Switching On:

1. Turn on the "LASER IN USE" sign above the door.

2. Turn on the mains plugs labelled 1 and 2

3. Turn on the mains switch for compressor labelled 0 (unless already on); the compressor is shared with the other microscope in the room and the switch is behind the other microscope.

4. Turn on the switches on the electronic rack. The switch of the PC lablled 4 is behind the door on which the sticker is pasted.

5. Log in as user

6. Start laser software and under Utilities press Connection and select COM5 port:

7. The window should look like this with laser being On and AOM Output set to External:

8. Start ScanImage software and in the subsequent pop-up window press Start ScanImage:

9. In the following dialogue type N

10. Make sure the objective is retracted (if not lift it up in the direction of the yellow arrows) and home the piezo focus drive:

Locating Sample Using Widefield:

1. Pull out the slider (yellow arrow) above the objective which switches the emission path to the eyepiece:

2. Select a suitable filter set by rotating the filter cube turret (cyan rectangle in the image above); for brightfield select Epi BF.

3. Make sure the objective is retracted and place the sample on the stage. Add water immersion on the sample and carefully lower the objective (in the direction of the red arrows). Never let the objective crash into the sample!

4. Turn on the epi-illumination lamp.

5. Locate your sample by moving the stage and focus it by turning the microscope focus knob. Make sure you do not crash the objective into the sample while focusing!

6. When you have found a suitable location in the sample, push in the laser mirror and cover the microscope with blackout cover before starting 2-photon imaging.

2-photon microscopy:

1. Select which channels you want to capture; only channels 1 and 3 are active. Channel 3 is for longer wavelength - refer to PPMS to see what filter is currently installed filter for each channel. The Display and Save checkboxes toggle whether the image for the respective channel is shown in live view and/or saved during acquisition, respectively.

2. Set the laser power - the power is set as % of the full laser power, which is 351 mW at sample plane.

3. Set the the scan parameters - scan format as number of lines and pixels/line (identical for a square scan) and speed of scanning (pixel dwell time).

4. Set Zoom (defines the size of the scanned area) in the main control panel; note that the image may be vigneted for Zoom smaller than 3.

5. Select the path and define the base file name (a number will be appended to the base name for each consecutive acquisition) for saving images.

6. To see a live image press FOCUS in the main control panel.

7. The image brightness can be adjusted for each channel. The double arrow button on the right-hand side adjusts brightness of the respective channel automatically according to the image histogram. If the image is saturated even for the maximum black-to-white range, the input range of the signal digitiser can be increased in the Channels window. Conversely is the black-to-white range covers only a small postion of the dunamic range, the input range can be reduced. The maximum range is (-1, 1), the smallest range (-0.25, 0.25).

8. To acquire a Z-stack, make sure stack is enabled in the the Main panel. Then set the range and slice spacing or nummber of slices in the Stack Control window. Important: Take a note of the Step size (slice spacing) as this information is not captured in the image metadata!

Uniform stack means a stack spanning equal range below and above the current Z position.

Bounded stack spans the range between user-selected start and end Z position.

9. To define a bounded stack start live scan and move the focus up and down using the Z piezo drive to find the upper and lower limit of the volume you want to image. The number (10 in the example) defines the step size in microns. Important: do not use the manual focus knob to search for the limits of the Z-stack - the piezo Z coordiante does not change when you focus manually and hence both upper and lower limit will have the same Z coordinate!

10. If you want to average multiple frames for each slice, set the appropriate number of Frames per Slice in the Stack Controls window and at the same time Enable Integration and set the number of frames to be averaged (# Avg) in the main control panel. If only the former is done, each frame is saved separately (e.g. if 10 frames per slice are set and the stack has 20 slices, single channel, the aquired file will consist of 200 images - 10 images for slice 1, 10 images for slice 2, ...).

11. When everything is set, start image acquisition by pressing GRAB in the main control panel.

Practical tip: If you take a 2-channel stack, the channels are interleaved in the TIF file (slice 1 - channel 1, slice 1 - channel 2, slice 2 - channel 1, ....). To deinterleave the stack and at the same time assign the Z-slice spacing to the stack you can use ScanImageUnscramble macro in FIJI.

Switching Off:

1. Make sure you have saved your data and turn off ScanImage and the laser software.

2. Pull out the laser mirror, retract the objective and remove the sample.

3. Wipe the objetive lens using the lens cleaning tissue. Always wipe the objective only once, in one direction. If this is not sufficient, repeat with a new piece of tissue. Never reuse the tissue.

4. Check in PPMS calendar if there is any user coming shortly (< 90 min) after you. If yes log off from PPMS tracker and proceed to point 9. If no, follow all the points.

5. Turn off the power switch (3) and the epi illumination lamp.

6. Shut down the PC.

7.Turn off the mains switches for the microscope (1 and 2) and for the compressor (0) - unless used by the uuser of the other microscope in the room.

8. Cover the microscope with the blackout cover.

9. Make sure you leave the microscope room clean. Spray with 70% ethanol and wipe any surfaces that could have been in contact with biological material. Do not leave any samples or any other belongings behind.