Transcript
Louise Cole Apr 2013
Zeiss Transmitted light, wide‐field Fluorescence and Deconvolution microscope
Start‐up
Uncover microscope. Do not put dust cover on the floor.
Plug in the correct camera. The default camera is the AxioCam HRm (monochrome camera) for fluorescence imaging (left hand side of microscope). The AxioCam HRc (colour camera) is suitable for transmitted light imaging (top of microscope).
Turn on the microscope (green switch on the power supply box; see right image, bottom unit) and round silver switch on the left hand side of the microscope (see below). The TFT display on the microscope will come on.
If require ApoTome.2, switch on ApoTome. 2 power supply (middle unit). NB. Important: If want to use “Smart Set‐up” in Zen Blue software. Switch on the ApoTome.2 power supply now, even if not using ApoTome.2.
Turn on the UV lamp (if required for fluorescence imaging)(top unit). Note HBO lamp hours (changed at 300h). Wait for the LAMP green light to come on. If it doesn’t, contact the Microscopy Officer.
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Turn on the computer (Login Name: User, password: decon).
Start the Zen Blue software (it takes a few minutes to
load).
ZEN ‐ ZEISS Efficient Navigation – is the single user interface that you will see on all new imaging systems from Carl Zeiss.
To set up the microscope for fluorescence imaging
Load microscope slide onto stage. Note XY movement of stage is MANUAL Use the touch screen on the RHS of the microscope to select objectives and filters. Always start with a low objective ie. X5 and move up and down in order of objectives. NB. The nosepiece that carries the objectives is MOTORIZED so must NOT be moved manually. Alternatively you can use the buttons on the focus wheel (filters on left, objectives on the right wheel). Note also the RL (reflected light) and TL (transmitted light) controls on the far right of the TFT (above right).
Looking down the microscope:
Push both pins in all the way to view your sample through the eyepieces (far right image). If the top pin is pushed half way in, half the light goes to the eyepieces and half goes to the colour camera. Check that the pin on the LHS is also out.
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Open UV light shutter via RL (reflected light) illumination button on right of the TFT display or the RL button on the right hand side of microscope (see below). Note you can use the attenuator (lower image on right) to cut down UV light reaching specimen if sample very bright or if it is bleaching rapidly. Position 1 = no attenuation. Find specimen and make sure it is in focus. NB. Close shutter when not imaging to reduce sample bleaching.
Image acquisition
Send the image to the correct camera on the microscope. Setup as follows;
Monochrome camera setup
Bottom pin all the way out.
Colour camera setup Bottom pin in and top pin all the way out. Top pin halfway = half to camera and half to the eyepieces.
You are now ready to acquire FL images using the Zen software
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Overview of the Zen screen layout: On the Left Tool Area the user finds the tools for image acquisition and image processing, easily accessible via the Main tabs. All functions needed to control the microscope can be found on the Locate tab. To acquire images use the Acquisition tools. Arranged from top to bottom these tools follow the logic of the experimental workflow. The area for viewing and interacting with images is centered in the middle of the Main Application window: the Center Screen Area. Each image (once acquired) can be displayed and/or analyzed with many view options available through "view tabs" which can be found on the left side of the image. According to the chosen view tab, the required view controls appear in View control blocks below each image. File management and data handling tools are found in the Right Tool Area.
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Select light/dark from design dropdown list in workspace configuration
To zoom in or out of the workspace move the slider to left or right
Left Hand Tool Area
The Left Tool Area contains all tools for system operation, image acquisition and image processing. The major functions are organized in the four Main tool tabs: Set exposure, Live, Continuous and Snap Locate: camera inspection in transmitted and epifluorescence light Locate Tab Click on Locate tab in the Left tool area. The currently set light path of the microscope is displayed. Note: In most FL cases, you can proceed straight to using the Acquisition Tab 5
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Depending on the level of motorization, the following parts can be controlled (Interactive control): Shutter Reflected Light: This turns the rear port mirror to guide the light to the specimen or to block it from the specimen. Reflector: Push and click reflector cube can be selected via graphical pop‐up menu. Objective: Objectives can be selected via graphical pop‐up menu. Condenser: Numerical aperture of the condenser is set via input box or slider. Turret position (filter) selected from graphical pop‐up menu (only for motorized condensers). Field Stop Filter: Opening of luminous‐field diaphragm (transmitted light) can be set via input box or slider. Transmitted Light Lamp: Transmission values for attenuation filter (transmitted) are set via input box or slider for the front or rear filter wheel in accordance with the available filter steps. Transmitted light is switched on / off via ON button in the Transmitted Light frame, setting of light intensity can be varied via input box or slider. 3200 K color temperature for photo documentation can be switched on via 3200 K button in the Transmitted Light frame. Shutter Transmitted Light: This function opens or closes the internal shutter for transmitted light. Under Camera Tab Under mode, select appropriate resolution. Standard frame size is 1300 x1040 pixels (2.75 MB) for fluorescence imaging. NB. You can crop image but at the expense of resolution. NB. Always check the scalings. Use slow live speed. This can be done under acquisition tab also.
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Acquisition
You can load a predefined acquisition configuration from the Experiment Manager field by selecting a saved configuration in the pull down menu. To delete a configuration press the Delete button.
Smart Set‐up
Use Smart Setup for rapid selection of single or multi‐channel set‐up. The tool Smart Setup is an intuitive, user‐friendly interface which can be used for almost all standard applications. It configures all system hardware for a chosen set of dyes (as well as TL setup) Note: Smart Setup offers you support when configuring multichannel acquisition experiments. Select the fluorescent dyes and contrast techniques that you want to include in your experiment from a large dye database. Smart Setup takes the configuration of your microscope hardware and the properties of the selected dyes into account. Based on this information it makes one or more suggestions for acquisition. You can adopt these into your experiment as required and make further changes to them there. Smart Setup also configures the motorized components of your system for the acquisition of multichannel images. It does not, however, influence any camera parameters (e.g. exposure time or resolution) and also does not change any parameters of other acquisition dimensions (e.g. Z‐stack, time series).
Simply choose the dye(s) used from the list dialog and assign signal display colour(s)
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Select Add, select Probe, Add and OK when finished Save method under Experiment Manager. Once asterisk disappears, method has been saved. Defining Channels: The Channel tool displays every Channel that is currently open in the Light Path tool. The Tracks name and the used Channels for that track are indicated. Each Channel that is highlighted will be displayed in the Channel Configuration display area, which allows for individual settings of each Channel. A Channel will only be active when the Snap button is pressed, if its check box is ticked. Changes in the selection of the Channels in the Channel tool will automatically update the selection in the Light Path tool and vice versa.
Action buttons Action buttons provide control for how the image is acquired. They have the following functions, regardless of the imaging mode: Set exposure optimises the camera exposure. Set exposure can be disrupted by pressing the Stop button, to which the set exposure button changes upon activation
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Live uses the maximum speed for a given image frame (and zoom setting) for all active channels. To disrupt Live acquisition, the Stop button has to be pressed into which the Live button changes after its activation. Click on Live button. The live mode will be activated. You will recognise the live mode by the green light signal and by the hatched tab in the document bar. In the Centre Screen Area you will see the camera live image. By default the live image shows a cross hair helping to navigate on the sample.
Select Set exposure button. Alternatively set the exposure manually using the slider
Select linear display mappings (reset) on the display window and find the optimal focus on the monitor. (NB. You can select Best Fit and Min/Max to alter your display settings if required.) Use the dynamic range indicator in the Dimensions tab to highlight overexposed (too bright) regions (indicated by red pixels) and underexposed (too dark) areas (in blue).
If you want to adjust the gamma; on the Display tab click the 0.45 button. The display curve will be adapted to a gamma value of 0.45. This will set the optimum colour presentation. NB. The gamma value makes it possible to correct the display of images on computer screens which do not allow the linear display of gray value curves. By changing the gamma value you can emphasize certain intensity ranges within your image when it is displayed on the screen. A value <1 emphasizes the ranges of medium pixel intensity (medium gray values), while a value >1 emphasizes the dark and bright pixel intensities and therefore increases the contrast. The recommended settings are 0.8 for fluorescence images, 1.2 for phase contrast or DIC images and 0.45 for true color images. Please bear in mind that a "correct" gamma value setting depends on numerous parameters, such as screen settings, ambient brightness, etc., and a universal setting cannot therefore be given.
Click on Snap to take the image
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Viewing images
The Center Screen area can be set up to hold 1, 2 or 3 Image Containers. Clicking the Expose Mode button in the top right corner of the Image Display opens a field of thumbnails in the image display with one large thumbnail for each open image. Whenever you lose track of your open images, open the Expose Mode and click once in the image you are looking for and it will be immediately brought to the foreground of the Image Display.
2D View: This function allows to: ‐ display a single image in frame mode, ‐ display multiple channel images in superimposed mode.
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Split View: This function allows to display the individual channels of a multi channel image as well as the superimposed image. NB. Cf. Use of splitter – see below image Gallery View: This function allows to: − display images (Z‐Stack, time series, combination of both) side by side in a tiled fashion, − add data relevant to the displayed images (Z‐Stack slice distance, time of acquisition) − extract a subset of images from the original stack and store the result as a new image (controls for this function are in the Processing tab function Copy → Subset). Ortho View: This function allows to − display a Z‐Stack of images in an orthogonal view − measure distances in three dimensions The Information View is a view type that shows a summary information sheet of all relevant image acquisition parameters. Parts of it can be edited – e.g. the Notes field.
The Dimensions View Options control block holds the tools to modify the image display with respect to the (multiple) dimensions of the image data set. For z‐stacks: The sections (slices) in the dimensions can be scrolled with sliders (Z‐Position) and also can be directly addressed with setting numbers in the spin‐boxes next to the sliders. The small button on the right hand side of the spin‐boxes (Z‐Position) starts and stops an animation in the respective dimension. This basic 11
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animation cannot be customized. For all other player‐functionality refer to the Player View Block.
Zoom In the Dimensions View Options control block, there are the Zoom and Tools function lines related to changing the zoom factor of a displayed image. Any changes on these elements affect the displayed image immediately. The zoom function can be performed online. In the Display View Option control block, the intensity minimum can set to the Black value and the intensity maximum to the White value of the image using the Min/Max button. Clicking on the Best Fit button sets the intensity minimum to Black and percentile value and the intensity maximum to White and percentile value. Activating the Auto check box sets the intensity minimum and maximum values automatically. With the spin‐boxes you can set the lower and upper percentile values. The Black, the Gamma and the White values of the displayed image can be adjusted individually. With the Ch buttons, the effect of the settings can be restricted to an individual channel or be applied to all channels. By default, the settings apply to all channels simultaneously. The parameters can be changed using the spin‐boxes – or directly by typing in numbers in the number‐field. With the Reset button, the original settings are easily re‐set. The settings in this View control block apply only to the Image Display and are NOT changed in the original data set. To write the changed settings to the image data set, use the Apply Display Curve in the Processing tab. Graphics: This function allows to − select from a set of drawing functions such as rectangles and arrows, − add a scale bar to the image, as well as text annotations, − use a set of interactive measurement functions for length, angle, area and size, − add a text box that displays the coordinate Functional Description: The graphics function uses a plane separate from the image plane (the graphics plane) and therefore does not change the content of the image(s). Any changes done with this function are effective immediately. The overlay graphics can be stored in the images or separately in a file from where they can be re‐loaded. 12
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Saving Images See right tool area for Data Management and Storage
Open Images: In the Right Tool Area the Open Images section is displaying the images which are loaded in the system memory. Double clicking brings the image to the front. Files can be saved and deleted/closed on this section. Multiple images can be selected by holding down the CTRL key on the keyboard and clicking on the list entries. The whole section can be hidden by clicking on the black triangle at the left side of the panel. Once hidden, a click on the right boarder of ZEN lets the Documents panel slide back into the application. All files can be displayed in three ways by using the buttons underneath the section. 1. Text View 2. Thumbnail View 3. Big Preview The Re‐use function is available in the ZEN Image Browser: Hardware parameters can be loaded without opening the files by clicking the Re‐use button. NB. Star ratings ***
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Multidimensional Acquisition Settings The settings for multidimensional acquisition are accomplished within the tool groups eg. Z‐stack, time series. If available the settings are displayed beside the function, e.g. how many images are taken for the time series. Tool bars within the tool groups only become available if the box of a multidimensional function is checked; otherwise the tool bar is not displayed. The graphical display field above the Start Experiment button shows a representation of the experiment type with its dimensions is show. It also lists the estimated size of the final multidimensional image.
Taking a Z‐stack
The Z‐Stack tool tab is only available if the Z‐stack checkbox is ticked in the Multi‐dimensional acquisition field. NB. Select All Channels per Slice if collecting multi‐ channel images The way in which the z‐stack is acquired can be selected by pressing either the First/Last or Center mode buttons. The modes differ in the way the first and last images of the stack are selected.
Press the First/Last mode in the definition panel. Press the Live action button and focus onto the specimen. Adjust the focus to the top of the specimen or where you would like the z‐stack to start. Select Set first. Focus to the bottom of the specimen or where you should like the z‐stack to stop. Select Set last. These focus positions are then indicated next to Set First and Set Last buttons. The Range display box shows the total height of the stack in micrometers.
The Slices input box displays the number of slices of the stack. This number can be edited by typing or using the arrow keys. Changing the value for Slices automatically adjusts the Interval in a way that the total Range for the stack is maintained.
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The Interval input box displays the z‐distance (interval) between slices in μm. This number can be edited by typing or using the arrow keys. Changing the value for the Interval automatically adjusts the Number of Slices while keeping the total Range for the stack within the possibilities of fitting the calculated number of slices into the Range. The total Range may therefore vary to higher and lower numbers around the initial value of the Range.
Pressing the Optimal button sets Interval to match the Nyquist criteria (2 fold over sampling) and accordingly adjusts the Slices to keep the Range within the limits defined by Set First and Set Last. The optimal interval is displayed in the button in μm.
Within the Keep display field (only available in Show all mode) you have the option to switch between Keep Interval and Keep Slice. If Keep Interval is checked, the number of slices is adjusted to a new Range defined pressing Set First or Set Last. Likewise, when Keep Slice is checked, the value for the Interval between the slices is adjusted to a new Range defined by pressing Set First or Set Last.
When Start Experiment is pressed the system will perform a z‐stack
Start Auto Z‐ configuration to define top and bottom of specimen for easy z‐stack selection
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Exporting images Export images with the Export dialog in the File menu, the acquired data can be directly exported to various file types • •
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Select the image to be exported. Click on the Export menu Item in the File menu of the menu bar. This opens the Export window or Processing Tab. Under Method, select the format to which the image is to be exported to eg. Single File Export Select the file type which the image is to be exported under Parameters. Choose a compression level. For some file formats lossless compression or various other compression levels are available. The degree of losses for the image quality is listed according to the type of compression. Next select export destination and file name. Click Apply to complete the export process. When stacks or time series are exported, each frame is stored as an individual image. Under Options and Saving: The Auto Save panel enables you to instruct the program to save the results automatically and continuously during the measurement process. All data recorded until a, for example, a software failure, will be available this way. This function is recommended when performing long measurements. If the Auto Save check box is ticked you can specify the directory in the Directory selection box, to which the measurement should be saved.
NB. .raw data files are still saved in the directory specified under Save raw data during measurement. However, if the Auto Save option is active, temporary raw data files will be saved permanently and not only temporarily and will be available if a software failure occurs.
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Batch processing and export
This is useful for copying all parameters for export from one image to another. See Copy parameters and paste parameters. Make sure appropriate image(s) output storage path is selected in Browse
Various file formats are supported for export in pulldown menu for Filetype. Under Method parameters, you can also select burn‐in annotations and create folders.
When ready to batch export, simply select Run Batch
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Processing Maximum Intensity Projection The Maximum Intensity Projection function generates a maximum intensity projection image along the z‐, time‐ or channel dimension of a multidimensional image data set. • To open the Maximum Intensity Projection Processing tool, select it from the list in the Processing main tab. • Click on the triangle buttons to show or hide the Method Parameters and Preview panels of the function. • Select the Input Image with the Select button and choose the dimension along which you want to generate a maximum intensity projection from the Coordinate pull down menu (x, time, channel) in the Settings panel. • Clicking the Apply button creates the maximum intensity projection in a new image document, the name which is specified in the Output image display of the Select field. The maximum intensity projection creates an output image whose pixels contain the maximum value over all images in the stack at the particular pixel location.
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Deconvolution software
Deconvolution (removal of out of focus blur) is done using a mathematical algorithm. It gives an image with less noise, better definition and higher resolution.
A Z‐stack of your sample taken using the optimal distance and with known appropriate scalings is required.
Select Z‐stack image that you require to deconvolve
Select Deconvolution in Processing Tab
Choose appropriate algorithm (see below) to use eg. Inverse Filter seems to work well in most instances. The deconvolution process can take time if there are a number of channels and z‐slices. Once process complete. Select Apply. Observe and save the result.
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Deconvolution (defaults) This method allows you to use 4 different algorithms for deconvolution.
Simple, very fast (Nearest Neighbor) button: Executes the fast Nearest Neighbor method using default parameters. To use it, click on this button and then on Apply.
Better, fast (Regularized Inverse Filter) button: Executes the Regularized Inverse Filter algorithm for image enhancement. To use it, click on this button and then on Apply.
Good, medium speed (Fast Iterative) button: Executes the Fast Iterative restoration method. To use it, click on this button and then on Apply.
Excellent, slow (Constrained Iterative) button: Executes the Constrained Iterative quantitative restoration method. To use it, click on this button and then on Apply.
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Glossary Nearest Neighbor: The Nearest Neighbor method uses the simplest and fastest algorithm (Castleman, K.R., Digital Image Processing, Prentice‐Hall, 1979). Its function is based on subtraction of the out‐of‐focus information in each plane of a stack, taking the neighboring sections above and below the corrected Z‐plane into account. This method is applied sequentially to each plane of the entire 3D stack. It allows you to enhance contrast quickly, even if image stacks have not been put together optimally. Regularized Inverse Filter: The inverse filter is a genuine 3D method and generally achieves better results than the Nearest Neighbor algorithm. It essentially involves dividing the Fourier transformation ("FT") of the volume by the FT of the PSF, which can be performed very quickly. In the real space this corresponds to deconvolution. In addition, a statistical method ("General Cross Validation) is applied, which determines the noise component of the image and automatically sets the restoration strength to the optimum level in line with this. This process is also known as regularization. The method is very well suited to the processing of several image stacks in order to preselect images for the application of the iterative "high‐end" method. Z‐stacks must, however, have been acquired at the correct (Nyquist) distance. The additional acquisition of Z‐planes above and below the structure of interest is recommended. Fast Iterative: The "Fast Iterative" method is an iterative restoration method that uses only one iteration per convolution step (see Meinel, E. S.: Origins of linear and nonlinear recursive restoration algorithms. J.Opt.Soc.Am, 3 (6), 1986, 787‐799). No regularization is used in this case. Due to the fast processing and convergence after just a few iterations, this method is suitable in particular for the processing of larger time‐lapse images. The results of the method can quickly lead to good results and remove most of the out‐of‐focus light. They do not, however, create quantitative brightness conditions in the image. If undersampled images are present, artifact formation may also result. Constrained Iterative: The best image quality is achieved using the iterative maximum likelihood algorithm (see Schaefer et al.: "Generalized approach for accelerated maximum likelihood based image restoration applied to three‐dimensional fluorescence microscopy", J. of Microscopy, Vol. 204, Pt 2, November 2001, pp. 99ff.). This algorithm is able to calculate light from various focal planes back to its place of origin. Consequently, with this method it is possible to derive the 3D structure from fluorescence images with the correct brightness distribution and to visualize optical sections. It is also possible for missing information to be partially restored from neighboring voxels. The spatial resolution can be increased without artifacts up to a theoretical limit (one voxel). It is essential for Z‐stacks to have been acquired in accordance with Nyquist. Acquiring sufficient planes above and below the structure of interest is also imperative for achieving good results. As this is a complex mathematical method, the calculation can take longer, depending on the image size and the PC being used.
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Transmitted light set‐up
Select TL brightfield set‐up in Smart Setup. Make sure that you have set up properly for Kohler illumination with the objective that you require for imaging. Check condenser aperture diaphragm setting.
Under Locate tab Select colour camera
White Balance: Only active if a colour camera has been selected. ‐ Auto button: Sets the white balance automatically. ‐ Pick button: Select a point in the image as a reference point for the white balance. ‐ 3200K button: Sets the color temperature to 3200K. ‐ 5500K button: Sets the color temperature to 5500K. Show Channels checkbox: Only visible if the Show All mode is activated. Activated: Displays 3 sliders to allow each color channel to be set precisely. 22
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Color Offset slider: Here you can set the color temperature. Saturation slider: Here you can set the saturation. Color Mode button ‐ RGB: Sets the color mode to RGB. ‐ B/W: Sets the color mode to B/W. Live Speed dropdown list: Here you can select the speed of the live mode. You can choose between Slow, Medium and Fast. Resolution dropdown list: Here you can select the camera resolution. Binning dropdown list: Here you can select the binning mode for the camera. Acquisition ROI section: In this section you can define a particular area that you want to be acquired.
After clicking on the Refresh Overview button, in the window on the left you will see a Preview of your sample under the camera. The Pixel Size below the preview window indicates the size of a pixel depending on the scaling set. The blue frame indicates the selected area. The entire preview window is highlighted by default. To change the area, move the mouse pointer over the blue frame. The mouse pointer will appear as a double‐headed arrow. If you hold down the mouse button and move the mouse, you can change the size of the frame and in this way highlight a certain area on the sample. To move the area, position the mouse pointer inside the frame. The mouse pointer will appear as a four‐headed arrow. If you hold down the mouse button and move the mouse, the frame can be moved. Maximize button: Maximizes the frame to the maximum size in the preview window. Center button: Positions a frame that has been changed precisely at the center of the image. Size input field: Here you can enter the size of the frame that you want. Depending on the camera type available, select from the predefined sizes in the dropdown list (1388x1040, 1024x1024, 512x512, 256x256, 128x128). Alternatively you also have the option of entering the Size in X and Y, and the top left Start position. You can change the values by increments using the arrow buttons next to the input windows or you can enter the desired values directly using the keyboard. To do this, you must activate the number already present using the cursor. Complete the entry by using the Tab or Enter key. Offset input field: Here you can enter the start position for the frame, measured from the top left corner. Refresh Overview button: Adopts the image from the camera into the preview window. Post‐Processing section
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Depending on which camera you are using, some functions are not visible. The most important functions are explained in the section below. Black Reference checkbox: Only active if you have defined a black reference via the Define button. Activated: Applies the measured black reference to the image. Deactivated: The measured black reference is not used. The reference image is retained. Define button: Automatically defines the black reference. To do this a reference image is acquired, from which the black reference is measured. The light path to the camera must be closed and the lamp must be switched on for the measurement. The measurement lasts for several seconds. The Black Reference checkbox is then activated automatically. Shading Correction Activated: Applies the measured shading correction to the image. Deactivated: The measured shading correction is not used. The reference image is retained. Define button: Automatically defines the shading correction. To do this you will acquire a reference image for the shading correction. An empty image without structures is required to measure the shading image. Move the slide to an empty position on the sample with no artifacts in the image. The Shading Correction checkbox is activated automatically after the definition. Enable Noise Filter checkbox: Activated: The Limit slider and the spin box/input field are active. Enter the limit for the noise filter using the slider or spin box/input field. Enable Unsharp Mask checkbox: Activated: Emphasizes edges in the image more clearly.
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Shutting down the microscope
Check microscope bookings. If there is another user coming within 1 hour then leave the system on. If not, shut down completely. If you finish your session earlier than planned please send an email to
[email protected] to say the microscope is free OR call the next user. If working out of normal working hours (ie. Mon‐Fri 9 am – 5 pm), please do not leave the system on for the next user unless you are sure they are coming!
Note UV lamp hours. Turn off the Hg lamp and log your usage on the sheet provided. Report any errors on sheet and to Dr Louise Cole directly (in person or by email:
[email protected])
Save all images and exit the software.
Remove slide from stage. If used an oil objective, please wipe excess oil from objective using lens tissue ONLY
Leave microscope with a low objective in place and clean for other users. Switch off microscope by the silver switch and at the power supply. Turn off ApoTome.2 if turned on.
Unplug both cameras. Shut down the computer.
Cover the microscope – rubber covers for eyepieces and transmitted light as well as the large blue cover for microscope.
Leave area clean and tidy. Turn any lights off in room as you leave.
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