Leica TCS SP5 Spectral Confocal Microscope

User Notes CONTENTS: START UP PROCEDURE FOR THE LEICA SP5 CONFOCAL...........................................................1

Using the Leica DMI6500B Inverted Microscope................................................................................3 Capturing images with LAS AF software.............................................................................................4

RULES FOR USE OF THE MERCURY LAMP.....................................................................................9 SHUTDOWN PROCEDURE FOR LEICA SP5 CONFOCAL MICROSCOPE.....................................9 COMMON FUNCTIONS.......................................................................................................................10

Setting up Sequential Scanning for multiple fluorophores .................................................................10 Zooming and using the Region of Interest (ROI) zoom .....................................................................11 Capturing a Z-Series (xyz)..................................................................................................................12 Additional Modes and Functions ........................................................................................................13 Specialist Modes and Software Wizards.............................................................................................13 Saving and exporting your data ..........................................................................................................14

TROUBLE SHOOTING.........................................................................................................................15 Frequently Asked Questions ...............................................................................................................15 Restarting after an error ......................................................................................................................16

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START UP PROCEDURE FOR THE LEICA SP5 CONFOCAL

1. Turn on the switches and key on the TCS workstation control panel in order as shown below;

------ wait until system ready to proceed ------

2. Logon to windows on the computer

3. Check that the objective turret on the microscope is at the lowest position and that the condenser is in its normal position well above the stage. The microscope stage pans around upon startup and it is essential that the objectives and the condenser are out of the way to prevent the stage crashing into them.

4. Open LAS AF software Click on the LASAF icon on the desktop, then click on “OK” in the box that appears (shown below). Do not go into the configuration or microscope stand. Only activate the resonant scanner if you require superfast scanning at 8000Hz for a specialist application.

The program will then commence initialisation of the microscope (you cannot use the microscope during initialisation).

After a few minutes the following message will appear:

You should have already checked that the objectives and the condenser are out of the way at step 3. above. Clicking “Yes” initialises the stage, which is essential for using stage position coordinates (eg Mark and Find and Tile Scan modules). If you have had to restart the software after a software crash, and you do not wish to lose your position on your sample, click “No”.

------ wait until system ready to proceed ------

1. 2. 3. 4

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The LAS AF program will open showing the “Acquisition” page; 5. Select the desired objective in the software before proceeding to the microscope

The current objective lens is indicated in the “Beam Path Settings”, however the immersion fluid specification isn’t shown, so even if this is the magnification you want you must check the objective type before proceeding.

“IMM” multi-immersion (glycerol, oil or water) “OIL” oil only “WATER” water only

Click on the “Objective: …….” to display the available objectives and select the one you want:

**** Important Notes ****

i) It is vital to the longevity of the objective lenses that the correct immersion fluid is used, please double check which objective is in place before adding immersion fluid.

ii) You must clean your slides and the lens’s between using different immersion fluids. iii) The “20x IMM” objective lens has a corrective collar which is adjustable according to the choice

of immersion fluid used with this multi-immersion lens. The default position on the corrective collar of this lens should be set for use with oil. If you need to have this altered for use with glycerol or water please ask to be shown how to do this.

iv) The “63x WATER” objective lens has a corrective collar to enable optical correction for coverslip thickness. If you need to use this objective please ask to be shown how to do this.

Choice of immersion type. When working with biological specimens, the choice between an oil immersion objective and water immersion objective depends on the specimen itself and its embedding medium. You will achieve the best resolution when the refraction indexes of the embedding medium and/or the specimen are matched to the refraction index of the objective medium. 6. Proceed to setting up your sample on the Leica DMI6500B Microscope (see below).

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Using the Leica DMI6500B Inverted Microscope The microscope should already be on. (This was turned on through the TCS Workstation Control Panel). 1. Turn on the mercury lamp (the power switch on EBQ100 box) if it is not already on. Log the lamp usage time in the Leica SP5 Log Book. To use the mercury lamp you must be familiar with the rules of it’s use, which are detailed on page 9. 2. Gently tilt back the transmitted light illumination arm (top part of the microscope) 3. Add a drop of the CORRECT immersion fluid for the chosen objective (see “Startup” protocol Step 5 on page 2). 4. Place slide coverslip-side down on stage. 5. Return the transmitted light illuminator arm back into position 6. Choose between bright field or fluorescence mode and select filter. The microscope LCD screen displays the current filter setup;

To select brightfield (TL, transmitted light), press the “Chg TL” button, the button at the top behind the focus dial on the left-hand-side of the microscope. Pressing the “Chg TL” button again switches between the available contrast methods;

TL_BF: regular brightfield (no contrast method) TL_DIC: Differential Interference Contrast TL_POL: Polarised

To select fluorescence mode, press the button for the desired filter-cube on the front of the microscope;

FILTER CUBE EXCITATION DICHROIC MIRROR EMISSION N2.1* (eg for dsRed, Alexa546, Cy3) BP 515-560 (green) 580 LP 590* I3* (eg for GFP, Alexa488, Cy2) BP 450-490 (blue) 510 LP 515* L5# (eg for GFP, Alexa488, Cy2) BP 440-520 (blue) 505 BP 497-557 A* (eg for Dapi, Hoechst, AMCA) BP 340-380 (UV) 400 LP 425*

* These are all long pass filters, and so the emission you will see is quite different to that you observe with the filters on the AX70, BX50 and IX71 fluorescence microscopes. # This is a new filter to be installed in the latter part of 2007, and is not a long pass filter

The Shutter button for fluorescence light is on the front of the microscope

The intensity of the fluorescence and white light can be adjusted with the INT buttons on the left-hand side of the microscope. The FD buttons on the left-hand side of the microscope control the field diaphragm for the fluorescence light (eg you can decrease the illuminated field size to restrict the area of your sample being subjected to (and perhaps bleached) by the illumination).

To view both brightfield in DIC and fluorescence, select the desired filtercube then press the lowest button behind the focus dial on the right-hand side of the microscope. 7. Focus your specimen. There are two focus modes; “fine” and “coarse”. Select focus mode using the “z fine” and “z coarse” buttons on the right-hand side of the SmartMove knob.

To set a desired objective position, press the “z↑” and “set” buttons on the right-hand side of the microscope simultaneously. The first press clears any previous setting, and the second press enters the current focus plane as 0.0mm. (Do not use the “z↓” button). 8. Proceed to image-capture capture in the LAS AF software.

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Capturing images with LAS AF software 1. Open the “Configuration” tab, then open “Laser” configuration and turn on required lasers ( ). For the Argon laser, specify the % power setting. We recommend you use an Argon laser power setting of ~ 50%.

Laser Excitation lines

405 nm Diode; 405 nm

Argon; 458, 476, 488, 496 & 514 nm

DPSS 561; 561 nm

HeNe 633; 633 nm Note: The Laser configuration is the only item in the Configuration menu that you should change. You may wish to view the objective configuration as this includes details of the numerical aperture, resolution, focal depth and working distance for each of the objective lenses. 2. Log the time at which you start the lasers in the Leica TCS SP5 Log Book. 3. Set or chose your desired “Beam Path Settings” in the “Acquire” tab. You may define your own beam path settings or use / modify a pre-defined setting. There are a couple of special requirements you must follow if you need to set up beam path settings for subsequent combination as a sequential setting (see below).

In the first instance you may require assistance to set your own Beam Path Settings for your fluorophore combination, which you can then save with your name as a prefix.

To load a pre-defined setting (laser combination, respective laser power, and the spectral range of PMT detection), simply select the setting from the list under “Load/Save single setting”. Settings for commonly used fluorophore combinations are available, which you can modify as desired (see below) and save with a new name.

To modify an existing setting you can adjust the power of the respective laser(s) with the vertical sliders, activate / inactivate additional lasers with their respective vertical slides (ensure the required laser source(s) is active (“UV” and / or “Visable”; red dot: active, grey

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dot: inactive), adjust the PMT detection spectra range with the horizontal sliders below the colour spectra chart, and activate the required PMTs. Clicking on the list of common fluorophores beneath each PMT shows the emission spectra of that fluorophore on the colour spectra chart. This is a handy guide for setting the detection spectra range, and avoiding detection “bleed-through” with multiple labels.

To define your own settings, it is probably easier to start with a predefined setting and modify it. However, if you wish to define your own settings from scratch; click the grey dot to the right of the “UV” and “Visable” lasers as required to enable the lasers, set the respective laser powers using the vertical sliders, set the PMT detection spectra range with the horizontal sliders below the colour spectra chart, and activate the required PMTs.

For multi-labelling experiments you can choose between simultaneous or sequential scanning of the laser lines. Sequential scanning is recommended as this prevents artefacts due to cross-excitation of the fluorophores of other laser lines and “bleed-through” of fluorophore emission into other PMT channels. Cross-excitation and bleed-through can occur in simultaneous scanning and can give erroneous data if the proper controls are not performed.

3a. For sequential scanning, it is advisable to first set up the beam path settings for each label as a separate single setting. There are two pertinent points for setting up beam path settings for subsequent use in a sequential scan. For further information on Sequential Scanning refer to “Common Functions: Setting up sequential scanning of multiple fluorophores” on page 10.

1. You must ensure that each of the 5 PMT detectors are set with identical start and end spectral positions in each beam path setting to be combined. The easiest way to do this is to first configure the detection spectra for each PMT, and then create and save multiple individual beampath settings with the respective laser and detector activated. Any subsequent change to a PMT spectra setting needs to be made in each of the individual programs to be combined in the sequential scan, irrespective of whether that detector is active or not. Similarly, the beampath setting for transmitted light detection (see below) also need to be configured with the same positions set for all 5 PMTs if it is to be used as part of a sequential scan.

Dropdown list of pre-saved settings

Required laser source needs to be selected (● on, ● off)

Adjustable vertical slides for the power of each laser line

Adjustable slides for each PMT detection spectra (right click to display λ range)

Assigned LUT for each PMT (arbitrary, right click to change To display the emission spectra of a given fluorophore, select the fluorophore from the dropdown list Click box to activate required PMT(s)

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2. If the beam path settings to be combined into a sequential setting comprise both the UV laser and the Visible lasers, both laser sources must be selected (Red dot ) in every beam path setting.

For simultaneous scanning, multiple beam path settings can be set within the same setting, simply activate the lasers and detectors desired.

4. Transmitted light. To capture images in transmitted light mode (simultaneously or sequentially), click on “Additional Channels” to reveal the transmitted light PMT detector “PMT Trans”. Do not attempt to use PMT NDD1 or PMT NDD2. These are only for use with the FCS system, and their use requires specific approved training.

Imaging in transmitted light mode also requires the use of a laser line, such as the 488nm laser line at low power (5-10% is usually sufficient).

The use of transmitted light imaging requires that the condenser is in the correct position. The condenser is set to the correct position when the large grey condenser knob on the left-hand side of the condenser is at the lowest position (this is sometimes raised by users who require more working space above their specimen, so do check that it is in the lowest position). The large black knob on the right-hand side of the condenser is intentionally locked in place and should not be altered. The field diaphragm (lever above the condenser at the front of the microscope) is sufficiently open for use with all of the objectives even when it is in the “closed” position. The aperture diaphragm may be adjusted by the “AP” buttons on the left-hand side of the microscope (in front of the focus knob).

A range of contrast methods for the detection of transmitted light are available.

The degree of contrast within contrast methods which utilise polarised light (pol and DIC) can be adjusted by rotation of a small black polarising filter wheel which is found on towards the back of the left-hand-side of the objective turret. Adjust this wheel whilst imaging to set to the desired setting.

Note that the contrast method is not saved within any saved beam path settings, and you will need to re-select the contrast method each time you open a transmitted light beam path setting, including when you open it up within a sequential setting.

Because transmission imaging uses a laser line (eg 488nm) it is appropriate to detect transmission simultaneously with the fluorescence from the same laser (one laser line on and both detectors on). Or, if desired, transmission beam path settings can comprise part of a sequential setting (remember to re-select the contrast method if desired).

Click here to select contrast method from dropdown list;

Click on box to activate the PMT ( )

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5. Choose the desired scan mode in the “Acquire” menu under “Acquisition” “Acquisition Mode”.

xyz (the default) is the most common mode and can be used for both simple xy scans as well as xyz scans (Z-series).

Most combinations of x, y, z, t (time) and λ (wavelength) are available in two, three or four dimensions (refer to the dropdown list).

6. Set the scanning parameters of pixel format, scanning speed, pinhole size, line averaging, frame averaging, accumulation and zoom in the “Acquire” menu under “Acquisition”.

Pixel Format. The default, 512x512, is standard and acceptable for publication, although in some instances 1024x1024 may be desirable for publication. Lower and higher resolutions are available in the drop-down menu, but beware, the higher the resolution the larger the file. The pixel size and image size are shown.

Scanning Speed. The default, 400Hz, is recommended. Faster scanning speeds may be required for imaging live cells or moving specimens. For even faster scanning, you can activate bidirectional scanning (“Bidirectional X”). This doubles the scanning speed, but requires “Phase” adjustment. Please ask if you need to be shown how to do this.

Pinhole size. The default setting of 1 Airy Unit (AU) is the recommended setting. (The pinhole setting of 1 Airy Unit will display a different physical pinhole aperture size on the control panel for each objective). For faint specimens, increasing the pinhole size may help, but the images you are capturing may no longer be truly “confocal” images.

Averaging. The Leica SP5 automatically averages the image detection during the scanning process, however it is advisable to include additional averaging, perhaps 3 to 8 times, to reduce the background noise. To determine whether you should average your image more times, try capturing with more averages and see whether the image is improved. Line averaging is faster, and therefore recommended for live cells. For fixed specimens, it

Click to reveal dropdown list;

Click on arrow to reveal the full menu

Refer to notes below on each of these functions

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doesn’t really matter whether you use line averaging or frame averaging or a combination, although frame averaging may be better suited for some applications to allow for a brief recovery from photo-bleaching.

Accumulation. Usually only required for faint specimens.

Zoom. Refer to notes on “Zooming and selecting a Region of Interest”.

7. Click “Live” to commence continuous scanning in preview mode.

Whilst scanning, optimise the gain and offset of the detection and, if necessary, the laser power. You will probably need to adjust the “Z Position” using the dial on the Control Panel. CONTROL PANEL:

For fluorescence: initially set the “Smart Offset” dial on the control panel at 0.0% and adjust the gain. The gain can be adjusted automatically by clicking on “Auto Gain”. Alternatively, you can set the gain yourself using the “Smart Gain” dial on the control panel. The recommended approach for this is outlined below. The optimal gain setting takes advantage of the full range of 8-bit intensity detection (0 to 256), with very few pixels registering the maximum or minimum values. The best way to set this is to switch the LUT (colour designation Look-Up Table) to “Glow Over Under” (“GlowOU”).

Clicking on the LUT icon (in the vertical panel of icons on the left-hand-side of the image monitor) switches between the designated LUT, GloOU and grey. In GloOU, pixels registering the maximum detection appear blue, whilst pixels registering the minimum detection appear green. Adjust the gain such that only a very few pixels register as blue. Using a lower level of laser excitation and higher gain for detection will help minimise photo-bleaching of your sample. You can also adjust the level of laser excitation (see “3. Beam Path Settings” above) whilst scanning in “Live” mode. You may chose to reduce the offset to a negative value (the offset should not have a positive value for fluorescence). For transmitted light the procedure for setting gain and offset is the same, however positive offset values may help to improve the contrast. Click on “Stop” to end the scanning. 8. To capture your image simply click on “Capture Image”. You may choose to alter the scanning parameters as appropriate, eg how many times to average etc.

Smart Offset Dial

Z Position Dial

Smart Gain Dial

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RULES FOR USE OF THE MERCURY LAMP

1. Only turn the lamp on when it is cool, ie the lamp needs to have been off for ≥ 30 minutes. The lamp must not be turned on if it is still warm from the previous use.

2. Once turned on, the lamp must remain on for at least 30 minutes. If you finish you session within 30 minutes, you need to wait or come back to switch the lamp off once it has been on for 30 minutes (providing that no-one else is booked to use it within the next hour – see 3. below).

3. Before switching off the lamp, check the booking sheets to see whether anyone is booked on within an hour of you finishing. If someone is booked on up to one hour after you finish, then leave the lamp on. If no-one is booked until ≥ 1 hour after you finish, then turn the lamp off.

SHUTDOWN PROCEDURE FOR LEICA SP5 CONFOCAL MICROSCOPE

• Save your experiments to your own image data folder on Drive D (see notes on saving and backing up of image data).

• Ensure you have removed the excess immersion fluid from all the objective lenses that you have used. If you have used anything other than oil on the 10x and 20x multi-immersion lenses, ensure you have cleaned them.

• If you are finish earlier than expected, and there is another user booked on after you, contact them (phone number on booking calendar) to let them know the machine is available.

• Check the booking calendar to see if there is another user on the machine with in the next hour.

Then,

• If someone else is booked on within the next hour; Return all settings in LASAF software to the defaults Go to the “laser” configuration in the “Configuration” menu and reduce the

power of the Argon laser to 0%. Leave the LAS AF software open Leave the Mercury Lamp on Finish filling in the Leica SP5 log book

or

• If no-one is booked on in the next hour but there is someone booked on later that day;

As above, but turn off the Mercury Lamp

or

• If you are the last user for the day; Close LAS AF (you don’t need to turn the lasers off in LAS AF first) At the TCS Workstation Control Panel;

Turn the “Laser Emission” key to the “Off-0” position Leave the “Laser Power” switch on (this operates a fan for the Argon

laser, and will turn off automatically after 5-20min when the laser is sufficiently cool. It doesn’t matter if this switch is left on overnight.)

Turn off the “Scanner Power” switch In Windows; shut the computer down, then turn off the “PC Microscope” switch

on the TCS workstation.

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COMMON FUNCTIONS

Setting up Sequential Scanning for multiple fluorophores

To do this you need to set up each of the individual beam path settings as separate single settings. There are a few special requirements for these if they are going to subsequently be used in a sequential setting: Firstly, if the sequential setting is going to use both the UV and the Visible lasers then both of these need to be selected (red dot) within each individual setting, even though only the desired laser line in that setting will have a power setting >0%.

Secondly, you also need to set up the same bandwidths and positions of all five detection PMT spectral windows in each individual setting, even though only one PMT channel will selected as active in each individual setting.

Also note that the pinhole size must be the same in each individual setting.

Ensure the gain, offset, and laser intensity is optimal for each individual setting, and re-save each optimised setting. Open the single setting which you wish to be scanned first (“Scan 1”).

Click on the “Seq.” icon. This will open the “Sequential Scan” menu.

“Scan 1” automatically sets to the currently loaded single setting (to change the single setting in “Scan 1” load the desired single setting then click on “Scan 1”). To load the second single setting, click on the “+” and “Scan 2” will appear and be selected.

Open the desired single setting, and click on “Scan 2” to enter it into this position. Repeat to add in all your desired settings. Once done, it is advisable to click on each “Scan” to double check they are entered correctly and the correct laser line and PMT settings are saved in each one. Changing the different scanning settings “between lines” is fine for most applications, and best for live cell imaging. Adjust any other scan parameters desired, eg averaging, and click “Start” to capture the sequential scan. The “live” function can also be used to preview sequential scans.

Sequential settings can be saved via the “Save” button in this window (to your own image data folder on Drive D), so in the future you can recall the setting by clicking on “Load”. Note however that recall of a saved sequential setting also recalls all other parameters under the “Acquisition” (zoom, format, etc). To exit the sequential scan, click again on the “Seq” icon. Do not save upon the popup message as the sequential scan setting closes (this save location is unspecified). Note also that sequential settings are linked to the individual settings loaded within it. These means that changes to the individual settings affect any sequential settings they are used in. It is therefore necessary that you check the settings of any pre-saved sequential settings.

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Zooming and using the Region of Interest (ROI) zoom The zoom factor can be selected in the “Acquire” “Acquisition” menu. This will zoom in on the center of the field of view. Click on the respective panning icon to move around within the field of view. To zoom in on a region of interest, perform a live scan, stop, tick “Zoom in” and a rectangle icon will appear in the vertical panel of icons on the left-hand-side of the image monitor. Click and drag on the image screen to select the region of interest. Upon clicking on “live”, the scanner will zoom in on the selected region, and display the zoom factor. The zoom factor and pixel size fields will be automatically updated.

*Note* the zoom function is an optical zoom (not a digital zoom), therefore zooming increases the resolution of the image. The maximum resolution is obtainable on a light microscope, however, is approximately 200nm, and thus there is no further benefit to be gained by zooming beyond a pixel size of around 60nm. Zooming to a pixel size smaller than 60nm only gives an image which appears blurred.

4. Re scan (“Live” or “Capture”) and the zoomed section will appear;

1. Tick “Zoom In” 3. Click and drag around your region of interest 2. Click on “□”

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Capturing a Z-Series (xyz) For fixed cells, use the default “z-Galvo” mode (this gives precision Z positioning using the Galvo stage). (If you are using the temperature controlled stage you must use the “Z-Wide” option instead). For accuracy of the start and end positions, it is recommended to set the “end” position of the Z-series first, then to set the “begin” position. To offset the effects of bleaching that may occur during the Z-series acquisition, and to offset the attenuation of laser power and signal intensity that occurs deeper into the sample, it is recommended to start the Z-series from the deepest desired position in your sample (furthest away from the coverslips and the objective) and end at the position closest to the coverslip and the objective. Following the instructions below automatically takes into account both of these recommendations. Setting the start and end points of the Z-series: Whilst scanning in “live” mode, use the “Z-Position” dial to scroll anticlockwise to the optical plane at which you wish to end the Z-series, ending the series at the closest desired point to the coverslip. Click on the black “End” arrowhead to set this as the end point for the series. Then scroll clockwise to the optical plane at which you wish to begin the Z-series (further away from the coverslip), and click on the black “Begin” arrowhead. Setting the number of steps or the step size: The ideal “system optimised” “z-step size” and number (Nr.) of steps will be shown, according to the Nyquist Theorem. This is excessive for most purposes. Instead, click on “Nr. of steps”, which enables the number of steps or z-step size to be entered. As a guide, it is appropriate to set the z-step size at approximately twice the xy pixel size (see “XY” dropdown). The number of steps will be automatically re-calculated.

Check the configuration of line/frame averaging. It is usually best to set averaging to 1 for taking a Z-series, otherwise it will take much longer and your sample may undergo significant bleaching before the end of the series. Click on “Start” to capture the series. After capturing a Z-series, you need to click on the begin and end arrowheads to de-activate them (brown; active, black; inactive).

Click for XY dropdown to look up xy pixel size

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Additional Modes and Functions Timeseries This is fairly self explanatory within the software. Chose the xyt or xyzt mode and a new window for configuring the time series will appear. If you need to decrease the minimum time between frames, go to a faster scanspeed. Note that with scanspeeds of 700Hz or faster it is necessary for the scanner to zoom in. Choosing a different pixel format, eg a strip such as 512x64, will also enable you to further reduce the minimum time between frames. Selecting bidirectional scanning will effectively double the scanspeed, but you need to check the “Phase Correction” (synchronisation) of the scans in each direction. The “Phase Correction” setting is at around -32. To fine-tune the “Phase Correction” set it as a button on the Control Panel (go to Control Panel under configuration and set a dial not normally used, eg Scan Rotation or Pinhole to Phase Correction instead), and adjust according to the image. To image very fast events, activating the “Resonant Scanner” upon startup of LASAF will enable a fixed scan speed of 8000Hz. See also “Live Data Mode” in “Specialist Modes and Software Wizards” section below. LambdaScan Can be used to determine an unknown emission spectra. Process tab The process tab includes a number of useful options, eg the crop function can be used to crop out unwanted channels from a multichannel image, or to crop out unwanted frames from a series (eg z-series or time-series). Similarly images can be recombined, eg adding in a single image in transmission mode to a fluorescence z-series, by using the merge function. To flatten a z-stack, go to 3D projection and click apply. To make a simple 3D rotation, go to 3D projection, tick “create movie” and then configure the rotation as desired. Quantify tab Enables quantification of a line or region of interest. Enables limited analysis of colocalisation.

Specialist Modes and Software Wizards

The normal mode of operation of the LASAF software is in the “TCS SP5” mode. Clicking on this tab at the top left-hand side of the screen enables access to specialist software modes, these include FRAP Wizard for measuring Fluorescence Recovery After Photobleaching. This wizard is fairly self explanatory. FRET AB Wizard for measuring FRET by Acceptor Bleaching. This wizard is also fairly self explanatory. FRET SE Wizard for measuring FRET by Sensitised Emission. This wizard is quite complex, please refer to the separate documentation in this folder on this application. FCS Wizard for taking fluorescence correlation spectroscopy measurements. This is very complex, refer to the separate FCS folder. Live Data Mode for live monitoring of data acquisition. This is fairly self explanatory, and allows for “macro” sequences of tasks to be set, and provides a live monitor of the intensity of set regions of interest, for example, for doing calcium imaging.

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Saving and exporting your data Saving your Leica lif file: Note: The captured images displayed in the “Experiment” menu are not safely saved until you save them. It is therefore strongly recommended that you start saving your images from the very first capture you make, and that you update your save after each image capture. After capturing your first image, right-click on the “Experiment” and select “Save Experiment “Experiment” as…” from the drop-down menu. Save your Lieca lif “Experiment” file into your own folder in the D directory of the computer, renaming as desired. Once you have specified the file directory for saving your Leica lif file, you need only update your file after each subsequent image capture by clicking on “Save All”. Exporting your Leica lif files: Leica lif files can only be opened using the LAS_AF program. To obtain images that can be opened in other applications you need to “export” your data. You can export your data into tif format (recommended, maintains pixel format and resolution), jpeg format (not recommended as this is lower resolution) or avi format (movie player format, eg to play an image series). To avoid using expensive time on the confocal microscope, please note that the Image Analysis computer in Room 4E423 has a offline version of the LAS_AF software (this is free to use), so time-consuming exporting of data should be done there instead of on the confocal computer. Images or series can be exported individually (right-click on the name of the image to be exported in the “Experiment” menu, and choose tif), or you can export all of the images/series in an experiment (right-click on the name of the image to be exported in the “Experiment” menu, and choose tif). There are several options when exporting your image as a tif: Selecting “Overlay” will save the merged image of a multi-channel image in colour. Selecting “Raw” will save the individual channels in greyscale will appear in the file name for the respective channel in multi-channel images). Not selecting either of these options will save the individual channels in their designated colours (also named as “chan000”, “chan001”, “chan002” etc). The lower panel of options enable a micron scale bar or time stamp (eg for time series) to be added to the tif, as desired. Note that there is a bug in the system where changing one of the options for exporting may cause the destination file to revert to that previously used, so double check the destination before exporting Saving and backing up your data: All exported data and your original Leica lif file should be saved to CD/DVD or memory stick. It is your responsibility to back-up your Leica lif file, and it is strongly recommended that you maintain a copy of this. Leica lif files can be left on the confocal microscope until you have had a chance to verify that your backup is successful. However, no exported data should be left on the confocal microscope.

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TROUBLE SHOOTING

Frequently Asked Questions Nothing is being detected on the screen. Check the lasers are on and that a detector is selected. Check the gain is above zero (usually the gain needs to be around 700-1000 volts, but the default setting is zero) The lasers don’t appear to be working. Check the lasers are on at the TCS control panel (switch and key), and that they were turned on here before being turned on within the software. Check a laser is selected in the beampath setting Confirm that no laser light is coming through the objectives (turn room lights out and start live scanning). If the lasers still don’t appear to be working, restart everything (see below). The objective turret can’t be lowered Either 1. the objective turret is slightly out of position or 2. a lower objective turret position has accidentally been set.

1. Check the LCD panel at the front of the microscope, if it reads “Obj ???” then it doesn’t recognise which objective is in place, most likely because the turret is slightly out of position. Gently manually re-adjust the rotation of the objective turret so that an objective “clicks-in” to position. The objective should now be recognised on the LCD screen, if not, restart the software, computer, scanner and microscope as described for “Restarting after an error” below.

2. Check the LCD panel at the front of the microscope, there are two sets of arrowheads near the bottom left-hand corner of the LCD panel.

The double facing arrowheads indicate whether an upper objective position has been set, with set positions indicated by a horizontal line; ie ▼ indicates no position is set, whereas ▼ indicates a position is set ▲ ▲ The setting of an upper objective position is a very handy feature, and how to do this is described on Page 3 point 7 “Setting a desired objective position”.

The single arrowhead ▼ next to the double arrowhead indicates whether or not a lower objective position is set, with set positions indicated by a horizontal line; ie ▼ indicates no position is set, whereas ▼ indicates a position is set Lower objective positions should not be set intentionally, as this prevents lowering of the objectives which could result in damage to the objectives upon stage movement and adding/removing samples. To unset a lower objective position, press the “z↓” and “set” buttons on the right-hand side of the microscope simultaneously. The horizontal line should now disappear and the objectives should now be able to be lowered. If not, restart the software, computer, scanner and microscope as described for “Restarting after an error” below.

My sample wont focus/went out of focus/appears to be moving You may have run out of immersion fluid, add more. If you are moving the stage a fair distance across your specimen you may need to add immersion fluid to your coverslip. Check for air bubbles in the immersion fluid, if present, wipe off excess immersion fluid and re-apply. You may have air bubbles in your preparation…..

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The intensity in the transmitted light detector has increased. Transmitted light beam path settings do not recall any contrast methods used, and you need to re-select this everytime you open a transmitted light beam path setting

The intensity of the in the transmitted light channel is uneven or has lines through it. Ensure the room lighting is off, as the room lighting interferes with transmitted detector resulting in vertical lines in the image. Check the condenser height and, if using polarised light or DIC, adjust the polarising filter wheel (refer to Transmitted Light section on page 6.). You may also need to make minor adjustments to the condenser height with the large grey dial to improve the image

Any other unexplained/strange happening in the system You did not imagine it! Very weird things happen with this machine. Please document the computer time of the error and the nature of the error in the confocal logbook, and generate an error report and restart as described below.

Restarting after an error Document the error: note the computer time of the error and describe the error in the confocal logbook.

Generate an error log: in LASAF, under the Help menu, go to “Create Error Report” to generate and save an error log. “Create Error Report” can also be accessed via the “Start” menu, via “All Programs” “Leica LAS AF” “Create Error Report”. If the computer is crashed generate the errorlog upon the restart. Click “Save” after is has been generated, it will automatically be zipped and compressed, then rename it with the date as the prefix, ie “YYYYMMDD_Logfiles” and save it to the “Local Drive (C:)” under “Documents and Settings” “General User” “Desktop” “Logfiles”.

Errors indicate something in the system is not communicating correctly, and may involve communication between the software, the microscope, the scanner, the confocal computer and the microscope computer. It is usually best to restart the software, confocal computer, scanner and microscope, to ensure everything is restarted with fresh communication. It is not usually necessary to restart the lasers unless the problem appears to have something to do with the lasers.

To restart: 1. Lower the objective turret at the microscope and remove your sample (if you don’t want to have to remove you sample see * below) 2. If possible, save your experiment then close the software (“X” in top right hand corner, or “Close” under the file menu) If the computer has crashed you may need to force quit by simultaneously pressing “Ctrl” “Alt” and “Delete” on the keyboard to go to the “Task Manager”, and going into “Processes”, selecting “LASAF” and clicking on “End Process”. If you are only going to restart the software and not the computer, then you need to also select “LCS” (software which runs in the background of LASAF) and “End Process”. 3. Shutdown the computer; for a complete restart of the computer, microscope and scanner chose the “Shutdown” option (not the “Restart” option). 4. Turn off the scanner at the TCS control panel 5. Once the computer has shutdown, turn off the “PC & Microscope” at the TCS control panel. 6. Wait a couple of minutes then restart everything as per normal startup procedure * If you have located an important position on your specimen and don’t want to loose it you may wish to try restarting the software alone, or restarting the scanner and the software, but not restarting the microscope, so as not to loose your position in your sample. In this case, you still need to lower the objective turret, and when the message appears to “initialise” the stage upon restarting the software, click “No” (refer Startup Procedure point 4 on page 1).