. method and cartridge files) used for an analysis. Currently, only Sizing analysis files are available. Sizing analysis For a Sizing analysis file, the Analysis tab contains information relating to the peak-fitting performed on the data and the calculation of the hydrodynamic radius and diffusion coefficient. Sizing analysis data The Results tab presents the results of the calculation and the related parameters. Sizing analysis results 81 Adjusting parameters If required, it is possible to modify the temperature, viscosity and capillary radius parameters that are used in the sizing analysis, and recalculate the hydrodynamic radius. Changing a field in the Analysis Parameters causes the hydrodynamic radius to be adjusted automatically. Analysis parameters Peak fitting The peak fitting uses a combined linear/gaussian least squares fit, and is designed to operate on samples with a single component. The All tab shows the original data and the resultant fit parameters for both channels. Sizing peak traces 82 The Channel 1 and Channel 2 tabs show each individual channel respectively, along with the residual errors between the fit and the data. Residual error traces Printed report The results of the sizing analysis can also be printed in the form of a sizing report. See also: Plot controls 83 Viewing frames In Offline Mode it is possible to view the data from a frame file captured during a capillary based experiment. To open a frame file, select File→Open... from the menu and navigate to location of the file. In the Open File... dialog, make sure Frame-Files type is selected. Click the image file and press the Open button. Frame files are an intermediate data format that is somewhere between image data and absorbance traces. They contain both spatial and temporal information, and represent the signal and reference zone information collected in capillary mode experiments. Viewing frame files INFORMATION: Frame files are primarily used for diagnostic purposes and are not required for standard applications. However, they can be used for advanced applications such as peak focusing and velocity optimisation. For further information about the use of frame files contact Paraytec support. See also: Frame files 84 Data reprocessing Several tools are provided for data reprocessing: • Process Image • Process Trace • Process Frame To use a reprocessing tool, select Tools→Process from the menu and select the type of processing to be performed. File reprocessing Input file type Image Frame Trace Output file type Frame, trace, csv, NetCDF Trace, csv, NetCDF Analysis (sizing) See also: Process image, Process frame, Process trace 85 Image to trace conversion In Offline Mode, it is possible to reprocess captured capillary images and convert them to trace files. If a sizing method is used, an analysis results page is also generated. Loading an image To open the image to trace conversion tool, select Tools→Process→Process Image... from the menu. Press the Load... button to open an image file. Loading image Setting up a method On the Method tab load a method file with an appropriate cartridge for reprocessing the image. Adjust the method and select the desired output data format(s). It is also important to set the experimental conditions under which the image was collected, i.e. flow rates and channel directions, or the trace data may be generated incorrectly. 86 Generating trace data On the Data tab, press the Preview or Start button to generate the trace from the image. Pressing the Start button causes the trace data to be written to disk in the format selected in the method. Data trace INFORMATION: Image reprocessing can be a complex process, and may require adjustments to cartridge files using the Advanced Cartridge Editor. Please contact your Distributor or Paraytec if you require further advice. 87 Processing frames In Offline Mode, it is possible to reprocess captured frames and output them a trace files. This can be useful if the trace data needs to be optimised for a particular flow rate. Loading a frame To open the offline frame processing tool, select Tools→Process→Process Frame... from the menu. Press the Load... button to open a frame. Processing frames Generating trace data To generate trace data, select the Method tab and choose the required output data format(s). It is also important to set the experimental conditions, i.e. flow rates and channel directions, or the trace data may be generated incorrectly. On the Frame tab, press the Preview or Start button to generate the trace from the frame. Pressing the Start button causes the trace data to be written to disk in the format selected in the method. Parameter optimisation Frame reprocessing allows certain parameters to be optimised to produce maximised peak responses. For LC methods the Flowrate can optimised, and for CE methods the Length and Separation start parameters can be optimised. INFORMATION: Frame processing can be used to optimize and even determine the flow rate of a sample through the detector. Please contact your Distributor or Paraytec if you require further information on frame processing and the optimisation tools. 88 Offline sizing analysis In Offline Mode, it is possible to reprocess traces captured during a sizing experiment and produce a new analysis report. Loading traces To open the offline sizing analysis tool, select Tools→Process→Process Trace... from the menu. Press the Load... button to open a trace file for each channel. Loading traces Adjusting analysis parameters On the Method tab, adjust the Analysis Parameters if desired. These can be further adjusted on the Analysis tab. Analysis parameters Selecting the data On the Data tab, the Start and End time of the peak fitting can be adjusted if required. This can be useful if the trace includes artefacts that are not related to the sample peaks. To select a new start time press the SHIFT key and the Left mouse button in the data plot window. The End time point can be adjusted by holding the SHIFT key pressing the Right mouse button. 89 Adjusting the selection region 90 Markers are placed on the graph to show the selected region. If no adjustments are made the all of the data is included in the peak fitting and analysis. Region selection Reviewing the analysis Select Analysis→Run Sizing Analysis from the menu to generate the sizing analysis results in the Analysis tab. Sizing analysis results 91 Folder settings The Offline Mode settings can be accessed from Tools→Settings menu. Select the preferred folders for opening and saving Data files. Offline mode folders settings See also: Default data folder 92 File types The ActiPix D100 uses several different file types: Configuration Configuration files are used to setup and run an experiment using the ActiPix D100. Method files determine the mode of operation of the ActiPix, and enable the configuration of experimental parameters. Method files are used in both imaging and capillary mode. Cartridge files are used in capillary mode. They describe the physical setup of a cartridge, including signal and reference zone locations and capillary dimensions. Data capture Trace files are absorbance data plots, captured in Online Capillary Mode. The can be view and post processed using Offline tools. Image files contain UV imaging data captured in Online Imaging Mode. Image files can be converted to absorbance image files using Offline dissolution tools. Frame files are intermediate files that are generated from images captured in a capillary based experiment. They can be used for advanced applications such as peak focusing and velocity optimization Absorbance data from a capillary based experiment can also be saved/exported in comma delimited .csv and .NetCDF format. Post processing ActiPix D100 analysis files contain the results of a specific data analysis operation. Currently, only Sizing analysis files can be saved. Additional Firmware files are used for upgrading the D100 Control Box software. Software and firmware updates are made available on the Paraytec website. http://www.paraytec.com 93 Analysis files ActiPix D100 analysis files contain the results of a specific data analysis operation. Currently, only Sizing analysis files can be saved. See also: Cartridge files, Method files 94 Cartridge files ActiPix D100 cartridge files are used in capillary mode. They describe the physical setup of a cartridge, including signal and reference zone locations and capillary dimensions. The software comes pre-loaded with a library of cartridge files for all types and numbers of capillaries. If you a capillary size is not found, it is not a size recommended by Paraytec for use in the ActiPix D100. However, this does not prohibit the user from making a cartridge file to fit their specific research needs. The default cartridge library can be found in C:\Paraytec\ActiPix D100\Cartridge. They are organized by ID and OD size and number of capillaries. See also: Method Files 95 Frame files ActiPix D100 frame files are intermediate files that are generated from images captured in a capillary based experiment. They can be used for advanced applications such as peak focusing and velocity optimisation. A frame file can be selected as an output file type in the Output Data section of the method. Frame file selection See also: Trace files, Image files 96 Image files ActiPix D100 image files contain UV imaging data captured in Online Imaging Mode. Image files can be converted to absorbance image files using Offline dissolution tools. See also: Trace files, Frame files 97 Method files ActiPix D100 method files determine the mode of operation of the ActiPix, and enable the configuration of experimental parameters. Method files are used in both imaging and capillary mode. There are four different types of method file used by the ActiPix D100: • LC method used for running liquid chromatography experiments. • CE method used for running capillary electrophoresis experiments. • Sizing method used for running protien sizing experiments. • Image method used for dissolution experiments. The first three methods are capillary based, and contain both experiment and cartridge settings (loaded from a cartridge file). An image method is a special case used for capturing image data directly from the sensor. The method folder comes with a standard method library. These methods are identified by the type and number of capillaries. The standard method library can be found in C:\Paraytec\ActiPix D100\Method. Capillary method file structure See also: Cartridge files 98 Trace files ActiPix D100 trace files contain absorbance data, captured in Online Capillary Mode. The can be view and post processed using Offline tools. A trace file can be selected as an output file type in the Output Data section of the method. Trace file selection See also: Frame files, Image files 99 Calculator tools The ActiPix software includes a number of calculation tools for helping with data conversion and other tasks. They can be accessed from the Tools→Calculators menu option in both Online and Offline mode. Currently four calculation tools are available: • Absorbance conversion • Extinction coefficient • Flow rate conversion • Hydrodynamic radius 100 Absorbance conversion Converts an ActiPix absorbance value (measured in mAU) for a given capillary size to an equivalent 1 cm curvette absorbance (measured in AU). Absorbance conversion tool See also: Extinction coefficient, Flow rate conversion, Hydrodynamic radius 101 Extinction coefficient Calculates the extinction coefficient and concentration based on the measured absorbance for a given capillary size. Extinction coefficient tool See also: Absorbance conversion, Flow rate conversion, Hydrodynamic radius 102 Flow rate conversion Converts between linear and volumetric flow rates for a given size of capillary. Flow rate conversion tool See also: Absorbance conversion, Extinction coefficient, Hydrodynamic radius 103 Hydrodynamic radius Calculates the hydrodynamic radius and diffusion coefficient based on the variance and time between two sizing peaks, for a given capillary size. Hydrodynamic radius tool See also: Absorbance conversion, Extinction coefficient, Flow rate conversion 104 Advanced operation The follow section describes various advanced topics that are not generally required for everyday use of the ActiPix D100. They include the following: • Updating the D100 Control Box firmware • Configuring the D100 Control Box ethernet connection • Diagnostic logging See also: Updating the D100 firmware, Configuring the Ethernet, Diagnostic logging 105 Updating the D100 firmware The ActiPix D100 Control Box firmware may require updating periodically as new features are added to the ActiPix software. Incompatible firmware message Whilst operating in Online Mode, if an incompatible firmware message is shown the control box firmware must be upgraded before data can be collected. Incompatible firmware dialog Updating the firmware To update the firmware: 1. Download the latest firmware from the Paraytec website, http://www.paraytec.com/downloads/software. The firmware is packaged in a compressed zip file, which also contains the latest PC software. 2. Unzip the software to a convenient location. 3. Switch on the ActiPix D100 Control Box, and connect it to the PC via the USB cable. Make sure that the network connection status is valid (i.e. the status reports Ready, or a green tick is shown next to the Network indicator). 4. Start the ActiPix D100 Online Mode application, and select Tools→Settings... from the menu to open the Settings dialog. 5. Select the Upgrade tab, click Browse... and locate the firmware file that was unzipped. The firmware file has a .d100_firmware extension. Select the file and open it. 6. Click the Download button to start the upgrade. This may take several minutes to complete. Do not close the upgrade dialog box or switch the ActiPix D100 Control Box off, or the update may fail. 106 Updating the firmware 7. When programming of the firmware has completed, the Upgrade completed message is shown. The ActiPix D100 Control Box should be switched off and on again, to enable the new firmware to run. Firmware update complete 107 INFORMATION:The software and firmware version numbers can be checked by selecting Help→About menu in the ActiPix D100 Online Mode. 108 Configuring the ethernet connection Communication between the ActiPix D100 Control Box and the ActiPix Online Mode software can be setup using a USB or Ethernet connection. The simplest method of connection is via the USB, however, it is possible to connect the D100 Control Box to a network using the Ethernet connection. Default configuration The ActiPix D100 Control Box is assigned a static IP address. The default factory settings are: Network settings Parameter IP address Subnet mask Broadcast address Description 192.168.1.1 255.255.255.0 192.168.1.255 These default settings may be restored at any time by holding in the reset switch while powering on the unit. The reset switch is accessible through a small hole in the front panel. The switch must be held for 30 seconds after power is applied. Resetting the ethernet configuration 109 Modifying the settings To update the network settings: 1. Start the Online Mode application and select Tools→Settings... from the menu. 2. Select the Connection tab. If the connection to the D100 Control Box fails, the D100 Ethernet Settings are not displayed. If the connection is good, the current settings are shown. 3. Adjust the settings and press the Update button to save the new configuration. 4. Switch the D100 Control Box off and on again to initialise with the new settings. Connection settings 110 Diagnostic logging The Online Mode provides a logging feature that allows different levels of diagnostic data to be captured when operating the ActiPix. The purpose of the log is to provide Paraytec support with useful information in diagnosing issues with the operation of the ActiPix D100. To access the log settings select Tools→Settings... from the Online Mode menu, and click the Diagnostic Log tab. Diagnostic log settings Setting log level Four logging levels are provided: • None - no diagnostic logging. • Basic - basic runtime status messages and method data is logged. • Medium - runtime status messages with frames and method data. • Full - full diagnostic log, including runtime status messages with image/trace/frames and method data. If activated, a log is written each time the Start button is pressed the detector collects data. Only a single log file can exist at any time. Any previous log will be overwritten each time a new run is started. Locating the log file It may be necessary to locate the log file, for example if you need to email it to Paraytec support. The log file is located in the users Application Data\Paraytec folder. It can be easily located by selecting the Open log file folder... link on the Diagnostic Log settings page. 111 Removing the log file The log file can be removed by pressing the Remove Log File button on the Diagnostic Log settings page. INFORMATION: Diagnostic log files can be very large, especially when full logging is enabled. No personal information is collected during the logging process. 112