Microscope Startup

Microscope Startup Protocol

Leave a note at the microscope and write an email to the lab (puchners-lab@googlegroups.com) if anything at the microscope is broken, modified or the adjustment is off.

Key points: Do not look into the laser or through the eye piece if lasers are on. Do not crash the x-y stage in the objective. Do not saturate the Camera.

Precautions:

When mounting samples onto the microscope, ensure that any and all lasers are signals are off; not switched off in the hardware, but off in the software. If the room is dark, the LED may provide sufficient lighting and will not feel like staring into the sun.

When moving the stage, used the “extra fine” setting and take care that it does not hit the microscope objective lens. This can occur if the microscope objective lens is raised to a high z position (vertical) and the stage x-y position is moved to an extreme.

The software must be closed BEFORE the hardware components are shut off. The software is designed to perform simple maintenance tasks – such as closing the camera shutter – upon exiting. Failure to do this may result in the camera shutter remaining open indefinitely while it is off.

1) Switching on the hardware:

First power on the required hardware:

  • Necessary:

    • The Andor iXon EMCCD camera. The button is pressed when it is on.

    • The Prior stage control. The switch is located in the back of the box.

    • The Nikon microscope at the lower right back side of the microscope

  • Optionally any lasers that will be used for the session. First turn on the power switches of the controllers but leave the keys in the off position until the sample is mounted.


Failure to start up required hardware components may result in unsuccessful startup or limited function of the software. The microscope may be switched on at any time before or after the software initialization (from the switch located in the back).

2) Starting the software:

- Double click the HAL4000 icon on the Desktop

- Load all camera settings that you will be using by dragging them into the left side of the HAL4000 window (e.g. for PALM/STORM STORM_256_20Hz.xml). Make sure to use the settings from the folder “dual_view” if the camera is not directly attached to the microscope.

- If you use shutter sequences for drift correction/two color imaging, click the button of the respective camera setting and load the camera settings file from the folder (e.g. Shutters_xy.xml)

- Choose your created folder to store movies by clicking “working directory”. The folder structure should be C:/Data/”yourname”/YYYYMMDD

3) Mounting the sample

- Make sure the Objective is lowered so you don’t hit it when moving the x-y stage

- check if the objective ring is set to the appropriate temperature and cover glass thickness (typically 23C)

- Switch the x-y stage to “extra fine” (with the default setting you risk moving to fast and crashing into the objective) and center the opening over the objective.

- Let a drop of immersion oil (only the Nikon brand) drip on the objective lens and quickly proceed with the two next steps so that the oil won’t flow away.

- Place your sample in the sample holder. Make sure the sample sits on the thin support of the sample holder and slightly clamp it between the two moving pieces. Make sure no sample liquid will spill.

- hit the PFS (Perfect Focus System) button at the front of the microscope (it will blink) and raise the objective by turning the knob at the microscope counter clockwise until the oil touches the cover glass.

- Carefully raise the objective further until your LED illuminated sample comes into focus or the PSF locks on by making a sound and the green light goes from blinking to on. Make sure that the objective does not touch and lift up the sample and that no air bubbles are formed the oil, which would distort the image. If you use the eyepiece, make sure all lasers are still OFF and the camera port at the front of the microscope is set to the top center position.

4) Preparing the data acquisition

- Turn on the lasers on by using the key of the controller. Never look through the when the lasers are turned on.

- Adjust and write down the setting of the automated filter wheel (typically setting 1 for OD=0 or 2 for OD=0.5). Adjust and write down the settings of the 405 nm laser filter wheel (typically starting at OD=3 and going to OD=1) and 488nm laser filter wheel (typically between OD=3 and OD=2 for 5Hz GFP/conventional fluorescence imaging).

- Set the camera port at the front of the microscope to the left position and open the camera shutter through HAL4000. Adjust the brightness of the LED manually with the knob starting at low values. Turn on the EMCCD gain (typically 30) and make sure the chip does not saturate (this would harm the electron multipliers).

- Move the stage carefully until you have enough cells in the field of view. Check the stage position and keep the sample center close to the objective so that the stage does not crash into the objective.

- Check if the emission filters are correct (only if dual view is not mounted) and that the magnification switch on front of the microscope is set to 1x and NOT 1.5x.

- Turn on the imaging laser with low power first (adjust slider intensity, click the button and update) and make sure not to look into laser shining through the objective. Check where the laser exits the objective so that you don’t look into it and if necessary adjust the TIRF stage to improve the signal/noise.

- Focus on the surface with the external wheel (once the PFS is locked indicated by the green light, the microscope wheel does not do anything), which becomes apparent by a blinking background signal. If necessary, bleach the background. The brightness of the background indicates the quality of the excitation/emission alignment. Change the focus to the height you want to image (typically a few 100 nm away from the surface so that the blinking fluorescent background is not detected but not too far away since S/N and sharpness will decrease).

- Once you are focused on the region of interest, turn on the 405nm laser at very low power (only for PALM imaging) to activate a few photoactivatable fluorescent proteins to check if the S/N is good, if the point spread functions are symmetric and round and if the illumination/activation area is centered. Move the stage to a new position to start the acquisition.

- Document any changes of the filter wheels in different movies you record.


5) Shutting down the microscope

- Close the shutter of the camera with HAL4000 and Close HAL4000

- Hit the “Escape objective” button at the microscope and remove the sample

- Open the sample holder and carefully remove the immersion oil ONLY with a thorlabs lens cleaning tissue. Do not put pressure on the objective lens while cleaning but use the folded tissue to soak and wipe away the oil. You can use your finger to remove oil from the objective rim around the lens. Put e.g. a box above the objective to protect it from dust

- Turn the laser keys to off. Turn off the microscope, camera,  lasers and x-y stage

-Transfer your data to the lab server and turn off the monitor


Troubleshooting:

The software is not controlling the laser.

Was the laser power switched on before the running the software?

Is the laser power switched on and is the key turned to the permitting position? Check the indicator lights.

Is the corresponding laser USB cable connected to both the laser control module and the correct port on the computer?

There is no image on the software.

Is the camera shutter open?

Is there any illumination? (e.g. laser, LED, ambient light, etc.)

The laser is on but there is no illumination.

    Is the beam’s path unobstructed?

PFS does not lock

Does the immersion oil form a continuous layer between the objective and the cover slip? Are there water bubbles? Is there a material with very inhomogeneous refractive index on the cover slip (the PSF works with a reflected IR laser beam)

External PFS focusing wheel does not work

Is the “Dichroic mirror in” button on the wheel turned on as indicated by the orange light? Is the PFS out of range? Make sure the objective is not lifting up the cover slip, turn off the PFS, manually focus and turn the PFS back on.

Comments