By: Christine Labno, Ph.D. Assistant Director, AB129 site Office: AB129 (Abbott Hall) Phone: x

Transcription

1 Leica TCS SP2 Laser Scanning Confocal Microscope Operation Manual (version 1.0) University of Chicago March, 2007 A.pdf version of this manual is available at By: Christine Labno, Ph.D. Assistant Director, AB129 site Office: AB129 (Abbott Hall) Phone: x Vytas Bindokas, Ph.D. Director, BSD Integrated Microscopy Core Office: AB120 (Abbott Hall) Phone: x Overview: The Leica TCS SP2 is a spectral confocal with 405 UV laser 3CH+DIC digital emission nm range designed to acquire high spatial resolution images of fluorescently labeled materials and for analysis of these images. The confocal principle utilizes a pinhole (confocal aperture) to eliminate out-of-focus light from fluorescently labeled specimens (i.e., provide optical sectioning ). Lasers provide intense, point illumination that is scanned over the preparation and the fluorescence at each point is quantified and used to construct a representation of the object brightness (an image). This method provides high resolution for the x and y planes as well as vertically (z plane). The SP2 is an advanced confocal system with nine laser excitation lines spanning the spectrum from UV to near IR. The following laser lines are available: 405nm, 458, 476, 488, 496, 514, 543, 594, and 633, all fiber coupled and controlled by AOTFs. It has a laser scanner with up to 2000-Hz line scans, frame scan rate of 40 fps at 512x32 pixels, scan resolution up to 4096x4096 pixels, zoom to 32x, scan rotation; interactive control panel with digital potentiometers. The scan detector is equipped with an acousto-optical beam splitter (AOBS) to select/introduce most excitation laser lines, eliminating the need for main dichroic mirrors. The system has three PMT detectors with 8 or 12-bit output, simultaneous operation and digital/graphic definition of emission wavelength content per channel. Spectral scanning / display and linear un-mixing are possible, giving the ability to separate largely overlapping, similar colored fluorophores (e.g., GFP vs. FITC or YFP). LCS Leica Confocal Software has a fully operator configurable user interface. Multitasking high performance Windows XPpro Xeon Workstation (HP X4000) with 2.4GHz CPU, 2048MB RAM, two 21"CRT Monitors, 60 GB SCSI hard disc, 16x DVD ROM, CD writer 16x/10x/40x. Software includes LCS 3D Volume rendering, 3D animations, 3D filter, Stereo images and animations, Average projection, Maximum point projection, SFP; LCS Multicolor Co-localization of 2channel and 3channel recordings. Cytofluorograms in 2D and 3D display. Histogram-segmentation and masking. The SP2 is an expensive system to maintain. Use of the confocal system requires in-person training to make sure the system remains available to Users. Laser safety must be followed! There is an hourly fee for using the SP2, and users will be billed to recover costs incurred in maintaining the system. The fee schedule is currently $37/hr for Cancer Center members, $38/hr. for Digestive Disease members, and $40/hr. for other UC staff.

2 2 To start Leica confocal: 1) Uncover the microscope 2) Make sure all electronics are OFF, then start Hg arc power supply (#1, left side, top) 3) Start Microscope Controller power (#2, left side, button near floor) 4) Start PC Stand Power (#3a, right side, red toggle); PRESS THE CPU START BUTTON (#3b, White, middle right side of computer) 5) Start Scanner Power (#4, red toggle) 6) Start Ar laser blower (#5a, red toggle) 7) Start needed lasers (405 on separate small box with switch and key, other lasers are keys on the main panel) 8) Logon to Windows XP 9) Click LCS Confocal Icon (initialization takes many seconds) Place sample cover slip down onto stage; use oil sparingly AND wipe objective off every other slide (very important). NOTE that there are two 63x objectives. NEVER use OIL on the 63x Glycerin lens, use glycerin. Plan your magnifications to avoid contaminating lens with inappropriate immersion liquids! To quit your session: 1) Be sure to SAVE your data before exiting the LCS software 2) Logoff the CPU If another user is coming within 4 hours, leave components powered up. If you are the last person, shut off the system: 3) 4) 5) 6) 7) Shut down the computer with Ctrl-Alt-Delete, then Shut Down. Turn off all laser keys, but DO NOT turn off the Ar laser blower switch. Turn off the Scanner Power (red toggle #4) and CPU power (red toggle #3). Turn off the Microscope controller power (#2) and Hg Arc lamp (#1). Cover the microscope, but be careful not to cover the warm, black lamphousing on the right-hand side. 7) WAIT at least 15min to allow Ar laser to fully cool (this is very important); then turn off the blower power switch (#3a).

3 3 Table of Contents Manual microscope functions Changing the objectives...4 Focusing through the oculars...4 Fluorescence color filters for the oculars...4 Using DIC and setting the Köhler illumination...5 Removing/adapting the galvo stage...5 The Leica LCS Software Starting the Software...5 Choosing an objective...6 Setting the bit depth (change to 12-bit grayscale)...6 Setting the focus control...6 Setting up the Beam window for laser control...6 Sequential scanning...7 Capturing a single image The quick look up table (Q-LUT) for brightness...8 Changing the Z-POS (focus) button sensitivity...9 Live averaging and frame averaging...9 Digital zoom...9 Saving and renaming your files...10 Overlays, Scale bars and other 2D display options...10 Taking a Snapshot (aka a screenshot or save as displayed )...11 Collecting a Z-series for 3D volume renderings...11 Collecting a time series...12 Changing the functions of the control panel dials...12 Setting the microscope back to oculars...12

4 4 The Leica TCS Inverted Microscope The Leica SP2 system is almost completely automated, i.e. the majority of its functions are controlled through the computer, but there are a few things that can or must be manipulated manually. These are described below: Changing the objectives There are six objectives available on this microscope: 10x /NA 0.4 dry, 20x /NA 0.7 dry, 40x /NA oil, 63x / UV oil, 100x / oil and 63x /1.3 glycerol. The objective magnification is displayed in the upper left-hand corner of the yellow screen on the front Read of the microscope beware the 63x oil vs. 63x glycerol!! Both are listed as 63x. The -outs glycerol objective has an orange ring around the top. If you are unsure which objective you are using, look under the objective button in the software. Objectives can also be changed through the software. focus step Focusing Dry objectives (the 10x and 20x) should not touch the slide, but immersion objectives (40, 63, 100x oil and 63x glycerol) should just touch the cover glass. It s easiest to bring oil objectives up and in contact with the slide using the buttons on the right-hand side, just behind the focus knob (see blue arrow in picture to the right). Go GENTLY so you do not crash into the slide and break the slide or the objective. Once an oil or glycerol objective is touching the slide, begin using the focus knob. To adjust the coarse/fine settings for this knob, use the step button on Fast the front of the microscope, just below the yellow screen (see arrow in picture focus above). The readout for step size is on the yellow screen. S0 is the finest focus, button S3 is the coarsest. The rest of this panel is covered with a plastic shield for a reason. Please do not remove the plastic shield!! If you focus on your sample before you start the software, the software startup will drop the objective, so re-focus may be necessary. For parfocality with the software, twist the oculars so that the silver line on the tube is silver just showing (see picture to the left). This way, when the sample is in focus for you, it is line probably in focus for the computer. We say probably because the computer has 20/20 vision, many of us do not. There can still be differences between what is the best focus for you and what is the best focus for the camera. Be sure to check that you are on the optimal focal plane once you send the image to the camera. Fluorescence Color Filters There are two separate light systems on this microscope. When you are viewing a sample through the oculars, the microscope is using arc lamp light and a set of conventional fluorescence filter blocks covering three ranges: * Blue -- A (UV/violet excitation BP ; long pass emission LP 425) for DAPI or Hoechst, not good for CFP * Green -- I3 (blue excitation BP ; long pass emission LP 515) for FITC, GPF. Will show autofluorescence * Red -- N2.1 (green excitation BP ; long pass emission LP 590) for TRITC, Cy3, TexRed, sometimes Cy5 Blue Red Green Blank Filter changers shutter To use a fluorescence color to find your sample, make sure you are in visual mode through the software. Then use the black arrow buttons on the front of the microscope to put the red light under the filter you want to use. Filters are labeled by fluorophore color on the grey tape above the lights. Use 1 for blue, 2 for red and 3 for green; 4 is a blank for the DIC, and is also used during laser scanning. Turn on the light with the shutter button on the front of the microscope. When you have found your sample, hit the shutter button again to turn the light off and prevent photobleaching.

5 5 Using DIC and Setting the Köhler Illumination DIC power wheel VIS/SCAN Field aperture Cond. focus Conden. aperture Centering screws To look for your sample under white light/dic, make sure you are in visual mode and use the filter changer buttons on the front of the microscope to move the filter setting to the blank (4) position. Flip the knob on the right-hand side of the condenser to VIS (see bottom picture). Turn the light on and increase intensity with the power wheel on the left-hand side of the microscope in front of the focus knob (see top picture). To get more contrast and see your sample better, it helps to push the condenser aperture all the way to the left (almost closed, see bottom picture). Remember to push it back to the center before imaging, otherwise your images will be too dark. When you are finished, roll the power wheel back until the readout in the yellow box on the front of the microscope says 0V (the wheel has no stop) and flip the knob on the right-hand side of the condenser back to SCAN. The white light is not under the control of the shutter, so opening and closing it will not do anything. Setting the microscope for Köhler to get evenly illuminated DIC images, it is helpful to set the microscope for Köhler illumination. To do this, first focus on your sample. When the sample is in focus, turn on the white light as described above. Close down the field aperture at the top of the condenser by sliding it all the way to the left (see bottom picture). Unless you are using the 100x, you should now see a bright circle of light in your field of view. Focus this spot using the sliver condenser focus knobs on the condenser (see bottom picture) so that the edges are sharp and it looks like a hexagon. Then center this spot using the centering screws on the condenser (see bottom picture). If you are using the 100x objective, the spot will always fill the field of view. Use the centering screws to move the spot off center slightly, focus the edge, and re-center the spot. Removing / Adapting the Galvo Stage The microscope comes equipped with a motorized z-galvo stage for fine control of the z-step size (depth). This stage is built to hold standard glass slides. Our homemade sliver adaptor plate can accommodate most 35mm dishes and many non-standard slide sizes. However, if you have large dishes or plates that do not fit on the stage, you may have to remove it. If you feel you need to remove the galvo stage, please ask for a brief demo. It is an expensive piece of the microscope that is easily damaged. To remove the galvo stage, tilt the arm back, unscrew the two black screws at the back of the stage and carefully lift off the stage holding the controller box. Place the stage off to the side and use one of the adaptor plates to flatten out the stage. If you take off the galvo stage, you must change the z-scan button in the software to z-wide, otherwise the focus knob on the control panel will be useless! To do this, see Getting Started Choosing an Objective, Setting the Bit-Depth and Focus Control on pg. 6. The Leica LCS Software All imaging for this microscope is done through the proprietary Leica LCS Software. To start the software, login to the CPU and choose the Leica Confocal Software icon: The software will open with a menu asking if you want personal company or basic company settings. Choose personal and hit start, or just wait for the software to do it for you. The software takes several minutes to start, and will check all the parts of the microscope it is supposed to run. First time users will not have a personal profile, and that is OK. The software will save the company profile as your personal profile at the end of your session. If you get an error message that the software cannot find all hardware values or something like it, exit the software, run through the startup checklist to be sure all the hardware is on, and then start up the software again. The most common cause of this error is the laser scanhead. Exiting the software and then turning the red power switch #4 (middle) off and on can help.

6 6 List of captured images Beam window goes here Settings and important info. Images appear here Objective BEAM button Bit and Z-scan buttons Once the software has started, you should see something filling both screens like this: If you do not see this, but instead have a big black box on the left monitor, grab the black box and drag it over to the monitor on the right. Do not minimize the big black box, you will need it later. Getting Started Choosing an Objective, Setting the Bit-Depth and Focus Control If you have not chosen an objective manually or need to check that you are using the correct objective, the Obj objective button in the lower left-hand corner of the software will bring up a menu of all the possible objectives. The one that is currently in use is checked. To change objectives, click the name of the objective you want to use. If you are moving to an objective that uses a different immersion media than the current objective (i.e. dry to oil or oil to glycerol) you will get an error message asking if this is ok. Be sure you have the right immersion media on your slide (or no slide at all) before hitting OK. If you took off the galvo-stage, go under the Z-Scan button and change the setting from Z-galvo to Z-wide. This will allow you to keep using the Z-POS (Focus) button on the control panel. If you would like to change the bit-depth on the images, now is the time. The default is 8-bit. Use the Bit button to change to 12-bit before you capture any images. 12-bit depth will give images with a broader pixel intensity range. These 12-bit images will open in MetaMorph and ImageJ, but not in Photoshop. Setting up the Beam Window Before you can capture an image, you must create settings in the Beam Path Settings window. The Beam window is the master control center for the lasers (excitation) and photomultiplier tubes (PMTs, emission detectors). To get the Beam window, click the Beam button, located in the lower left-hand corner of the left window. You will get the window pictured at right, which you can put in the big blank space in the middle of your left screen. Setting the Beam window is challenging; fortunately these settings can be made once and saved for future use. 1 Steps for setting the beam window (an example for DAPI, FITC, Texas Red): 1. Start with the blue fluorophore (DAPI). Check the active box in the ArUV laser window. Set the % power slider to 10-20% laser power. MORE LASER POWER = MORE BLEACHING, so try to keep power low. A line will appear in the rainbow window where your excitation will be. 2. Go to PMT 1 under the rainbow window. Use the white pulldown menu to find the name of the fluorophore you are using. This will bring 2

7 up the appropriate reference emission spectrum in the rainbow window as shown (right). 3. Set the grey PMT bar under the peak of the reference spectrum. Do not set the PMT directly under or too close to any of the laser lines (must be a minimum of 3nm away). To see the exact wavelengths the PMT covers, double click the bar. Exact values can be typed in. PMT bars cannot overlap Set the color you would like for your display by clicking on the color bar and make sure the active box under the color bar is checked. 5. Repeat this process with your other fluorophores (FITC and then the Texas Red in 3 this example). 6. To get the DIC image, simply check the active box under PMT Trans. It helps to have set the microscope up for Köhler illumination (see Using DIC and setting the Köhler illumination pg. 5) SAVE this setting using the Save button at the bottom of the Beam window. The name you give your setting will appear under the User menu in the upper right-hand corner of the Beam window. To use the same settings during another session, double click on the name and all the settings will be restored to the Beam window. If you make changes to your settings over the course of the experiment and you want to keep them, click this SAVE button again and either overwrite or give the new settings a new name. 7 7 User defined settings You do not have to use all the PMTs. PMTs can be used more than once if you are ambitious and have >3 fluorophores. To go beyond 3 colors + DIC ask for advice, there are some tricks that will make life a little easier! Sequential Scanning when you are concerned about bleed-through and crosstalk If you look at the settings below, you will notice a large overlap in the emission spectra of the DAPI and the FITC, and a smaller overlap between FITC and Texas Red. This overlap is known as bleed-through. Overlap in the excitation spectra of two or more fluorophores is known as cross-talk. Both of these phenomena can give false signals. This microscope is equipped to minimize bleed-through and crosstalk using sequential scanning. Sequential scanning on this system is easy to set up, save and re-use, but difficult to change. Steps for setting sequential scanning (sticking with the DAPI, FITC, Texas Red example): 1. If you haven t already, set up a base sequence with all your colors (and DIC if you want that). 2. Turn OFF all but what you need to excite and collect the first color (here DAPI). Be sure to turn off PMT Trans too. Save this setting again with a new name (we use something like seq DAPI{FITC TexRed} or DAPI only ). 3. Repeat this process with your other fluorophores (FITC and then Texas Red. Here is shown the FITC only or seq{dapi}fitc {TexRed} setting). Save each with its own unique name. To have DIC, keep the PMT Trans box checked with your last fluorophore. 4. Open the Sequence window with the Seq button. Drag the only or seq settings that you made into the sequence window in the order that you want to scan them. Do NOT drag your Save or Load a sequence Seq button Saved settings

8 base setting into the sequence window; it defeats the purpose of setting up the sequence. SAVE your sequence by hitting the Save button in the sequence window. LEAVE THE SEQUENCE WINDOW OPEN to keep running in sequence. Just tuck it in behind the Beam window, it will be OK. To use the same settings during another session, double click on the base (all colors) setting you want to use. This will put the PMTs in the right positions, which is very important. Then open the Sequence window with the Seq button, hit the Load button in the Sequence window and choose the sequence you want to use from the new menu that pops up. Hit the Load button at the bottom of this window and your sequence parts should appear in the Sequence window. LEAVE THE SEQUENCE WINDOW OPEN. Some Rules for Sequential Scanning: ϖ ϖ ϖ ϖ Keep the Sequence window open. If you close the Sequence window, the microscope will go back to running in parallel using whatever settings are in the Beam window. This may cause false positive signals. If you change something in the Beam window (% of laser power, false color assignment, etc.) but do not make the same change in the affected part of the sequence, it will not be registered by the software. The only exception to this rule is the positions of the PMTs. These can be adjusted on the fly without re-saving the affected part of the sequence. The Save button on the Beam window is for settings only. It does not save data. To save images/data, use the Save button at the top of the main window (we know, they look the same) or File Save/Save As. If you are confused, don t feel bad. This is normal. We don t like this process either, we just know how to cope. Ask the staff for help. Taking an Image Once you have your Beam window set and your sample in focus, it is finally time to capture an image. For image capture you will be using the buttons along the bottom of the left-hand window and the dials on the control panel located below the monitors (both are pictured below). Hit the Continuous button to preview your image. The laser light should come on and begin scanning over the sample. If this does not happen, check to make sure that all the lasers you need are turned on (keys up and yellow lights on) and that the appropriate lines are activated in the software (boxes checked and % laser power set). The right screen is still blank because the voltage on the PMTs is 0, therefore there is no signal amplification. Begin turning the dials on the control panel labeled PMT 1 PMT2 etc. These will increase the brightness of your images, and each color channel is set independently. PMT voltage only goes to 1000V, beyond that your signal will not get any brighter. The readout for actual PMT voltage is in the Settings and Important Info box just to the right of the Beam window. To control the DIC brightness, change the setting on one of the dials to control Gain PMT Trans (see Changing the Functions of the Control Panel Dials pg. 12 for step-by-step instructions). A useful tool when looking at image brightness is the hot lookup table under the QLUT button to the left of the image window (pictured below). Clicking QLUT will assign colors to your images based on pixel intensities, with blue = full range and green = blank. You do not want all blue or all green, but mostly oranges and yellows with some blue pixels. In the example below, image 1 is too bright, image 2 is too low and image 3 is just right. Click on the QLUT button two more times to return the colors to the ones you assigned. 1 Q LUT 3 2

9 Averaging buttons Z-series buttons 9 The focal plane you are on can have a big impact on the apparent brightness of your sample (this is because the confocal pinhole blocks out-of-focuslight). Use the Z-POS knob at the right end of the control panel to adjust the focus/focal plane of your sample and help optimize brightness. The coarseness of this focus can be changed by right clicking on the Z-POS value setting on the bottom of the left-hand window and setting the step size. We find that 1_m per turn is good for fine adjustments and 10_m per turn (the default) is quick and good for zipping through large volumes. Once you have your image looking the way you want it, you are ready to capture the image. To capture the current focal plane, first stop the continuous scan by hitting the STOP button. Then click Single Scan. Once the image is captured, it will appear as a listing in the window to the left of the Beam window. If there is a grainy background in your image caused by detector noise, this can be cleaned up and the image smoothed out using line averaging (Li. A. button) or plane averaging (Aver. Button). These functions do the same thing in slightly different ways. Plane averaging takes X number of images of a single focal plane and averages together the pixel intensities. Random noise pixels average to zero, your signal intensities average up until they start to photobleach. Line averaging also averages pixel intensities on a single focal plane, but by line rather than whole frame, putting the finished picture up immediately. Both work equally well for fixed cells, use line averaging for live cells that move a lot. Either way choose one or the other, NOT BOTH. Using both is too slow on this system and will photobleach your sample rather than giving you nice results. The example image below compares no averaging to an 8 frame average. No line averaging Line average (8) Digital Zoom Once you have an image on the screen, you can digitally zoom in on part of that image and capture just that region. This gives better detail and resolution than cropping out part of a larger image and increasing the size because with digital zoom the size of the pixels (measured in nm) decreases. You can read out pixel size (sometimes called voxel size, a voxel is a 3D pixel) on the Settings box to the right of the Beam window. There are two ways to zoom: To zoom in on the center of the image a defined amount, click the Zoom button along the bottom of the Beam window (yellow box, right) and set the magnitude of the zoom. Non-listed values can be set by selecting Others and typing in the desired value. You can zoom on the fly while continuous mode is running, or you can stop the continuous mode, set the zoom and then start continuous mode again. To zoom in on a defined region of interest, click the Z. In button, on the bottom of the Beam window (gray and red box, right). Draw a box around the object you want to zoom in on. Again, you can do this on the fly while continuous mode is running, or you can stop the continuous mode, No frame draw your box and then start continuous mode again. To read out the amount of zoom you have averaging achieved, either click the Zoom button and look at the last value listed, or look for the readout in the Settings box to the right of the Beam window. If you want to take all your zoomed images with the same amount of zoom, you can standardize the zoom by first drawing your box, allowing the microscope to zoom, then clicking the Zoom button and choosing one of the listed zoom settings or choosing other and typing in a value. BE AWARE that if you try to set the zoom value without drawing a box or before allowing the microscope to zoom on the box, the default zoom-in-on-center mode will override your box and you will not get an image of the area you want. To undo zoom, click the Zoom button and choose 1.00.

10 10 Saving and Renaming Your Images Once an image has been captured it will be listed in the box to the left of the Beam window. Captured images can be renamed by right clicking on the name, then rename. Image names are limited to 20 characters and must all be unique. Captured images can be deleted using right click delete. A captured image is not necessarily a SAVED image. To save your images, click the Save button at the top of the left-hand window, or use File Save As. Make sure you are saving to your folder on the local SRF server (the R:\ S:\ or T:\ drive) using the pulldown menu at the top of the save window. At this point the save for the Leica software is different from normal Windows saving. When you enter a Filename, the software creates a folder with that name and puts all your images in that folder. So the filename is NOT renaming an individual image, it is creating a folder for all your images. To name an individual image, right click on the image name and choose rename. DO NOT try to rename the Experiment1.lei folder with a right click or double click. This will corrupt the file and you may not be able to get your data back. Images are automatically saved in two formats, the proprietary Leica.LEI format that will only open in the Leica software and as.tif files, one.tif file for each channel/time point/z-plane. If you chose to collect 8-bit images, these.tif files will open in any software; if you chose 12-bit, these images will NOT open in Photoshop, PowerPoint, or other Microsoft products because they do not support 12-bit grayscale images. Images can be converted from 12-bit to 8-bit using ImageJ freeware. Along with your images, the Leica software will automatically save a.txt file containing information on how your images were collected. This file contains valuable information for scale bars, 3D reconstructions, false coloring, etc. The information in this file can be accessed through the Notepad software on Windows/Mac or in the Leica software by right clicking on the image name and choosing Properties. Overlays, Scale Bars and Other 2D Display Options Now that you have captured your data, you can view and annotate it in several different ways. Keep in mind that these views and annotations will not be saved for you unless you use the snapshot option described below. For an overlay image, click the Ovl button on the channels toolbar on the left-hand side of the image window (see Scale bar box overlay picture). This will create a new image with all of the channels overlaid. To turn LUT off one or more of the channels, use the numbered channel buttons from this same toolbar (see picture). Scale bar adj. To make the overlay image fill the whole screen, click the image so that the white dashed bounding box is around it, then choose the Single button from this toolbar. You can still toggle channels using the channel buttons. To save an overlay, take a snapshot of it, described under Taking a Snapshot or Screen Capture on pg. 11. To add a scale bar, check the Scale Bar Box under the Display tab to the left of the Beam window (see picture above). A scale bar will appear on your image that is calibrated to the objective magnification and the zoom that you are using. Often this scale bar will be über-accurate, with decimal places. To make this into a nice round number, click the Scale button. A window will appear where you can adjust the characteristics of your scale bar. You can also move the scale bar to another location in the image at this time. Scale bars are only saved in snapshots, not in the raw data, so see Taking a Snapshot or Screen Capture on pg. 11. There is information stored with the images that allows us to re-create scale bars after images are taken, so don t worry if you forget. To change the color of an image after it has been captured, use the LUT button (not the QLUT button, see picture above). This allows you to choose a new color based on the image channel highlighted at the top of the window. This will not be a permanent change unless you take a snapshot. The next image that you collect will revert back to the colors that are assigned under the Beam window.

11 11 Taking a Snapshot or Screen Capture When you save your images, the software automatically saves the single channel images, but NOT overlays and NOT annotations like scale bars. The only way to save these things is by saving a screen shot or snapshot. This is equivalent to the Save Display mode in other software programs -- it saves an image exactly the way that it appears on the screen. To create a snapshot of the entire screen, right click on the image and choose Send To Experiment All Snapshot. To create a snapshot of part of the screen (say, one color channel) click on that part of the image to get the white dotted bounding box around it, then right click on the image and choose Send To Experiment Selection Snapshot. Your snapshots will appear in the list to the left of the Beam window with rainbow icons. Captured snapshots can be saved and renamed just like the raw data images. They are automatically saved as 24- bit RGB color.tifs which will open in color in any program, including Photoshop and PowerPoint. Collecting a Z-Stack for 3D Volume Images Confocal microscopy is well suited to imaging in 3D because the confocal slices can be easily reconstructed into a volume. To create a z-stack, you will use the Series buttons pictured to the left. Start by adjusting the brightness on the slice of your sample with the highest signal intensity (this is often the middle of the object). Then set the beginning and end of your series. To do this, it helps to open the Series window using the smaller Series button (window is pictured left). While the Continuous mode is running, use the Z-POS dial at the far right end of the control panel to focus to one end of your sample. You will know when you have reached the end of your sample when the pixel intensity starts to get very low. When you have reached one end, click the End button or check the End box in the Series window. If you have the series window open, a red line will appear in the cube to represent your end position. Then focus back through your sample to the other end. If you have the series window open, the yellow line will move to mark your current position. Click the Begin button or check the Begin box in the Series window to get the beginning of your stack. Once you have set the column for your series, you will be given a readout of the total volume of you stack at the bottom of the Series window. You can also see the total volume of you stack under the Sect button. To set the step size, click the Sect button and type in a step size. You can choose anything from 0.1 _m to 2.0 _m or larger, but the bigger the step size, the more information you could potentially lose. The number of images to reach the total column at that step size will be calculated and displayed. When all of that is set, click the larger Series button to start collecting the series. If you have set a line of plane averaging that will stay and each z-plane will be averaged. As the series is being collected, you can keep track of how long is has to go with the indicator on the far left side of the screen under the captured images box. When the whole series is collected, it will be displayed in the captured images box with a stack icon. When the stack is saved, it will be a series of individual.tif files, one for each z-plane/channel. To see the various z-planes, flip through them with the Prev and Next buttons on the panel to the left of the image window. To display this series in 3D, click the Process tab under the Beam window. Choose the Projections with Animation feature. Projections made with this feature can be applied to the data, which creates a new stack in the image window. This stack can be exported as an.avi (PC only) movie by right clicking on the animated stack s name in the captured images box and choosing Export. If you have the raw stack data, 3D projections can also be done through offline Leica software, MetaMorph, or ImageJ. gallery Prev/Next buttons

12 Collecting a Time Series Laser scanning confocal is a slow process, but it can be used to take time lapse images, if you time points don t need to be too close together and you sample is not too sensitive to photobleaching. To make a time series use the Mode button on the Beam window (see picture at left) to change the mode form xy or xzy to xyt or xyzt. This will enable the use of either the Basic Time button (top) or the Advanced Time (bottom) button (pictured at left). The basic time button allows you to set the time period between scans, the total time for image collection and/or the number of scans to collect (pictured below, any two of the three and the third is calculated for you). If you check the minimize box for time interval in this box, the software will automatically collect images as fast as it can. If this is still not fast enough for you there are several things you can do. Try decreasing line or plane averaging, reduce the number of pixels per image under the Mode button, zoom in on a smaller area, and/or increase the scan speed under the Speed button. All of these things will decrease the amount of time it takes to take a single scan. The advanced time window is more difficult to set up, but allows you to string together varying time intervals and/or do a bleaching routine. There is also a fluorescence recovery after photobleaching wizard under the Applications tab that guides you through the FRAP process. Feel free to ask the staff for a brief demo on either Advanced time or the FRAP wizard. There are several options for displaying time series data. You can see all of the time points in any or all channels using the Gallery button on the toolbar to the left of the Image Display window (pictured on pg. 11). You can export the series as an.avi (PC only) movie by right clicking on the stack name in the captured images box and choosing export. A dialogue box will appear asking you how many frames per second to run the movie. The larger the number, the faster the movie will run. We like 3 fps, you can choose what you like. 12 Changing the Functions of the Control Panel Dials The functions of the control panel dials located below the monitors are set through the Leica software. The most popular settings have been set as defaults and are labeled below the dials. However, it is easy to change the settings during a session and/or to save a customized profile for yourself that will appear every time. To change the function of a button during your session the most common reason for changing the function of a button is to give yourself control over DIC signal intensity and offset, so this is what we will use as our example, but all other available functions for buttons are changed the same way. Remember that the buttons on the control panel correspond to the settings listed below the scan control buttons (Continuous, Single Scan, etc.) at the bottom of the left-hand screen. Go to the fourth option from the left, which is set to Smart Offset and corresponds to the fourth button from the left, the only button that is not labeled (see picture above). Right click directly on Smart Offset. A new menu should pop up. Select Customize, then Gain PMT Trans. The value of the option should change to Gain PMT Trans and the button will now control the brightness of your DIC/transmitted light image. To set a customized control panel as your personal default once all the control panel buttons are assigned to the values that you want, you can save these settings and get them back automatically when you start up the software. To do this, you will use the tiny buttons located in the bottom right corner of the left-hand screen. To save settings, click the far left button that looks like a brown floppy disk. Give your settings a name ( Joe s Settings) and hit save. Then go to the far right button that looks like a blue pick axe (I did not choose these icons, so give me a break here). Choose Joe s settings from the menu, right click and choose set as default. That s it. Your custom settings will appear every time you log in. If you make changes that you want to keep, remember to re-save and re-set the defaults. If you are very ambitious, you can have multiple configurations saved under different names for different imaging projects. Setting the Microscope Back to Oculars Save custom settings Set custom as personal default The MicCtrl microscope control button in the lower left-hand corner moves the light from the oculars to the computer and back again. The default start position is visual so that you can find your sample in the first place. When continuous is clicked to enter focus mode, the software automatically changes to Scan mode. To go back to looking through the oculars after taking an image, click the MicCtrl button and check Visual from the pulldown menu.