GENERAL INFORMATION

Excitation lasers: 405, 491, 561, 640 nm (AOTF in the control panel, MPD should be set to CSV)

Emission Filters, Wheel A: 460/50, 525/50, 545/30, 590/30, 600/40, 620/60, 700/75

Emission Filters, Wheel B (slave camera): 660/20 (currently not mounted), 690/50

The detection is done by commercial spinning disk confocal microscopy Yokogawa CSU-X1. The system has an integrated dichroic mirror which prevents excitation wavelength to pass through (FLUORESCENT TAGES HAS TO BE CHOSEN CAREFULLY) and an iris with which one can adjust the incoming illumination on the sample.

The bright field acquisition is not motorized, so the bright-field image has to be acquired manually before or after fluorescence acquisition.

 

SET UP

1. Turn on power supply at the bottom of electronic rack
2. Turn on power supplied from top to the bottom for all devices including computer (Yokogawa power supply switch is on the back)
3. Turn on spinning disk with the key and turn on the light supply from thorlabs.
4. Turn on power with voltage: 24V the mina power supply, 18V to keep the window between microscope and the objective lens dry (2V are not needed it is only for plunger)
5. Turn on power on PI controller on top of power supply for freezer.
6. On the PC: Start up labview code for the stages, run it.
7. start uc480 viewer this is low magnification camera (LMC) for alignment. Start the camera.
8. Start andor software, if one camera (wheel A) to be used get “cryolight master time lapse” if both (wheel A+ B) open andor software again and start “cryolight slave 2”.
9. Check that the wheel A reads out camera du897_bv5157 and wheel B reads out du897_bv2035.
10. Make sure that all cameras are working (make dark current acquisition). Setup wheel positions to open. Set up trigger control to free run. 

WARM TEST

1. Take an empty glass capillary, fill it with rainbow beads and put it in the sample holder by hand.
2. Open sample loading port (with the switch) and push forward sample manipulator (switch forward).
3. Pre-align capillary with the manipulator by adjusting X and Z to the black marks on the sample port.
4. Put capillary down mechanically (not motorized).
5. Setup LMC for bright field acquisition: set Filter wheel to 5, close iris, laser off. Light can be adjusted with Thorlabs manual controller.
6. Find capillary in X and Z again, put the tip of the capillary to the middle of the sensor (motorized Y). Mark position on the screen. Write down X,Y,Z positions of the stage
7.  Move 1100 towards high magnification camera (HMC).
8. Setup imaging for high magnification: Filter wheel to 1, iris open. If necessary setup trigger (#3 written for bright field) in Andor control to “low”, i.e. high magnification.
9. The capillary should be in the middle of the FOV and in focus. 
10. Setup a new protocol on the camera.
11. Warm up required lasers, setup filters and exposure time, z stack spacing, etc.
12. Adjust capillary height such that it will be for sure in with a new one (not a the tip), put in Low Mag and when take it out

COOLING DOWN

1. Connect Helium and liquid nitrogen pipes. Open He tank. Open In and Out valves to the zeolite canister to flow. 
2. Fill thermal plastic white box with liquid nitrogen to cool zeolite bottle.
3. Turn up DC power supply to 24V, 18V to keep the window between microscope and the objective lens dry
4. Flow liquid dry nitrogen through cyo-LM by opening front valves (two back scales at the back of the microscope) to flow.
5. Fill dewar with liquid nitrogen to 3-4 inches deep (10cm).g
6. Wait until the temperature falls down to -162 degrees. Let it settle down for a bit.
7. Meanwhile, disconnect all plunger stage. It should be on the kinematic mounts. 
8. Bring sample transfer device from the beamline. Connect it to cooled Helium. 
9. Add propane to the propane holder.

LOADING SAMPLE

1. Make sure to check Liquid Nitrogen level and refill propane. 
2. Start flow of He in the transfer machine, add liquid nitrogen there.
3. Meanwhile adjust flow of dry nitrogen in the microscope (left one -bottom flow- should go to zero).
4. First grab capillary with the grabber (without He flow as it gets very steamy and one can not see anything). Detach it from the crocodile clam and start He flow. 
5. Put it to the kinematic mount on the cryo light, make sure it is positioned correct! 
6. Open iris on top, put capillary down such that the top part is still accessible, close iris and dismount transfer machine. 
7. Grab it with the specimen holder, open iris and put it down. 
8. One can not see the capillary in the Low magnification, so start to move to the high mag with steps of 100 (it should be ca 550 in the end), find capillary.

IMAGING

Confocal z-stack acquisition

1. Close iris, put filter wheel to 1, open shutter for lasers (AOTF control panel), open shutter in Yokogawa. 
2. Go to LVPZT control panel (that is scanning motor for z stack), move it to 25. Its range is from 0 to 53. 
3. Insert the channel of interest in the control panel. Start to edit it, set up the right wheel position depending on the fluorescence of choice. Start live acquisition. Adjust EM gain to minimum of 10, exposure time of 200ms. If you want to proceed faster set binning to 2×2. 
4. Adjust Z position in labview panel until you are in the middle of capillary. 
5. Now you are ready to setup your scan for saving. Make sure to close the shutter on Yokogawa while you think not to illuminate the sample.
6. Start with any protocol. From top to bottom it should be set: Image, Camera binning- Use Current, Wait allocation-ON, Camera Selection – Use current, Events, Repeat T- 1 times, Repeat Z (um range in X planes), move channel of choice, trigger out – Out3 High, snap, end Z, move scan to 25, endT.
7. Move scan to 25 assures that you always align with whole range of motion available. 
8. Any camera and microscope setting can be adjusted now within this protocol. For sensitive samples it is recommended to put Yokogawa shutter in open-closed mode.

Confocal 2 cameras z-stack acquisition

1. Follow protocol above to set up camera with wheel A.
2. Change the position of wheel B in this protocol for the fluorescence of interest, typically red.
3. Setup protocol for camera B. This one should be: Image-Protocol1, wait allocation-off, camera selection- Use current, Events, Repeat T- number of planes in camera A settings, Move channel – no settings required, snap.
4. Make sure in camera setting go to ext trigger. 
5. The actual readout between camera A and B is almost 2.5x longer. Thus it is crucial to adjust the intensity in camera A, either electronic gain, binning or decrease the laser for camera A (if different lasers are used). Similar image quality should be on both cameras with similar exposure time.
6. Check the actual frame rate by running both cameras simultaneously.
7. If the frame rate is different. In camera A control pane add after snap – wait time. Put as many seconds as the difference in frame rate.
8. Setup save directory for camera B, make it auto. Set up saving directory for camera A make it auto.
9. First run protocol for camera B, it will wait for trigger signal, so start camera A as well. If you set up them correctly, you will have the same number of images in folders you specified. 

SHUT DOWN

1. Move the capillary to low magnification, then remove it. 
2. Remove propane with venturi pump, 2 minutes later disconnect liquid nitrogen line and close the walve on the dewar.
3. Close both valves to zeolite bottle.
4. Turn off voltage on heaters to 12V
5. Close all the camera programs and lab view
6. Shut down the computer
7. Switch off all the power supplies in the electronic rack and spinning disk. 

For each specimen

Lasers in use	Filters in use (order as on the acuisition)	EM gain and exposure time	Folder where the data will be stored	Remarks

Capillary:
FOV	Time	X	Y	Z