Manual 14552335

Transcript

UC BSD Light Microscopy Core Facility Vytas Bindokas, Director Olympus IX70 Multi-parameter Fluorescence Microscope Operations Manual (version 1.0 February ‘09) The IX70 microscope is a manual stand with the ability to collect multi-color fluorescence, differential interference contrast (DIC) and total internal reflection fluorescence (TIRF), as well as provide optical sectioning (z-axis stepping) and time lapse imaging. It is equipped with a high sensitivity, high resolution chilled CCD camera, a green/red or blue/red emission beam splitter for simultaneous two color capture and MetaMorph + MetaFluor software to control the system. Examples of types of images that can be captured range from simple image capture (bright field or fluorescence), sequential capture of up to 4 fluorescent probes, time lapse imaging, (relatively) high speed imaging, CFP-YFP FRET studies, ratiometric indicator dyes such the calcium probe Fura-2, etc. Software provides a wide range of image capture and image processing capabilities. MetaFluor is optimized for real-time (ratio) image capture with real time region of interest (ROI) intensity/ ratio/calibrated data display. MetaMorph is a more general image capture, processing, morphometry package. Both packages allow images to be automatically collected and annotated. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 2 Turning on the System 1) fire the arc lamp under the microscope table (make sure nothing else is on) #1 always! 2) turn on the brightfield bulb power switch on the microscope base (#2) #2 #3 3) turn on the filter changer box (#3) 4) power up the computer (#4) 5) ONLY if you are using TIRF, fire the Ar laser 6) log on and start either MetaMorph or MetaFluor 7) write down your start time on the log sheet #4 #5 for TIRF ONLY Shutting down the System The arc lamp must be on for at least 20-30 min prior to turning it off, you must wait that long before turning it back on, too. Firing the arc greatly shortens the lamp life, so it’s best to leave it running during the day when the schedule shows other users are coming—check the schedule, the last person MUST shut down the system. If there is another user after you: 1) Clean any oil or water immersion objectives you used during your session. 2) Log off from your Windows session 3) If you used the TIRF laser (#5), turn that off; leave everything else (microscope and computer) on. 4) Write down the time on the log sheet. If you are the last person scheduled: 1) Clean any oil or water immersion objectives you used during your session. 2) Log off your Windows session, then do another cntl-alt-del and choose the SHUTDOWN button. 3) Turn off the TIRF laser (#5) if used, the filter changer box (#3), brightfield power (#2), and the arc lamp (#1). 4) Cover the scope with the plastic cover. 5) Write down the time on the log sheet (how else will the next person know if system is cool enough to restart?) PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 3 System Anatomy The IX70 microscope is a manually operated microscope with automated digital image capture. The objectives, emission filter cubes, DIC prisms, and focus are all controlled manually. Image capture and excitation filters are controlled through the software. DIC shear adjustment screw
Objective turret Filter turret Brightfield button and intensity slider (on front left) Brightfield power switch (on back of base)
Viewport knob Focus knob Fig. 1 Controls on microscope base To change the magnification, first lower the objectives by focusing the turret all the way down with the focus knob (so as not to disturb the sample or bash the objectives into the stage). Then turn the turret from the base. The objectives are arranged clockwise in order of increasing magnification. Be aware that when you change the objective MetaMorph will not register the change unless you choose the magnification from the taskbar as well, making scalebars and measurements incorrect. We now have more objectives than positions in the nosepiece. Objectives listed below as Available are not normally kept on the microscope but are available for use, just ask the staff to put it on for you. DO NOT try to change objectives yourself. We must enforce the China Shop Rule of “you broke it, you bought it”… Dropping a lens is very bad. NA refers to Numerical Aperture, the resolving ability of the lens; bigger NA = higher resolution. Available 2x/NA0.04 dry. This lens yields a field of view over 5mm and yields useful fluorescence images for very large preps. The brightfield condenser will require defocus (upwards) to PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 4 produce illumination over the entire field of view. Be careful when trying to focus this lens on materials high above the stage—you may cause the high power objectives to push up on the stage plate/specimen and risk damage to optics. Ask for advice on how to use this lens. Available 4x/0.16 dry. 10x/0.3 dry 20x/0.5 dry. NOTE this lens has 3 correction caps available to improve images through #1 coverslips, 1-mm thick glass dishes (or a glass slide), and also for plastic (1.1-mm thick). Caps are unscrewed and replaced with the desired cap. Be sure to place the cap in a plastic holder; holders are located on the shelf to the right of the scope). Note this lens has poor performance in UV (lousy DAPI signal). 40x/0.6 LWD dry objective. This has a correction collar for glass/plastic and thickness (0-2 mm). (not on scope routinely; place into 40x oil position when needed). Available 40x/1.35 oil UV-optimized. This is a very high resolution objective the best for use with UV dyes, especially the calcium dye, fura-2. Moderate magnification and high NA make this an especially bright objective. 60x/1.45 oil, TIRF. The very high NA produces the highest resolution under regular epi fluorescence. This objective is also optimized to permit the specialized illumination method called Total Internal Reflection Fluorescence (TIRF; AKA, evanescent wave). TIRF uses laser light to illuminate only the footprint of a cell attached to a glass coverslip (i.e., the bottom 40 to 200 nm), showing nothing of the portions above that zone. The high NA yields optical sectioning that approaches that on confocal microscopes. Available 60x/1.2 water. Use a hanging drop of water, not oil, on this objective. It provides a greater working distance than the 60-oil, and when viewing thick preparations it minimizes depth-related optical distortions. 100x/1.3 oil. Not as bright as either the 46x or 60x oil objectives, but still a nice high mag objective. Available 150x/1.4 oil, TIRF The Rolls Royce of objectives. This is a high resolution, high mag objective that can also be used in TIRF applications. You will need to manually calibrate pixel sizes when you use this lens. Brightfield and Fluorescence modes There are four positions for fluorescence filter cubes in the manual turret below the objectives. The filter position is changed by rotating the wheel under the objective turret (see Fig. 2, “Filter change wheel”). We have four cubes permanently stationed in the turret: DAPI, FITC long pass, the Quad mirror and Cy3. The DAPI, FITC long pass and Cy3 cubes will work for both the camera and the oculars (they have both excitation and emission filters built in) but the Quad mirror works for the camera only!! The Quad mirror (ID tag= “4”) is for collecting multiple color channels with the Acquire multiple wavelengths menu in MetaMorph. It will collect DAPI, FITC (narrow), TexRed and Cy5 channels only when the appropriate filter button in the taskbar is pushed. The cube in the light path is indicated by the colored ID tab, Manual shutter Filter wheel Filter list Mag changer Fig. 2 right side of scope PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 5 decoded by the sheet taped to the side of the scope. If you need filters not currently installed, please ask for assistance in swapping out the cubes (we have more choices than available turret positions). A manual shutter slide is also located on the side (Fig. 2 “manual shutter”). Push toward the wall to block fluorescence (but be aware this will prevent you from snapping a picture too!). There is a 1.5x tube lens that will enlarge the image destined for your eyes (see “mag changer” Fig 2). However, the image quality is poor and a bright reflection renders images in the camera port in need of post hoc corrections. Use the 1-X (pushed in) position for all quality work. A brightfield lamp is available (above the scope) and is controlled via the square button on the front left-hand side of the scope base plus the voltage up/down slider (see Fig 1). DIC: The microscope is equipped with Differential Inference Contrast (DIC) optics to generate contrast in unstained materials. To setup DIC or use is in ocular mode, one needs to use the analyzer slider (must be inserted under the filter turret) and the optical path should be optimized (Kohler illumination, described below). The DIC analyzer (polarizer) for image capture is mounted in the emission changer. The visual DIC analyzer is NOT normally present (i.e., by request) since users tend to leave it in the optical path and cut fluorescence intensities by mistake (must be pulled out for all work apart from visual work). DIC uses a pair of prisms, one above the slide, one below. The upper one is found in the wheel on the condenser (Fig. 3), and should be changed manually to best match the Brightfield lamp housing objective in use. Due to space+cost limitations we have only 3 (upper) prisms: 10x (use for 10x), 40x oil (use for 20x, 40x and 100x), and 60x TIRF/oil (use with 60x lenses). The prisms match the objectives they are named for, so DIC quality will be best with 10, 40, 60x lenses, and acceptable with others. The Field aperture lower shift prism is constant for all objectives. It has a knurled screw that allows you change the contrast and apparent direction of light hitting your sample (also called the shear). Adjust the screw to get a uniform background and details (see Fig. 1 “DIC shear adjustment screw”). Before you display the Centering Focus knob DIC image, you should optimize the optical path to get the screws most even illumination possible. To achieve Kohler illumination, first find your sample on the slide and make sure Prism it is in focus. Then focus the condenser onto that plane by selection Condenser closing the field aperture (Fig. 3) and using the condenser aperture focus knob to get the sharpest image of the closed iris leaves (should be a bright hexagon of light). Use the centering screws (Fig. 3) to center the iris image, then open the field aperture Fig. 3 The condenser iris until it just vanishes from view. The iris on the condenser itself (Fig. 3 “condenser aperture) can be closed to enhance the image contrast (at the price of brightness; you will also see more dirt on your slide the more you close it down). Note that plastic degrades the DIC effect, and while you may get enough contrast for an image, the quality is far worse than for cells on a glass coverslip. It is also best to remove the lids from plastic dishes, especially if there is condensation. A plastic lid with built-in glass window is available. Silicon oil PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 6 can also be layered on top of the media to prevent evaporation without image distortion. DO NOT USE SILICON OIL WITH THE WARNER DISHES! It will ruin the rubber gaskets and cause leaks. We have added a transmitted light shutter to gain better control of DIC capture and fluorescence sequences. The brightfield bulb can be turned on with the power switch and button and set to the desired brightness with the intensity slider, and then controlled with the shutter. The shutter opens automatically when the DIC button is selected and closes when a fluorescence button is selected or once an image is collected. There appears some resistance of program control vs. manual microscope control where you may have to hit the power-on button and scroll voltage up and then try the power button again to get the lamp to behave (it will perform once you show it who’s boss, I guess; It’s a MM bug. This isn’t an issue in MetaFluor software). Fluorescence: It’s important that no other electronics be powered up when firing the arc lamp; it can create a power surge that will fry other components (a very bad thing). Since this is a manual microscope, choices in the software require changes to the microscope that must be made by hand. We have added pop-up window prompts to MetaMorph to guide you in changing filter cubes, etc. Fluorescence uses excitation filters to excite your probe(s) with a narrow band of wavelengths, a dichroic mirror to reflect the excitation onto your sample while allowing the longer fluorescence to pass trough, and a barrier (AKA emission) filter to block stray excitation light and limit the output color. Most filter cubes use dedicated excitation and emission (barrier) filters. This is the case with the DAPI, FITC (wide) and Cy3 cubes in the turret. These filters will work for both the camera and the oculars (by eye or by image). A few cubes (like fura-2, and the cubes in the “FRET” set) require proper exciters or barrier filters to be set by the filter wheels (Sutter controller) through the software. These filters also work by both by eye or by camera. However, the new quad mirror requires the filter to be changed thought the software. It will image DAPI, FITC (narrow), Cy3 or Cy5, depending on the taskbar button selected, but it will ONLY work through the camera. You will NOT see anything by eye but a lot of very bright light!! Use the single color cubes to find your sample and only use the quad if you need Cy5 or if you are capturing multiple wavelengths with the Acquire Multiple Wavelengths dialog. Regardless of the filters used, it’s highly recommended that changes of all filter cubes (for the color of fluorescence) be initiated through the taskbar. To choose a color channel, click the taskbar button for the wavelength you want, then follow the on-screen prompt or filter list to rotate the manual filter turret under the objectives to the correct position (see “filter wheel,” Fig. 1 and 2). Press “Continue” on the pop-up window to toggle the shutter (if necessary). Alternatively, you can learn the settings of the Sutter Lambda 10-2 filter changer box located to the right of the microscope (long, bottom-most controller with keypad). Be aware that not setting the exciter or emitter filters correctly can blast your sample, or worse, your poor eyes, with the full arc lamp output. The Sutter filter changer operates a shutter to limit illumination to actual image collection (to minimize sample bleaching and phototoxicity), a high-speed excitation filter changer, and a high-speed emission (camera-side) filter changer. {optional reading /details} By default, it is under computer control (parallel port). Manual mode requires you to press the “Local” 1% slider button on the top right of the keypad. Press either “F1” of “F2” to select a 25% slider filter changer to command. Note that the shutter is on F1; pressing the “shutter” will toggle the light on/off. The other keys address the ten filter positions per wheel (numbered 0 through 9). Fig. 4 Neutral density filters PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 7 Importantly, there are two filter sliders to attenuate light (ND = neutral density filters) one on the filter changer just in front of the arc lamp housing, and one in front of the filter changer (Fig. 4). The filter changer (back) slider imposes an ND2 (100x less light, or 1% illumination), and the scope (front) slider produces 25% illumination. You should use as little light for live cell imaging as possible (both sliders pushed in) to forestall photodamage. Most imaging can be done with only the 25% slider pushed in (well suited for your eyes, too). You can use full light for dim samples, but be aware that you are probably getting high autofluorescence and probes will bleach quickly. And remember to always check your controls with the same illumination intensities! Position 0 1 2 3 4 5 6 7 8 9 Whl A (F1) (excitation) Closed Open 340 380+nd1.0 440/20 480/40 560/40 640/20 Open Open Whl B (F2) (emission) Open 450/65 485/40 535/30 polarizer colorRED colorGRN colorBLU 630/60 682/22 {optional details} For those who want to know the details, the following chart reports the contents of the 10 filter slots in each wheel: The Whl-A 340/380 positions are used to conduct calcium imaging studies with the indicator fura-2. The 440 exciter and Whl-B 485/535 emission filters are used for real-time FRET probes such as cameleon sensors. Both of these methods will require the proper dichroic mirror (filter cube) to be placed into the microscope (not routinely installed at this moment). We are swapping them to the #3 filter cube position since few high-speed studies require combinations of fura-2 plus FRET plus DIC. ColorRED is (600nm LP), colorGRN (500-575 BP), and colorBLU (500nm SP) filters are intended for (additive) RGB color capture, but can be used in custom filter designs as well. Need special filters? Ask the staff about them… we have more filters than turret positions, so CFP, YFP, Chameleon FRET, FURA, TexRed and Cy7 cubes are available but not installed. How-To Do (non-ratio) Fluorescence Image Capture Much of the configuration of the microscope is manual, but the software will queue you with pop-up windows to change what needs to be changed by hand. Turn on the computer and logon; start MetaMorph. MetaMorph (see Figure below) is configured to always start with the 20x objective in place and cubes set to DIC. The software will pop up a window reminding you to change the objective to the 20x to start. The TASKBAR that pops up contains most of the buttons to select an objective, rotate in filter cubes, select light paths, operate the camera, etc. If the taskbar does not open, you can find it under Journal
Taskbars
Load Taskbar. Choose the “main” taskbar. When you choose a function on the taskbar, a small window will pop up to tell/remind you if you need to change something manually. 1) You should always use the main menu (top bar) to open the ACQUIRE
acquire menu. This menu gives you access to camera controls. Other ACQUIRE menu options include acquisition of multiple wavelengths, Z series, timelapse, etc. Many of those menus close after you use them, so you’ll have to go back and re-open them for more data. If you are collecting multiple PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 8 fluorophores, you can make the task easier by using the Acquire Multiple Wavelengths menu. This automates collection of up to 6 “channels” of data, each stored with a fixed/different exposure (or autoexposure). To use the Acquire multiple wavelengths menu, pick the number of probes to collect and assign the identity and order in the lower section. Pressing the upper right camera icon collects the entire sequence and stores it in one memory stack. For this to work, the microscope has to be set up with the quad mirror in place in the lower filter turret. If you want to use the individual color cubes and change cubes between captures, pressing the cameras next to each probe collects only that probe. To collect color (histology) images, make sure the order is colorRed. colorGRN, colorBLU. Gain is assigned from the main “acquire” window and is applied to all channels. Independent exposure times can be assigned by the user or calculated for every capture by checking the “autoexpose” box. Autoexposure takes time since a series of exposures are tried and evaluated. You may want to disable the auto feature after the first pass and used fixed times thereafter. You certainly do NOT want to autoexpose everything if you doing a study where staining intensity is important (e.g., treatment vs control). If you want to time-lapse this sequence and/or do a zaxis collection, then use the APPS menu routine Multi Dimensional Acquisition. Again, for multiple wavelengths, the quad mirror cube must be used in the filter turret. 2) Pick one of the illumination settings from the taskbar (2-column menu). There are two columns: the righthand side is all buttons that work with the “quad” mirror (camera only). Change the manual filter turret to position 4 and flip through the channels with these buttons. The lefthand side is all buttons for the single color cubes (use for either oculars or camera). Pushing one of these buttons will pop up a window to tell you which cube to position in the bottom filter turret and then “Continuous” on the pop up window will open the shutter. You will be able to scan for your sample with one of these cubes, but be sure the light path is set to EYE (check that the knob on the right-hand side of the scope is set to the eye image). If it set to camera (SP, or the icon for the non-existent manual camera) rotate the wheel until it clicks and you see the eye. Focus upwards (CCW, ‘fingertips up = up’) to find your samples, but keep in mind there is NO limit in place to prevent you from crashing into things and breaking your prep, or worse, an objective. If you are having trouble here, ask for help. Unstained cells may be easier to find with DIC, but I prefer to use fluorescence mode for stained materials. If you have a DAPI stain, this works particularly well. 3) Find the cell(s) you wish to image and place them just to the right of center. The camera sees roughly the center/right 50% of what you see. You should then verify that the camera sees the same focus that you did by pressing the “Show Live” taskbar button (middle left column)—this reminds you to change the light path by rotating the knob to the “SP” side port setting and then starts the camera+screen display. You can use the Acquire menu “autoexpose” button to set the brightness (this can take some time with dim samples), or enter numbers directly. With no binning and gain=1, 100-200 msec should give a decent image (on average). (exposure testing is set to max out at 10 sec). If there is no image on the screen, double check that you remembered to send light to the camera. Note: There are pulldowns at the top that also allow you to change “illuminations” and objectives. Be aware that these menus do LESS than the similar buttons in the taskbar. Changing a filter, for example, only tells you to change the filter, while the task button would also then open the shutter… PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 9 Troubleshooting Guide: • No Image by eye, no illumination of slide. If there is no illumination when using the software to open a filter setting (verify by looking at slide), check to see that you have turned on the mercury arc power (be sure to power down all electronics before firing it, if this is the case!); 2) check that the Sutter controller is turned on (turn on); 3) check if the fluorescence/TIRF plunger (behind scope, see picture on pg. 11) is pulled UP to the fluorescence position. 4) Are too many ND filter sliders pushed in? Try with only the 25% ND slider in, Fig. 4. 5) Is filter turret (manual) shutter closed? Pull toward you to open. 6) Is the filter cube seated properly? Try turning the manual filter turret wheel and see if it settles in. 7) Is there an objective lens in the position you are using? • Ugly image, but looked OK by eye? 1) Check the Display/AdjustDigitalContrast menu to see if the last user has left some strange setting. Note the RESET button—press it to undo any custom display values. You can check/uncheck the “use display settings for activated images” box to control display of data. This tool only affects the appearance of the data, NOT the data itself. Images will not save with these settings. You can use the GAMMA slider to (nonlinearly) emphasize the bright or dimmer parts of the image. Using a gamma=1.3 or so tends to make the data look more like what our eyes see (we favor less bright parts and squint at brights). You can also set display color here. Camera data is 12-bits (4095 brightness values), the computer display is 8 bits per color (255 levels), our eyes see less than 100 shades per color. So you have much ability to manipulate displays. You want to use the same settings for controls vs. treatments. Also, while MM will correctly restore any settings you save to images, most other software will not use this custom information. (Use the Edit/Duplicate/AsDisplayed menu to create an exact likeness for other applications). 2) check the display scaling range (two ways to do this). While it’s useful to use autoscaling to find focus, it won’t actually give you an idea of how bright the data actually is (for this, see the live histogram along the side of each image). Using big values to control (autoscale) display mapping can make the image really look wrong! (use 0.0 or 0.1, 0.1). Using a fixed range (0 to 4095 is full range) works until the sample gets too dim. The Display/ScaleImage menu gives you display mapping range control too, PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 10 and also allows you to directly enter the same display ranges for images meant to be shown identically. There is a taskbar menu item (top Right-side Column) that forces displays to full range (“scale12bits”). The “autoscale” taskbar button reverts/sets autoscaling. 3) Did you use the taskbar “Show Live” button? This activates the camera+screen display; the Acquire/Acquire menu ‘show live’ ONLY starts the camera. You still need to direct light to the camera vs eye path. 4) Try an autoexposure. If you are collecting too few intensity values, the image will look grainy. If screen is solid color, you are probably too bright. Use the autoexposure button. If you didn’t send light to the camera, it will stop trying after testing a 10-sec exposure maximum (hit ESC key to abort an autoexposure). 5) Press the “Full Chip” button to make sure you are getting the full image vs some tiny portion no longer applicable. Total Internal Refection Fluorescence (TIRF) SAFETY FIRST! Know all operating procedures before attempting TIRF! TRAINING IS MANDATORY! Total Internal Reflection Fluorescence Microscopy (AKA evanescent wave) uses laser light to create a narrow band of excitation limiting the view of fluorescent materials to the space immediately above the glass coverslip. The depth of illumination can be 90-200nm thick, a region that limits your view to just the cellular contact to the glass. It allows you to view the cellular footprint and can be used to obtain high-speed images of changes in surface membrane, especially exo/endocytosis. It requires a laser spot be aimed at a critical location in the objective so as to bounce the light off the glass/water interface. This optical slice is more narrow than a confocal, but is limited to the space just above the cover glass. You must use the special 60x, 100x or 150x oil objectives to obtain the critical beam angle (via the high NA). You can use regular glass coverslips and regular oil with these lenses. Since this technique uses laser illumination, you MUST have special training. Laser safety is important; why risk damaging your retinas?? The laser source is a 10mW argon laser producing a 488nm beam. The laser is activated by rotating the key beyond “on” to “start” and releasing the key. It begins to produce light10 to 60 sec after activation. Select the laser illumination path by pushing the slider (behind the scope, see pg. 11) down. The beam can be moved to the critical angle by rotating the micrometer screw counterclockwise (above, right). Clockwise rotation moves the beam to the center of the objective, the non-TIRF position. Be sure to use the TIRF filter cube (called “green FITC wide/TIRF”) in the software. This is a long-pass FITC set that PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 11 will allow even red probes to be used provided they can be excited at 488nm (perhaps fura-red or RFP). You can narrow the emission by using the 535 barrier filter in the camera filter changer (FITC (535BP) TIRF filter definition in taskbar menu). Be aware that CFP will not be excited very well at 488nm (in fact, it’s peak emission is near this wavelength). See below for protocol. By turning the micrometer CCW you are marching the beam downwards (toward the periphery of the objective back aperture), and when the external beam disappears, you have reached the critical angle for TIRF. It’s convenient to set the depth of the TIRF illumination while observing the live image on the monitor. You may wish to use a camera sub-region, bin pixels, or both, to get fast image capture rates. The frame capture rate can be read off the Acquire window under the Live Bin box. Fast acquisitions may require using the “stream acquisition” feature (Acquire
Stream Acquisition) where data is grabbed as fast as possible without any active display to slow things down. By pulling up the illumination path slider (behind the scope) up, you can switch to the conventional mercury arc illumination to capture a standard image. Be sure to use the software TIRF illumination setting since this places the excitation filter into the path (as well) and prevents you from blasting your sample. Push the slider back down to return to TIRF mode. HOW-TO TIRF: Use the Plexiglas reflection shield (fix on scope just behind the eyepiece mount) Watch out for beam reflections! This is more likely if you use a metal chamber. Protect other users from stray beams as well. Never stare at a laser beam. The beam is low intensity, but eyesight is never worth even a small risk. The procedure is to first find your candidate cell using regular fluorescence (plunger slide UP behind the scope), then switch to camera, live view, TIRF illumination (plunger slider DOWN behind the scope), adjust the field depth of illumination (micrometer on TIRF arm), and start image acquisition. Note the illumination is constant (unless you switch back to mercury arc via the slider) or use the manual shutter on the filter cube turret. There is no automatic shutter for laser light (yet). TIRF is generally far less toxic and bleaches less than regular illumination. You can insert a ND filter with the slider on the TIRF tube (see left image on pg. 10) to decrease TIRF laser intensity, if needed. Power can also be modified to some extent by the trim pot on the front of the laser power supply if you use the non-standby mode. Generally there is more than enough light in standby mode for GFP work in shallow illumination. DsRed (and other RFPs) can be excited by 488 and you can use the colored filter to restrict capture to only the red component if desired. You can sequentially collect the green-only and red fluorescence by setting a protocol to switch the emission filter wheel using Multi-dimension Acquisition sub-application (see below). The filter can change in about 55 msec. Alternatively, you can use the image splitter tube and the split image capture mode in MetaMorph to simultaneously grab green and red signals, although the region you can capture will be smaller. Check the setup with ShowLive. How fast is it going? Check the Acquire/Acquire menu bottom left side (in show live mode) for the frames per second (fps). To capture data use the Acquire/StreamAcquisition menu item (this stays open) and stream to memory or disk. It will warn you if the requested data won’t fit into memory (but only when you actually try to collect it). Note that the stream mode does NOT give a live image. You won’t see what’s happening while streaming (it’s designed to go as fast as possible) until you play back the stream. Don’t collect full images (approx 1.4 MB each) when your target is only a tiny fraction of the image. Draw a box around the bit you are interested in, and then use the Acquire/Acquire/Use Active Region button to keep only the important bits. This saves memory and allows for faster capture (less to read back). The Center Quadrant button may be PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 12 useful here; the Full Chip buttons reverts to keeping the entire image. Do all of this setup in live mode. Still too slow? 1) Shorten the exposure time; you can’t run faster than the exposure. 2) Change the binning to 2x2 or higher. This decreases the amount of data to pass (by reading 4 pixels vs one {for 2x2}) and also increases the sensitivity by a factor of 4 {for 2x2 case}. You need bright images to get enough light during fast streams. 3) Change the choice of “shutter” in the Stream menu to NONE. This effectively keeps the software from trying to operate the regular light shutter during imaging. There is a built in delay to allow the shutter to open/close, and we are NOT using the Hg lamp for anything right now (laser path is separate). ‘Shutter’ here means choice of illumination. You can be explicit (a TIRF setting), set as ‘Use Current Shutter’, or pick ‘None’. Finally hit the acquire button on the lower right. You can abort a stream acquisition by pressing and holding the ESC key. Once you acquire a stream, there will be a pause while the software writes data from some buffer to another buffer. Grab another or save at this point. If you save the stack, it will take some time to write it to disk, so decide whether to collect another stream or to take a pause while data gets written. You have no choice but to save to disk if you’ve used up all the memory already. Use the Acquire/AcquireTimelapse menu option (top menu bar) to generate a live image stream and/or slow the data collection rate (i.e. collect an image every second/minute/ hour/etc.). TIRF Shutdown: The laser should be turned off by rotating the key to “off.” The blower will stop automatically when it’s cool. Remove the key to prevent unauthorized operation (place on shelf). Be kind; return the illumination selection slider to the mercury (Hg) position (UP). Troubleshooting Guide: If you cannot see the cell when you enter a shallow TIRF mode, it may mean that the cell is not firmly attached to the glass interface. Find another cell to study. If the sample is bleaching, decrease the laser power setting (trimpot) or add a ND filter to the TIRF laser path (via slider). Dirty surfaces, water, air bubbles in the oil will result in strange illumination patterns. If there is a problem, let us know and we will check the focus/alignment while you clean up your prep. No TIRF beam. 1) Did you turn the laser key to START, not just ON? (is the yellow light on the laser box on? Is there blue light in/around the laser tube?) 2) did you use the TIRF filter setting? (wrong mirror cube in path?) 3) Is the TIRF beam path slide (behind scope) set down for laser? 4) Are you using the 60x NA1.45 oil or 150x lens? Is the Oil OK? Is your dish leaking onto the objective? 5) Are cells on a single coverslip? …It was OK until I grabbed a stream or timelapse… Check the “shutter” to make sure it’s set to ‘none,’ ‘use current shutter,’ or one of the TIRF settings. Ugly image? See the Fluorescence Troubleshooting (above) section regarding image display options. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 13 MetaMorph Screens Fluorescence shutter state/toggle on/off Save image Close all Main (left) screen The MM window spans across both monitors, only the left window content is shown here. Please note that you should NOT maximize the MM window since a program limitation will make it fill only one monitor. By stretching the corner of the MM window, it should span both monitors. This allows you to keep the (big) images on the right monitor while leaving menus on the left. You may want to hike the bottom of the window up a bit so that the bottom row of the MM window is visible above the Windows footer. The bottom row of the MM window has information about pixel size, XY location and pixel intensity, memory use, etc. You can make the Windows footer auto-hide, if you prefer. Major pulldown menus are located in the top bar and organized to task. A variety of toolbars appear beside/below the main menu. You can move the toolbars along the top of the window by dragging them around. The Illum and Mag boxes show current settings (you can also change settings here). Region Of Interest (AKA, “ROI”) tools are available to limit operations to portions of images. The default is to have the arrow selected (tool to pick and ROI activation/operation). The next 3 shapes allow you to draw rectangular, circle/ellipse, or freehand selection AREAS. The next 3 line tools only define line/line segment ROIs, NOT areas (even if they are drawn to enclose something). The penultimate tool is an autotrace wand (works with thresholded images; not so good on regular images). PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 14 The Z display reports the current position of the objective and the arrows allow you to move the focus up down remotely (however, it’s easier to focus the scope directly). This readout will tell you if you are focusing up or down. MetaMorph TaskBar Switch light path Turn fluor on/off Snap image Display SHORTCUTS Scale12bits set display to full camera range; autoscaleDisp live reverts to scaling between data min/max Activate camera + show Select filter + illuminate {Must exchange filters} Select OBJECTIVE {narrow green} Repeated shutter control Brightfield lamp ON (prompt user to change lens and load system calibrations) REQUIRES filter exchange Close without asking/saving {preset for 5 color overlay} clearROI Grab RGB color image Taskbar buttons are actually “journals” (mini programs) that automate routine operations. If you have special functions that you would like to automate, ask the staff how to add functions. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 15 MetaMorph Main Menus File Operations New creates a blank canvas (not especially useful) Open (cntl O shortcut) opens image data (single images or multipage files in tiffs or stk format, as well as Zeiss confocal files, etc) The BuildStack submenu allows you import many single files into a single memory stack The Browse function is not installed since the SQL engine posed a security risk Note that once an image is loaded, you can use the cntl leftarrow/rightarrow to load the previous/next image from the same disk folder. It will ding at you when you hit the last image in the folder. The last few files that were opened are available in the bottom listing. You can click on them to reload easily. Use standard Microsoft cntl-S to save a file without having to use the menu or the floppy disk icon (equivalents). WARNING keeping any ROI active (an active ROI has a hatched appearance) will limit all operations to the area inside the region selected—that includes what gets saved! Either click somewhere inside an image with ROI defined, get rid of the ROI (use Regions menu), or be content. EDIT menu Cut/Paste (use standard Microsoft cntlC = copy and cntlV = paste keystrokes) Note: you will limit copy/paste by setting/using ROIs. Duplicate has submenus to replicate images or stacks. ALSO the AsDisplayed menu creates true color (RGB) copies of the appearance of any window chosen. It’s literally a what-yousee-is-what-you-get—any ROI, color schemes, ZOOM setting are preserved. This output is Photoshop and Office compatible. Be aware that you should create scale bars before this operation when you change the zoom to something other than 100%. Scaling data are NOT updated by the duplicate operation. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 16 Regions submenus -> shows ROI toolbar -> load/save ROI for later use -> use to copy exact ROI+locations to other images -> allows to manually move ROI to follow moving/drifting objects -> converts ROI from overlay to part of image (obvious) -> draws series of ROI along a drawn in line (for measurements) -> use this to create ROI from Object measurement output overlays Stack submenus Time and 3D operations -> looping and speed controls -> delete a plane at a time -> set Z calibration; note XY scaling is under Measure menu -> keep/delete range of planes. Caution: always clear the old ranges before operations (it remembers garbage) -> meant for motorized stage (not used presently) Movie creates a screen version (cannot save); MakeMovie creates AVI or MOV output that will save as a movie -> semi-automated way to align objects drifting over time -> fade/bleach correction (not so good) ->plot intensity change over time (or Z) for line ROI 3D operations PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 17 Acquire submenus You should open the Acquire/Acquire menu each session to access camera controls You can use the AcquireZseries menu to execute Z stack collection. You will need to set limits using the Devices/Focus menu first. This menu closes after you use it. AcquireMultipleWavelengths permits automated collection of up to 6 data channels. This menu stays open. StreamAcquisition collects data as fast as the system can run. Data is not shown live, but streamed to memory. You can review it as soon as a run stops. AcquireTimelapse permits slower image capture and gives a live stack display. This menu closes after you use it. The last two items are not general user items. Please don’t alter settings. (setting camera to 8-bit mode can speed data capture, but be kind and restore 12-bit mode IF you use this option). Devices submenus Most submenus are not meant for general user modification. An exception is the FOCUS menu. You can use it to alter the sensitivity of the focus knob on the microscope. Choosing an objective does input a preset sensitivity, but if you find the action is too coarse or fine, select another value from the pulldown list under the SPECIAL tab. You can use this menu to set the limits for a zseries collection via the Acquire/AcquireZseries menu. The Apps/AcquireMultiDimensionalData routine has its own built-in tab for setting Z limits. The Configure menus define the microscope settings and should not be altered. If you need a special setting, ask the staff to create/modify a custom setting for you. Display submenus Most functions to alter the appearance of images are located here. The AdjustDigitalContrast and ScaleImage menus control the appearance on the screen (as opposed to data in the image). See/use the Taskbar shortcut buttons for common operations such as setting scaling, color, etc. “Contrast Shortcuts” HERE is archaic and most operations do NOT work for 12-bit image data Graphics has annotation tools (see below). USE THESE for scalebars and other calibrations (resist using arrows and text—they are poor quality) IntensityProfile creates 3D histogram plots of intensity as height Use OverlayImages to combine dyes and DIC images ColorCombine is an alternate tool for overlays, esp. good for making RGB images for true color captures. ColorAlign corrects chromatic aberrations PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 18 Process submenus Tools to analyze and massage data. The top section lists common image filters. Many operations use “kernels” that pass over data and do operations. You can create/use custom filters (Kernel tools). FFT performs operations in frequency space such as periodic noise/line removal (very math intensive). Remove haze requires z stacks and subtracts off a portion of info averaged from planes above/below the one you are working on. 2D deconvolution is more optically precise, requiring information about the optics used (should be already known for data collected here) to de-haze based on optical theory. No-neighbor is pure theory, nearest-neighbor uses data from above/below in addition, to clear blur. Binary images are only black+white. Mainly used for special analysis operations. Arithmetic calculates many operations between images/stacks or constants (e.g. subtracting a background image) StackArithmetic does same but WITHIN a stack RatioImages creates MetaFluor-like output FlattenBackground corrects for uneven lighting when no real background image exists CorrectShading flattens illumination defects based on a bright reference image. ProduceBackgroundCorrectionImage attempts to create a background from your image. MorphologyFilters are used to smooth specific interests, find bright/dark areas, etc. This works on all images, not just binary. UseRegionForBackground subtracts off the min/avg/max from a ROI defined as background. SubpixelShift allows image or ROI movements/alignments in steps of 5%. SegmentImage is an automated method of separating objects in images (for analysis). You can have it find stuff based on intensity and of texture. It can be hard to get correct output. Log submenus Output results to text files and/or Excel Most measurements go into a data log Object logs are for Morphometric Routine output Summary Logs (morphometry) yet another output option (no detail, just the means, StdDevs, etc) LogPixelsInRegion lists intensity data for every pixel within a ROI EdgeLists (generally not used) PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 19 Measure submenu ->Assign XY calibrations (images collected here are generally pre-calibrated) -> for OpticalDensity work Thresholds define signal vs non-signal for counts/measurements; auto method good starting point Cut/join allows manual splitting/joining of objects for counts/etc Object standard used to define average object (to calc objects that are touching w/o split) IMA sophisticated measure/count tool (more later) Use to define objects in objects Active display of active ROI (or window) stats Measure one/all ROI in one/all planes all at once Displays intensity plot for ROI (can average) Journal submenus Keystroke recorder to automate mind-numbing tasks Command strings are called “journals” Menu to select and execute journal (tools to help create complex protocols) (Taskbars are main menus) PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 20 Applications submenu (why? These are extra-cost items) MDA collects multi-wavelength/time/z data Review- opens analysis portion (see below) Colocalization tool reports % overlaps Track objects automatically tracks/measures objects (speed/angles/location/etc) Graph intensities klutzy tool to plot 1+ ROI in stacks or live images (obvious) Window submenus All open windows are listed in LEFT Column (can be used to locate a buried window by name) Toolbars opens/shows the little icon menus (like top bar); check to show, uncheck to hide PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 21 Multi Dimensional Acquisition MDA setup Timelapse MDA main Be sure to select YOUR directory and a name; images automatically save there! (can enter “0” for number of timepoints to run indefinitely==until stopped manually) Pick dimensions to run (other tabs don’t appear unless you check those functions here) * Camera parameters (gain and offset, yes; NOT exposure) are those from Acquire/acquire dialog EXCEPT use these buttons to set binning and camera subregions A B C D set #wavelengths desired, then “current wavelength” to work on (pulldown will list 3 items in this example), THEN define current identity and exposure for that setting. Repeat for all illuminations. (each can/should be different) do LIVE, focus to top and bottom, setting limits with appropriate buttons. Set step size and/or number of slices to collect. This routine will NOT import limits from FOCUS dialog You can have software autoexpose a wavelength {never, first time, every nth, always}, and you can have it find focus (defined as highest contrast) similarly as long as you are not also doing a Z series. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 22 Review MDA data: Do SelectBaseFile, SelectDirectory, check one or more to view(at once), then VIEW Select channels to view Play selected data as movie (T or Z) Check for color overlay (or else you will get separate stacks) Only combines as RGB (choose what goes where) Matrix shows time and Z (not this example) data. Click data to show piecemeal, or rightclick row or column number to do all (each), or RtClk upper Left corner for all PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 23 Hit LoadImage(s) to extract data into regular image stacks (for analysis, etc). You can make Z projections and rotations with appropriate data input. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 24 MetaFluor This is the ratio imaging optimized sister program to MetaMorph. It allows you gather high-speed images used to calculate real-time ratio plots used to convert dye form (bound/unbound) and/or convert the ratio information to calibrated concentrations. Real-time plots or either/both output are available per region of interest (== “ROI”). You can collect up to 5 wavelengths and form two ratios, though most applications would just run two wavelengths. Launch using the MetaFluor icon (in the Meta Imaging Folder on the desktop or in Program Menu). Use the left menu to install a protocol (a set of parameters to acquire) or create one from scratch (see below). It will automatically reload the last protocol used (it may not be what you want AND watch out for the camera bit-depth bug, see below). Usual source is c:\MM\apps\mfluor Brief description of current choice Initial interface A) select protocol, recommended!, B) hit NEW PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 25 Screen after hitting “NEW” Shortcuts to common tasks are located in the default menu bar on the Left side. Main menus are in the top toolbar (all options vs common options). If you are making a new protocol, you need to define what to collect by using the Configure\ConfigureAcquisition menu (or Cfg Acq button on sidemenu). Pick wavelength to config, select an “illumination” from list, pick an exposure and binning; repeat as needed. Note the “allow items to differ” checkbox—it enables different gains/exposures for each channel. Do use binning to decrease image size and increase sensitivity (a must for live cells). 2x2 binning increases sensitivity of the camera 4X, 4x4 increases it 16X. I recommend 4x4 and use either all or at least the ND3 neutral density filter to limit light damage to cells (you should get thousands of intensity values this way). Running gain at 30X works without penalty for this camera (default is 1X). If you need a custom “illumination”, there is a menu under the main CONFIGURE menu. Please create new names for custom setting vs alter the defaults. You can also save them in your own directories to avoid other users messing with them. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 26 Refer to the MM section on filter definitions currently used. Create a new name, select correct filter cube position from list. This example is not correct since the Sutter Lambda wheel positions are not defined (and must be). Wheel B is the emission wheel, Wheel A is the excitation wheel. If this was 380 setting for Fura-2, Wheel A should be checked and set to 380/nd1 filter. Wheel B should be set to OPEN. (confused? Ask for help!) Finally, set the Lambda shutter A to ‘active’ (open only for actual image collection). Now hit Add/replace. Once defined, the values are stored until someone alters them. If we have problems with users altering main definitions, MF can be locked into Administrator/multi-user mode to prevent this. PLEASE create versions vs replace! Now you may want to setup the experiment details using Cfg Expt sidemenu or main Configure menu. You can set the collection intervals per wavelength, save intervals and screen update intervals. To run fast, you might display (a slow step) every 3 image. To run even faster (takes about 50msec to change filter positions) you can collect one of the wavelengths (like 340 for Fura-2) every other or every ten F380 images (software uses old value until refreshed). Highest speeds require streaming (no display at all), short exposures (will limit rate) and locking the shutter open (see config acquisition, config illumination) Run the experiment using the Control menu (open it using top sidemenu button if missing). You probably must use the Focus routine to turn on illumination, find cells by eye, MANUALLY SWITCH TO CAMERA PORT, then hit Start Focusing. Hit ‘start focusing’ to activate camera (or toggle shutter to find cells by eye). Note you can change binning and exposures in this dialog (in case you guessed wrong), test the stored values, or store the current values into the ‘cfg acq’ settings with buttons here. Check other wavelengths by clicking on the list entries. Note subarray controls. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 27 NOTICE: MF can sometimes ‘forget’ the camera is 12-bit (senile code) and the images will come up as 8-bit (see scale on side of image or histogram); it’s a bug. This happens if you don’t load a protocol before hitting NEW, or actually create a new protocol. To correct it, you need to close the experiment, use the File/select video channel/camera and re-select Retiga monochrome-12 bit (even though it looks selected). You will be back to normal. So, the first task in creating a new protocol is to go to FILE\select camera and re-pick 12-bit mode. You should set the sampling time you desire in the Expt Control Panel button ‘Set Timelapse’. You can enter the total number of points you desire or the duration. Entering “0” for # points makes the collection run until stopped; you still want to tell it how often to sample, though. You can check the parameters by using the Acquire One sidemenu button (it grabs ONE SET). The other buttons below it grab only the associated wavelength (1-5). Be sure to check the boxes for logging ROI data (we’re getting there next) and images otherwise it runs and you save nothing. The first time you check a box, MF will ask you for a filename and location. Data can be sent to MS Excel and/of a text file. The Save images enables storage of anything (and at interval) you set in the ‘Cfg Expt’ menu. The little lights to the right will be colored for data channels that will be saved. MF hasn’t evolved from DOS days, so naming schemes still have to be limited to 7 characters (it appends a ‘d’ to the ‘inf’ name and a 1-5 to describe the wavelength to images, and the file descriptor is sequence #)(the inf file contains timing info, event marks, etc; don’t get rid of it). Next define ROIs and any calibrations and thresholds. Hit the Regions button on the sidemenu. The next dialog lets you pick the channel to define CLEAR all ROIs on and to collect an image if you haven’t done that yet. Pick a drawing tool from the icons at the top (I like circle), start at upper left of where you want to draw and you can stretch the size by holding left mouse button and dragging down and right. You can right-click over ROI icons to set default size (make this smaller than you normally want, maybe 5ROI 10 pixels, since it’s easy to stretch it during creation). The closed, squiggly icon is the freehand draw; you can draw while holding the left mouse button down (continuously) or tack down a few points to have linear spans connecting the ‘dots’. You need to double click to stop the ROI. ROI can overlap. You can save the ROI locations for reuse, else the location of the upper left point is stored in data logs (but no shape/size info). Set thresholds on one or more channels to exclude background from ratio calculations. Only data above (each) threshold are used in calculations. Only data that are below the top limit are used as well (so saturated pixels drop out giving wrong impression that average ratio is dropping; a good reason to save images and check for this). Press the 3bar icon, pick autothreshold for light objects and adjust blue triangles, if needed, to delimit “signal”. Zero the elapsed time clock (Expt Cntl Panel), then hit Acquire or the F2 key (F4 pauses). PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 28 Open the event mark menu by pressing the Events sidemenu button. You can enter any text you want associated with an event in the entry box, then either log it immediately or store it to the reuse list with buttons at the bottom. Hit the F5 button to speed mark an event (vs futzing with the mouse); F6 advances through the list. The list is stored with your protocol if you save it on closing an experiment (it will ask you each close). Graphs can be configured by pressing the Graphs sidemenu button. You can set line styles and axis scales. Defaults use the ROI color to identify data per ROI and solid vs dotted to ID the pairs used per ratio. For Fura-2, you expect the F340 to rise and the F380 to drop if calcium goes up; if both change the same direction, it’s something else like focus drift, bleaching, etc. You get ratio plots straight away. You need to calibrate values to get the real-time calibrated value graph to work. Calibrate the system, if desired, using defined buffered solutions and one of several methods offered. You can use two-points (fully unbound indicator and fully saturated to generate minimum and maximum ratios, Rmin, Rmax, respectively) and the equation method, or create a titration curve using many buffered solutions. I prefer an in vitro, multi-point approach for Fura-2, though there are advantages/drawbacks to all methods their discussion is beyond the scope of this simple manual. Ratio data are always right but calibrations can yield junk if you’re not careful. There is value in saving raw image data in that you can later apply various calibrations and other corrections (like background subtractions and flat-field corrections). Enter the dialogs via the top menu CALIBRATION. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 29 Current acquired image or reloaded data Use the Acquire Standards Dialog to create or load calibrations into the system. Place the standard on the scope, enter the value on a line (note double >> indicating ACTIVE ENTRY LINE, change by clicking on line to use). Enter the values in proper order low to Pick high. Hitting load/save “Acquire” stores locale the buffered standard image. Repeat for all standards (at least two are required always). Save the calibration for future use (recall using LOAD button). You can exit the dialog (“OK”), and see a titration curve by clicking on “Titration” (even if it’s already checked). It’s good to make sure the system can give back the reference value for a standard following calibration. It’s ideal to correct images for uneven illumination and backgrounds. This can be done using the REFERENCES sidemenu button. A blank chamber or area from your sample can be grabbed for background using the “Acquire” button (IMAGE mode). You can also use a ROI as background, if drawn, changing the pulldown list. This is useful if drugs might alter background during an experiment. Flat-field or SHADING correction eliminates differences in illumination quality by dividing images by a correction standard. You need a uniformly fluorescent standard, like a drop of standard, and set the camera to near saturation (but nothing saturated) for each wavelength. You might get by with one reference if the illumination pattern is similar for two wavelengths close together. Be sure to check the ‘do it’ boxes to actually turn on corrections. Corrections will alter the image data you save/use, so do set this up correctly. Ratios tend to decrease illumination artifacts, you really should do these for single wavelength dyes or experiments where you want to compare values across images vs across time at the same location. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 30 If cells move during your experiment, you can drag one ROI on-the-fly to rack data while collecting images. You can adjust all ROI at once (if you bump the dish slightly adding a drug) using the RunExperiment Menu MOVE REGIONS. This jogs all ROI 1 or 10 pixel increments up to the point where one of them hits the edge of the image. You can cheat and manually move the limiting ROI to then allow all others to move again. If you save the images, you can analyze the data and step through frameby-frame to track objects, logging good data later on (this is always time consuming, however). There are routines in ImageJ or MetaMorph to automatically track objects as an alternative. The Scale16 and SetDisp buttons determine image appearance (not values). You can display fixed ranges or autoscale each wavelength using Scale16. The Ratio appearances set using ranges in ImageDispalyControls. MF doesn’t have the ability to show true ratio numbers so it scales the colors according to the range you specify (no floating point image type). You can insert a color bar for calibrations of intensity (or calibration) values such as the one shown. You need to close an experiment to analyze it, play it back as a movie (only possible if data were saved). If you are doing another run, it is easier to use the GRAPHS menu item clear graphs and ROI, then FILE, CloseMeasurementsFile, and CloseImageSave. This returns the software to the state where it will ask you for a new name for ROI data and images. Remember to check the “save data” and “save images” boxes in the ExperimentControlPanel (this is where it asks for names). PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com 31 APPENDIX Filter cube specs: DAPI (quad) DAPI (oly MNU2) CFP FITC (wide) FITC(narrow) YFP Reds (quad) Reds (oly MNG2) Cy5 (quad narrow) Cy5 (wide) Fura-2 Cameleons 2 DIC exciter 380 360-370 436/20 480/40HQ 480/40HQ 500/20 530-550 530-550 640/20HQ 620/60HQ 340; 380 440/20 dichroic Quad 400 455 505 quad 515 Quad 570 Quad 660 400 455 emission 450/65 420LP 480/40 510LP 535/30HQ 535/30HQ 630/60 590LP 682/22/75HQ 700/75HQ 510/40 535/35; 485/40 analyzer (polarizer) Stage heater and perfusion. We have a homemade heater block that accommodates a standard 35mm dish. The heavy aluminum shell maintains the dish at set point with constant circulation of water from the bath. This is normally set to 37°C or as high as 39°C to account for thermal loss between the bath and stage. Use a digital thermometer to check the actual dish temperature, if critical. Heating the dish will increase the rate of evaporation, a headache for long timelapse studies. If you are doing DIC imaging, you need to avoid plastic since it degrades the DIC effect. One solution is to use a dish that is fitted with a coverslip as the bottom. You will need to seal the top for long experiments and you may then need a cover also fitted with glass windows. Moisture will condense over time, so a double pane design may work better. Another possibility is to perfuse the dish using a pump or gravity-feed system. It is critical that you consult us on how to set this up so as to avoid flooding the system. If you have a spill, let us know right away so that it can be cleaned up before damage to the microscope can occur. Do not think you can still finish the experiment—it’s finished! We need to track down leaks quickly since the salts will seep in and corrode/ruin components. You can use a J-tube to maintain bath levels and to help safeguard against overflow leaks. PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com