Getting started v. 1.7  September 12th, 2011   

Getting started with the confocal microscope Leica TCS SP5 Matrix, HCS A

TABLE OF CONTENTS Switching on the microscope and the scanner………………………………………...……………………2 Opening the scanning software LAS AF……………………………………………………...……….…2-3 Activating the lasers……...…………………………………………………………………………………3 Using the microscope…………………………………………….………………………………………4-5 Field of view vs. scan area………………………………………………………………………………….5 Arrangements for scanning Choosing the Beam Path Settings………….……………………………………………………………….6 Settings for basic scanning……………………………………….……………………………………….6-7 Start scanning……………………………………………………………………………………………….7 Modifying emission detection range……………………………………………………………..…………8 Setting the dynamic range………………………………………………………….……………………….9 Averaging optical plane………………………………………….…………………………………………9 Bidirectional scan……………………...…………………………………………………………………..10 Zooming………………………………...…………………………………………………………………11 Saving images………………………………………………………...…………………………………...11 Leica TCS SP5 shutdown procedure……………………………..……………………………………11-12 Troubleshooting Nothing but blackness in microscope eyepieces…………………………………………………………....5 No scanned image on screen…..……………………………………………………………………………7

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Getting started v. 1.7  September 12th, 2011   

Getting started with the HCS confocal microscope Leica TCS SP5 Matrix Located in the room 2209, at the Department of Biology, Åbo Akademi University, Tykistökatu 6 A, FI-20520 Turku, BioCity building, 2nd floor. With this guide you should be able to turn on the confocal microscope, scan fixed samples, and close the system after use. In case you consider live-cell imaging, please contact responsible personnel well in advance. Note that gas- and ion-lasers prefer longer running times compared to continual switching on – switching off. It is better for the lifetime of the lasers that they run at least one hour after switching on. Do not switch off the laser key, in case the next user has a reservation during the next three hours after you. It is your duty to check the electrical reservation book before you shut down the system. Switching on the microscope and the scanner First, tilt carefully the illumination arm of the inverted microscope backwards (fig. 1.) to prevent any collision between the condenser and the moving motorized stage in initializing phase.

Then, press the left green button “PC, Microscope” (fig. 2.) under the right corner of the table. You will here sound of the microscope as the motorized stage starts to initialize, and the stage moves in pre-defined area. Next, instantly after the first button, press the middle green button “Scanner Power”. The scanner starts to hum. Finally, press on the right green button “Laser Power”, and turn the Laser Emission key to “On-I”-position; the yellow indicator lamp will be lit (fig. 2). At this point you have switched all the lasers only to standby-mode.

Opening the scanning software LAS AF The operating system Windows XP opens automatically, after you have switched on the left green button “PC, Microscope”. Choose TCS-User, no password, in case you don’t have your own account. Double-click the icon LAS AF on the desktop. A message “Initializing LAS AF, Please wait …” appears on the left screen. In case you accidentally clicked the Leica AF Hardware Configurator, let this software open, until you shut it down again. Thereafter start the LAS AF scanning software. Both softwares can not be active concurrently. The next window to open is Leica Application Suite window (fig. 3), where you simply click the option OK. In case you want to check them, default options Machine (not SimulatorSP5) and DMI6000 (not DM-Manual 6) should be selected in Configuration and Microscope Stand options, respectively.

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Getting started v. 1.7  September 12th, 2011    The next window “Microscope Stand” opens in less than a minute (fig. 4). Click “Yes”, in case you will use the LAS AF MATRIX M3 application, or the Tile Scan option. Click “No”, if you’re going to use conventional scanning (like in most cases).

The scanning software LAS AF v.2.5.1, build 6757 opens with scanning options on the left screen, and the scanned image to be seen on the right screen. The Control Panel with six rotating knobs is behind the keyboard (fig. 5).

Activating the lasers The first step is to activate the laser(s). Click “Configuration”-tab (fig. 13), and click “Laser” in Hardware Configuration window (fig. 14). The view with currently available lasers opens. Fill in checkboxes of the laser(s) to be used: Ar, HeNe 543 or HeNe 633. In case of Ar-laser, you will here the air-cooling start after you filled the checkbox. Raise the excitation power to 20% with the slider. Return to scanning options by clicking the “Acquire”-tab (fig. 13).

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Getting started v. 1.7  September 12th, 2011    Using the microscope The microscope stand is an inverted Leica DMI6000B. Place a microscope slide on the specimen stage (fig. 6.). Here is assumed that you are using a fixed sample. Place the slide on the shallow notch, cover glass facing down. In case you’re using an oil immersion objective, add a small drop of oil on the cover glass, and turn the specimen round before placing it over the objective. In case you used gloves when handling immersion oil, you are not allowed to use same gloves for operating the microscope or using the keyboard. Use the specimen holders to better keep the sample in place. Turn carefully the illuminator arm of the microscope (fig. 1) back to place.

ATTENTION! Read this revised paragraph thoroughly. Switch on the external light source (fig. 7).It stands on the table between the microscope and the left screen. When you switch it on, both green (Power) and yellow (Shutter) indicator lamps will lit. Please, do not touch the intensity knob. NOTE! The lamp must be switched on at least one hour from ignition. It must cool down at least 20 minutes from switch off. Do not switch the lamp off after your session in case there are other reservations after you. When scanning a time-lapse series lasting hours, switch the lamp off, because even the transmitted image is scanned with laser excitation. Caution! The cover of the lamp housing gets warm or even hot during prolonged use. Never place anything on top of it! The light source itself is a metal halide bulb.

Choose the light microscopy method to use. Choose fluorescent cube I3 or N2.1 for fluorescence microscopy of “green” and “red” samples, respectively. The cube I3 has a band-pass filter of 450-490nm, a dichromatic mirror of value 510, and a long-pass suppression filter of 515nm. For the cube N2.1 these values are 515-560nm, 580, and 590nm. The buttons for selecting cubes are facing you above the LCD, on the front of the microscope (fig. 8).

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Getting started v. 1.7  September 12th, 2011    In case you need transmitted light, press the lowest button “TL/IL” on the left side of microscope stand (fig. 9). To open the light path completely you must also press the button “Shutter” in front of the microscope, (fig. 10), in case you chose a fluorescent mode.

Focus the image in microscope with motorized and manual fine focusing (fig. 11). Z ↑ raises the objective towards the specimen, and should stop to 0-position, when set right. Z ↓ lowers the objective. In case you want to reset the 0-position where the motorized upwards focusing stops, find the focus, and press simultaneously the middle (Set) and the top (Z ↑) buttons more than a second, and repeat this procedure. Use the hand wheel for manual fine focusing. For moving the stage in X- and Y-directions, use the SmartMove remote control module (fig. 12). The preciseness (fastness) of movement can also be defined to either “fast” or “precise” by pushing either one of the black buttons on the left side. For focusing, z-movement, you can use either the focusing knob (z) of the SmartMove device, or the manual fine focusing hand wheel of the microscope.You can choose between Z-fine and Zcoarse with the single top right button of the SmartMove module, and the chosen option is visible on the right low corner of the LCD of the microscope.

Nothing but blackness in microscope eyepieces – troubleshooting: -is the external light source for fluorescence mode (fig. 7) switched on? -do you have the right filtercube (fig. 8) in use for your fluorescent sample? -is the shutter (fig. 10) on in case of fluorescence mode? -have you pressed the TL/IL –button (fig. 9) in case you need transmitted light? -is the sample in focus? -is the sample facing cover glass down, and sits nicely in the shallow groove? Field of view vs. scan area The scanned area is always a smaller square or rectangular in the middle of the field of view (table 1.). Table 1. Field of view and maximal scanning area with available objectives in TCS SP5.

 

10x dry

20x dry

40x oil

63x oil

ø 2.50 mm

ø 1.25 mm

ø 0.625 mm

ø 0.397 mm

1.55 mm x 1.55 mm

775 µm x 775 µm

390 µm x 390 µm

250 µm x 250 µm

  Find a presentable area in your sample and centre it in the field of view. This is, however, not critical as the sample can be moved also while scanning. Note! Do not choose the best area of the sample first, because some preparing scanning is required to achieve suitable scanning conditions for the first scan. In this way the best area will not get any “pre-bleaching” before the actual scanning.

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Getting started v. 1.7  September 12th, 2011    Arrangements for scanning

Choosing the Beam Path Settings In the “Beam Path Window” you modify the light path suitable for your sample, from excitation laser all the way to the detector. Choose the correct dye (identical or nearest match) from a list of “Leica Settings”, in “Load/Save single setting” window (fig. 15). Here we prepare settings for Alexa 488 dye. Therefore, choose FITC (the nearest match) in the list of dyes (fig. 16). After clicking FITC, the excitation line 488 is activated with certain percentage of laser output, certain beam splitter is chosen, as well as certain emission detection range is chosen. These are pre-defined manufacturer’s settings, which can be further modified, but are good to start with. It is advisable to choose first some saved (factory or user) setting to start with.

Settings for basic scanning In the Acquisition-window on the left side of the screen are the most settings for scanning. Small, one row windows can be opened by clicking the triangle in right end (fig. 17).

In the top window “Acquisition Mode”, the acquisition mode can be changed from xyz to some other, if needed. The defined mode is xyz, when the scanning software is opened.

Scan format, speed and area can be chosen in the next window. Predefined settings are format 512 pixels x 512 pixels with scanning speed 400 Hz and maximal scanning area (i.e. zoom factor 1, without any zoom in). Note - the maximal scanning area gets smaller when you change to larger magnification objective (table 1.). Image size and pixel size with current settings are also visible here.

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Getting started v. 1.7  September 12th, 2011    There are 13 different predefined scan formats, most of them square, but some also rectangular (fig. 18). In case you choose the option “More”, a new window opens for setting the x- and y-format with sliders. The single file sizes become quickly fairly large in formats larger than 1024 pixels x 1024 pixels, where the file size is roughly 1Mb.

There are seven predefined scan speed options ranging from 10 Hz to 1400 Hz (fig. 19). In option “More”, you can type the wanted speed between those values. Remember, that scan speed 600 Hz is the fastest speed, which still can use the maximal scanning area. In case you choose speed 700 Hz, the zoom factor will grow to 1.4, and the scanning area will be somewhat smaller. With maximum speed 1400 Hz the zoom factor will be 6.0, when scanning area is clearly smaller than maximal scan area. This is normal, technical “speed limit”, which prevents overheating of critical parts of the scanner.

Start scanning Click the “Live” button on the lower left corner of the left screen. You will hear mechanical sounds from the microscope stand, as mode is changed from viewing to scanning. You will also here a “click” sound from behind the table, where the system interlock opens to let laser beam through the scanhead into the sample. The scanned image also appears on the right screen.

No scanned image on screen – troubleshooting: -are the correct excitation lasers activated (figs 13 & 14)? -have you chosen the correct dye in “Load/Save single setting” window (figs 15 & 16)? -is the sample in focus (fig. 11)? Use the rightmost knob of the Control Panel (fig. 5) to move in z direction, and the SmartMove remote control module (fig. 12) to move in x and y directions. In scanning it is advisable to use the panelbox knob rather than SmarMove module for z direction, as it is far more precise. The first will move the specimen stage, but the latter will move the objective. Now you have a scanned image of your sample on the screen, and the scanning continues as long as you click the stop button. This is where the work only starts. Next step is to set optimal laser excitation power and emission detection range for the sample. Preferred laser excitation power is such, that specific staining is bright, but not saturated. In a saturated image area you will not be able to detect any details, but it is homogenously all too bright. Furthermore, in most cases, it is not a bad idea to show also some unspecific areas in the image.

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Getting started v. 1.7  September 12th, 2011    Note! The image on the screen is rotated 90º to left in contrast to view in microscope. Type the value 90 in the numerical window on the right side of the slider “Rotation” (fig. 17), in case you want to have the image orientation on the screen to be identical with microscope view. Modifying emission detection range To collect effectively the specific emission signal to detectors, you can modify the emission detecting range, in case you are not satisfied with predefined settings. For our fixed sample stained with Alexa 488, we first select Leica / Alexa 488 dye in a list under the active detector, in this case PMT 1, i.e. Photomultiplier tube (fig. 20). An emission curve for the chosen dye appears (fig. 21). Note! This emission curve does not affect scanning in any way, as scanning can be also performed without it, when “None” is chosen in the list. It is intended simply to help you in setting the emission detection ranges, especially with two or more active channels in use simultaneously. The bar representing emission collecting range for the active detector is connected to it with a ”shade”.

You can affect the emission detection range bar while scanning, and see immediately possible change in scanned image on the screen. The emission bar can be broadened or narrowed in both ends (fig. 22), or moved without affecting the broadness by dragging it in the middle (fig. 23). Furthermore, emission range can be set numerically, after you double click the emission bar (fig. 24).

In case you have a bright staining, try also to reduce emission range in the right end to fit under the top of the emission curve, like in figure 21. This also reduces unspecific staining to be collected in detector, and thus betters visibility of the specific signal in the final image. You can choose the colour of the final images by clicking the colour bar next to word PMT (figs. 20 & 21), which opens a new window. Remember, however, that confocal images are always pseudo-coloured!

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Getting started v. 1.7  September 12th, 2011   

Setting the dynamic range To use the whole dynamic range of the detector (PMT), click the Quick LUT-button on the left side of the scanned image in the right screen (fig. 25). There are six rotating knobs on the Control Panel (fig. 5). The first in the left is for setting the “brightness” of the image, or voltage of the detector. The second is for setting the offset value, to acquire, for example, nicely black background for areas without any sample in scanned area. Now, scan the sample with Quick LUT-button activated. Rotate the first knob in left to get brighter or dimmer image. The intended value is such, that there is little blue to be seen somewhere in the image (fig. 25). The blue pixels are saturated, and represent the maximal value 255 in a digitalized 8-bit image. Note! While scanning, move also in z-direction with the first knob in right to reveal any brighter areas in differing depths of the sample. Continue scanning, and rotate the second, offset knob. In case you have a totally orange-brown image, rotate the knob counter clockwise until the image turns to green. Green colour is black pixels, representing zero values in digitalized 8-bit image. The intended value is such, that there are some green pixels somewhere in the image (fig. 25). Quite often, however, the green pixels cover large area of the image, and what is more, the limiting value between no green and a green cover can be hard to detect. Eventually you have both blue and green pixels in scanned image (fig. 25). Click once the Quick LUT-button, and the image turns to black-and-white. Click once more, and the chosen scan colours appear on the screen. Always use as low laser power as possible. When setting the dynamic range, try also reducing laser excitation power when setting the voltage of the detector. Usually, the best values for PMT voltage are between 800 and 900, which means quite reduced laser power with bright samples. Use the slider of the specific laser excitation line in top right corner of the left screen to control laser power. The optimal brightness of the image is set with both laser excitation power and PMT voltage. Neither should ever be in, or even near maximal setting!

Averaging an optical plane Try also averaging function to get better image/noise-ratio in blacker background and brighter specific staining. In practice averaging means how many times a single optical plane is scanned before it will be the final image. Scanning the same optical plane twice, for example, reduces some 25% of unspecific brightness and noise. This is worth using especially when there is clear “stardust” in background. In best case background becomes black after averaging enough. Frame averaging is the mode for fixed samples, line averaging for live cell imaging. Choose a value for “Frame Average” (figs. 19 & 26), and click “Capture Image” scan option on lower right corner. Chosen number of scans will be performed on the one optical plane visible on the screen. Choose a different value to try another amount of averaging, and compare results of the scanned images in “Experiments” to find out the best option.

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Getting started v. 1.7  September 12th, 2011   

Bidirectional scan You can also use “Bidirectional X” option to double scan speed. Usually it does not deteriorate image quality without further modifications, but always check quality when changing to bidirectional scan mode. When bidirectional option is activated, also a “Phase correction X” slider is visible (fig. 26). It is a bit too rough tool for phase setting, though. So, first click words Control Panel (fig. 27), and a USB Control Panel window will open (fig. 28). With this tool you can change the status of each rotating knob in the Control Panel (fig. 5). You need to change function of one of the knobs to “Phase”, for setting the optimal phase for bidirectional scan. The Pinhole knob is converted to Phase knob in figures 29 & 30.

Start scan by clicking “Live”, and move in z direction in your sample to choose a bright image to be scanned. Click a few times the magnifying glass (+) on the left side of the image on the screen, and check shape of a contrastingly bright area. Turn the phase knob to see modification on the screen. The bright edge will turn from even to more saw edged the further you go from the optimal phase, and finally even forms double image. Try phase setting also to the other direction to find the best position, which produces the most even image. Click 1:1, and continue scan procedure.

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Getting started v. 1.7  September 12th, 2011   

Zooming Scanning can be concentrated on an interesting, smaller area of the image even on the fly. Just tick the “Zoom in” box (fig. 26), and draw a square on the image screen. The scan area and scan information will immediately be updated on the screens. Although a confocal microscope is an excellent optical instrument, the limits of resolution must be taken into account. In practice this means using no smaller pixel size than some 100 nm, even with a high-resolution, N.A. 1.40 objective. Zooming above this limit will only get “larger view”, but the resolution will remain the same, and image becomes the fuzzier the more you zoom.

Saving images LAS AF scanning software creates a folder named “Experiment” where the scanned images are saved. This is only a buffer memory, though, and you have to rename and save the images to defined storage yourself. In case you use “save” command, the image format will be dot-lif. In case you export the images, you can use other formats, such as TIFF, JPEG or AVI. It is advisable to rename scanned images right away, as well as delete needless images. In that way you can save or export the whole Experiment folder whenever. Experiment folders can be found under Experiments-tab, but the Acquisition-tab is in use while scanning.

Known bugs The beam splitter 488/543/633 is chosen in factory defined Beam Path Setting for FITC. However, it is better to use the RSP500 beam splitter for single green emission dye.

LEICA TCS SP5 - SHUTDOWN PROCEDURE After your scanning session always log your reservation as used in the Reservation Book, for example through the link that was e-mailed to you in context your reservation. It is a four-step process where the submit command is showed on the last page. Do not forget to inform on any technical problems or malfunction of the system. Make sure you have transferred all your important images to external memory device from the buffer memory. Check the Reservation Book for any reservations after your session. In case there is a reservation during the next three hours: I)

Configuration Hardware Configuration Laser (figs 13 & 14) Decrease Ar-laser power to 0% with the slider. Remove ticks in the boxes of lasers in use:

II)

Exit the scanning software LAS AF: click the cross on top right corner of the left screen. A warning message for unsaved images appears on the screen, in case there are any.

III)

Log off Windows WP.

Leave all other instrumentation running. This way the computer is in XP-user accounts view, and hardware ready to be used. In case you are the last user of the day: 1)

Switch off the external light source (fig. 7.).

2)

Configuration Hardware Configuration Laser (figs 13 & 14) Decrease Ar-laser power to 0% with the slider. Remove ticks in the boxes of lasers in use:

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Getting started v. 1.7  September 12th, 2011    3)

Turn the Laser Emission key to “Off-0”-position (fig. 2.). Yellow indicator light goes out. NOTE! Leave the right green button “Laser Power” on until the air cooling of the Ar-laser automatically goes out after a few minutes. You will hear the difference, once it is silent. In any case, five minutes is long enough.

4)

Exit the scanning software LAS AF: click the cross on top right corner of the left screen. A warning message for unsaved images appears on the screen, in case there are any.

5)

First log off Windows XP!

6)

Thereafter turn off the computer.

7)

When the screens become black, press off the middle green button “Scanner Power” (fig. 2). The scanner becomes quiet.

8)

Press off the left green button “PC, Microscope” (fig. 2).

9)

Remove the sample. In case you used oil immersion objective(s), clean the lense(s). First wipe away the remaining oil from the lens with a dry lens tissue. Take a new lens tissue, dampen it with isopropanol (bottle on the table), and wipe the lens again. Use lens tissue only for cleaning objective lenses. What is more, clean all other stains of oil or cell medium etc. on microscope, tables and everywhere. Use the roll of Katrin tissue for these cleaning purposes. Always keep the microscope facility clean!

Fig. 31. A cleaned oil-immersion objective lens after use.

Fig. 32. Partially dried oil on immersion objective lens some 48 hours after use.

10)

Press off the right green button “Laser Power” before you leave the room, in case some five minutes have passed since you turned the Laser Emission key to OFF-0.

11)

Place the Zeiss eyepiece cover on.

12)

Place the “Please take care on microscopes” note on the specimen stage. Make sure the room is as tidy as it was when you entered. Don’t leave any of your belongings lying around on the tables. Sharp waste into special waste dishes, vinyl gloves to the bucket, and other waste to trash bin.

Fig. 33 Not a pretty sight. Specimen stage smeared with cell medium and oil immersion objective lens not cleaned. Note that continuous carelessness will lead to sanctions in usage.

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