OPERATING INSTRUCTIONS

Transcription

1 Zeiss LSM 510 M eta Confocal M icroscope OPERATING INSTRUCTIONS Starting the System: 1. Turn the black knob on the laser box one-quarter turn from Off to On. You will hear the laser cooling mechanisms engage. 2. Flip the two toggle switches on top of the CPU from O to. 3. Press the power button on the front of the CPU in the top, right corner. Allow the system to load. 4. When the log-in box appears on the computer screen, use your Histology Core Account Number and password to login. Allow Windows to load completely before opening any software. 5. Double-click the Zen 2007 software logo in the center of the screen and allow software to load. 6. When the Zen 2007 Log-in box appears, choose Start System for laser confocal imaging or Image Processing to work with previously collected image files. Allow software to load completely. 7. Click the blue Laser menu bar and make sure the pro option is highlighted. a. Turn all the lasers On if they are not already. i. The Argon/2 laser must be turned to Standby, and will change to On when it has warmed up. b. Highlight the Argon/2 laser and click Laser Information. i. When the laser status says Ready, increase the output to 50%. Imaging & Light Path Setup: 1. Open the Imaging Setup and Light Path panels by clicking the blue menu bars. If you cannot open both at the same time, right-click the blue bar and remove the check mark from in front of Auto Close Mode. a. Under Light Path, select the appropriate settings for your fluorescent dyes you need to know the excitation and emission spectral data for all your dyes. ii. Laser place checks in the boxes for the lasers you want to use. Only check the ones you need. You can adjust laser output here, if necessary. iii. Beamsplitter HT directs specific wavelengths of light from the lasers to the specimen. Choose one that includes numbers to match all the lasers you are using. iv. Beamsplitter NT1 splits fluorescent emissions from specimen to the different collection channels wavelengths below the number selected are reflected, and numbers above are transmitted. If using channel S1, choose the filter with a number lower than the emission peak for the dye you will image at channel S1, but higher than the peak of any other dye you are using. If you are not using channel S1, select the mirror Nils Hasselmo Hall 312 Church St. SE Minneapolis, MN

2 v. Beamsplitter NT2 splits fluorescent emissions from specimen between channels 2 and 3. If using both channels, select a number between the emission peaks for the dyes you are collection on each channel. If only using one channel, choose a filter that will either reflect or transmit your light to the desired channel. vi. Emission Filters for channels 2 and 3, select a long-pass (LP) or band-pass (BP) filter that will transmit only the emission wavelengths you want to capture. LP filters transmit all wavelengths above the number listed. BP filters emit only wavelengths between the numbers listed. If imaging multiple dyes simultaneously (explained later), you must choose filters for channels 2 and 3 that do not overlap. If imaging multiple dyes sequentially or only one dye, simply choose the filter that best fits the emission curve for your dye. vii. Channel S1 can be used as a customizable filter, allows you to specify the exact range of wavelengths to be collected. Move the sliding triangles together to set the range, and click the check-box to activate the channel. Use this only when collecting three or more dyes, or when there is no pre-set filter at channels 2 or 3 appropriate for your dye. viii. Assign Pseudocolors click the ChS1, Ch2, and Ch3 icons and assign a different color to each. ix. Activate Channels click the check-box next to the channel icons (ChS1, Ch2, Ch3) you want to use, and a white check mark will appear. See simultaneous and sequential modes, below, to determine how to use the different channels. Simultaneous and Sequential Imaging Modes: 1. When collecting emissions from multiple dyes on a single specimen, you must decide whether you want to excite all the dyes at once (simultaneously) or to excite and collect emitted light for each dye individually (sequentially). Either option will generate a multicolored image, but the two options are affected by non-specific excitation and color bleed-through. If your dye emission spectra are close together and you cannot configure the light path to adequately separate the collected emissions, or if your dyes are excited by more than one laser, you will want to excite each dye sequentially. If these problems do not apply, you can image more quickly using the simultaneous mode. a. Tracks: i. Simultaneous mode uses only one track, and one or more of the three collection channels are used in that track. All of the lasers you selected will turn on and excite the specimen at the same time. All dye emissions will pass through the beamsplitters at the same time and are guided to the appropriate collection channel according to the configuration you ve created. ii. Sequential mode uses multiple tracks one for each dye you are imaging. For each track, only one laser is turned on, and only one collection channel is activated. If all the tracks are activated when you scan your specimen, you will get a multicolored image where each color was collected individually Nils Hasselmo Hall 312 Church St. SE Minneapolis, MN

3 b. Simultaneous Mode setup: i. In the Imaging Setup panel, make sure there is only one track, and that it is activated by a white check mark. If other tracks are also present, click on them to highlight one at a time and delete them by clicking the minus sign button at the bottom of the panel. ii. Follow the instructions above to set up your lasers, beamsplitters, filters, etc. in the Light Path panel as appropriate for your dyes. When finished, your Imaging Setup panel should resemble the figure to the right. The white vertical lines represent the lasers you ve selected, and the colored bars represent the wavelengths of light that will be collected by each channel. The panel to the right is an example of a simultaneous mode setup for Cy3, FITC, and DAPI in channels S1, 3, and 2, respectively. c. Sequential Mode setup: Laser Scanning & Image Collection: i. First configure the Light Path panel just as you would for simultaneous mode you have only one track at this point. All beamsplitters and filters must be identical for each of the tracks you will use, because the microscope cannot switch filters during image acquisition. All channels will appear under Track 1, and your configuration will be the same as for simultaneous mode, above. ii. In the Imaging Setup panel, click the plus sign button to add a second and third track. When you do add a track, it duplicates the exact settings you ve chosen for the first track. The configurations must be identical for each track because the microscope cannot switch optics during image acquisition. iii. Click the Track 1 bar to highlight it in the Imaging Setup panel the track-specific light path settings will appear in the Light Path panel, below. Configure Track 1 to image the dye with the longest emission wavelength. 1. In the Light Path panel, select the laser that will excite the dye with the highest emission wavelength, and unclick all other lasers. 2. Activate only the collection channel for this dye, and remove the check marks from the other channel icons to de-activate them. iv. Configure the other track(s) the same way, using Track 2 for the dye with the second-longest emission wavelength, and Track 3 for the dye with the shortest emission wavelength. Make sure all tracks are activated with a check mark in the Imaging Setup panel. The panel shown above is an example of a sequential mode setup for Cy3, FITC, and DAPI in Tracks 1, 2, and 3, respectively. 1. At the top right corner of the Zen software window, there are three buttons: Ocular, Camera, and LSM. a. Ocular Mode: you can view your specimen through the binocular lenses, illuminated by a mercury bulb for fluorescent reflected light, or a standard halogen lamp for transmitted light. The Light Path panel shows the configuration for reflected and transmitted light. Use this mode to find your region of interest and choose desired magnification. You cannot do confocal laser scanning or view your specimen on the computer screen in this mode. b. Camera Mode: currently, no camera is attached to this scope. Ignore this function. c. LSM Mode: you can scan your specimen with lasers to capture confocal images. Laser safety shields prevent you from viewing your specimen through the binocular lenses in this mode. You must be in this mode to view the confocal light path configuration and optimize laser-scanning settings Nils Hasselmo Hall 312 Church St. SE Minneapolis, MN

4 2. Find your region of interest: a. To find your region of interest on the slide, click the Ocular button. In the Light Path panel, click the light source button on the lower left corner this controls the reflected light. Click the On button to turn on the mercury lamp, and then increase the light intensity to a desired level. b. Use the buttons on the microscope to open the shutter, change light filters and magnification, and navigate the slide in x-y directions. c. When you have found the region you want to image at the desired magnification and it is in focus, close the shutter and then click the LSM button in the upper right corner of the screen. You will hear the laser safety shutters moving into place. The Light Path panel will change to show the confocal laser configuration you created. 3. Optimize signal intensity: a. In the Acquisition Mode panel, make the following adjustments: i. Frame Size: adjust change manually, use a pre-set ratio by clicking X*Y, or click Optimal to find the best resolution for your objective. This setting determines the number of digital pixels calculated for the scanned area, and is limited by the objective s numerical aperture. ii. Speed: this is how fast/slow the laser scans across your specimen. Slower speed gives the most smooth, crisp detail but can take extremely long, especially when doing Z-stacks. Faster speeds produce slightly more pixelated images but are faster. Pixelation can be smoothed by averaging (explained below). Additionally, fluorescent molecules respond better to (and do not photobleach as easily with) short bursts of excitation repeated several times (fast speed w/ averaging) compared to long, single periods of excitation (slow speed w/o averaging). We recommend you click Max and use that speed. iii. Averaging: when you first start scanning your specimen in order to optimize laser settings, do not average set the number to 1. Once you have adjusted the gain, offset, and laser intensity to the desired parameters, use averaging to smooth out your image and remove any unstable fluorescence signals. Increase the number from 2 to 8 (above 8 is not valuable) the higher you go, the more times the laser will scan over the same area. Change the mode to Line this means the laser repeats the multiple scans line by line, rather than doing the whole frame at once. This reduces the effect of any slight movement or light intensity changes. iv. Bit Depth: this determines the range of grayscale values that can be assigned to your image. Default setting is 8 Bit, but 12Bit is recommended for publication-quality images (caution: much larger file size!). v. Leave all other settings as they are shown in figure, above, unless you have had advanced training. b. In the Channels panel, make the following adjustments: i. Pinhole: This controls the size of the diaphragm through which your specimen s fluorescent emission is collected, and is a function of the wavelength of the emitted light and the numerical aperture of the objective. It must be adjusted any time you change objectives. Click the tab for the dye with the longest emission wavelength. Then click the 1 AU button this automatically calculates the appropriate pinhole size for your objective given the wavelength of your dye. Look at the measurement (above shows 12.7 µm section ). Then click the tab for the other dyes, and change the pinhole size until the section thickness matches that of the longest wavelength dye Nils Hasselmo Hall 312 Church St. SE Minneapolis, MN

5 c. Click the Find button in the upper left corner of the Zen window this function estimates the approximate gain and amplitude offset necessary for your specimen. The lasers will scan your specimen one time, adjust these two controls, and produce a multicolored image. The light levels may be close to correct, but they will most likely require further adjustment. d. To make fine adjustments to your image s signal intensities after using the Find function, click the Continuous button. The lasers will repeatedly scan your image, and you can make changes to different parameters and watch the effect in the image window. You want to do this quickly, especially if your specimen is sensitive to photo bleaching. You can also adjust the focus at this time using the focus knob on the TFT screen. i. Split View: - click this tab along the left side of the live image to see each color channel and the merged channel in four separate panels. ii. Signal Intensity range indicator: Once the continuous scanning begins, go to the controls panel under the image and click the 3-color square under the Merged button. This changes the imaged from color panels to grayscale panels with minimum and maximum intensities indicated by blue and red, respectively. Do not click the drop-down menu arrow this will change the assigned pseudocolor. iii. Laser Transmission [%]: Increase laser output slightly above You do not want to increase this too much, or it will photobleach your signal. Use the controls below to further adjust intensity. iv. Gain: adjust level individually for each dye by clicking the dye s channel tab and then increasing or decreasing the gain value. Optimal level will show positive signal approaching white, but with very little to no red pixels. Red pixels indicate over saturation, which is considered false data. v. e. Amplitude Offset: adjust level individually for each dye as above. Optimal level will show background levels approaching black, with very little to no blue pixels. Blue pixels indicate lack of background signal, and if this is reduced too much, it is considered false data. Once your parameters are set and your specimen is in focus, click Stop. If you are scanning at the highest speed possible, you now want to turn on the averaging function, as described above. When you are ready to collect your image, click Single. Your image will appear in the display as a pseudocolored image Nils Hasselmo Hall 312 Church St. SE Minneapolis, MN

6 4. Save Images: a. In the Open Images panel on the right side of the Zen window, click the box containing your image and then click the Save button under the image thumbnail, or select File and Save As from the Zen menu. b. Save your file as a.lsm document this automatically saves all acquisition parameters including objective and magnification, light path settings, laser intensity, gain, amplitude, etc. However,.lsm format is not compatible with software other than Zen, so you will also need to export your image to.tif format see below! c. Save your document to your own removable USB device or to your AHC-network server. CD and DVD discs are available for purchase from the HMCF staff. You may temporarily save your work in the data (D:/) drive of the confocal computer, however these images are not backed up and are not guaranteed to be protected they could be lost permanently if you do not transfer them! d. If you want to open your image in software other than Zen 2007, you must also export the image as a.tif file. 5. Shut Down System: i. In the Zen window, double-click the image you want to export, so that it appears in the center image panel. Click the Split tab so that you can see all individual channels separately. ii. From the Zen menu, click File and Export. In the Export window, choose Format: Tagged Image File and Data: Full Resolution Image Window Single Plane (or choose Series for Z-stacks). Click Select file name and save. Make sure the file name and destination are the same as what you used when you saved the.lsm file. a. Turn off all lasers. b. Close Zen software. You should see a warning message that the lasers must cool down for 5 minutes before you can shut down the rest of the system. After the cool-down period, you will hear the cooling fans turn off. c. Shut down the computer. d. Flip the two toggle switches on top of the CPU from to O. e. Turn the black knob on the laser box one quarter turn from ON to OFF. f. Turn off the room lights and make sure the door locks behind you Nils Hasselmo Hall 312 Church St. SE Minneapolis, MN