Nikon sora introduction protocol

Introduction protocol

Type of microscope and its strengths

• Spinning disc microscope for fast confocal microscopy (optical sectioning)
• Suitable for live-cell imaging experiments
• Superresolution optional, to achieve (ideally) ca. 150 nm resolution in green.
• Two-camera option for capturing two fluorescent colors at the same time (dynamic processes)
• Fluorescence Recovery After Photobleaching (FRAP) to study molecular diffusion times

Switch-on procedure (link)

• Make sure to also start the software already and start Single-cam mode

Stage-top incubation system for live-cell imaging (switch this on before starting a live cell experiment)

• Component 2 is the "Warming box" and needs to be switched on as well as the "Heater" button on its front panel
• Component 3 needs to be switched on and is already on the right settings.
• Look at the stage-top incubator itself - it has a lid that should be used during the experiment
• There is a plastic removable frame covering a "canal" (basin) of water around the edge of the sample area
• The "canal" should be filled with MQ water using the attached tubing - the tubing sticks out of the front of the microscope
• Unclip the tubing clip, attach a syringe with MQ water fill stage-top incubator "canal" with MQ water
• Clip the tubing clip again, remove the syringe, and cover the canal with the plastic frame. It is ready to use
  

First manual operations (moving things around)

• Operate the lens switcher at the front of the microscope and select the 100X / Oil lens.
• Operate the joystick in various directions as an example. You may have to switch its movement speed to fast.
• Center the stage above the objective.
• Operate the focus knob on the side of the joystick to see the objective move up and down (also may have to switch the movement speed to fast).
• Lower the lens as far as possible down to prepare for placing the sample.
  

First brightfield observation (finding the sample)

• Mount a demo sample (or user sample) in the appropriate sample holder without placing it in the microscope yet
• Put oil on the lens
• Put the condenser arm in the backward position (access to sample area easier)
• Mount the sample holder with sample onto the microscope stage
• Make sure the condenser arm (top arm of the microscope) is set to "O" (brightfield shutter = Open) and not "C"
• Restore the condenser arm to the normal operating position (upright, not backwards)
• Switch on the brightfield lamp (lamp icon, left side of the microscope body) and demonstrate adjusting light intensity (left side rotatable adjuster knob)
• Make sure the eye-piece is active using the "eye" button at the front of the microscope
• Confirm with the user that light is visible through the microscope and that it responds to intensity adjustment
• Visually point the brightfield-lamp-spot onto a relevant sample area, where we expect to see structures
• Let the user raise the objective while looking through the eyepieces, until the sample becomes visible

Epifluorescence mode by camera (quick checking fluorescent signals)

• Go to the computer and make sure everyone can see the screen
• Point out that the software looks complicated but we will discuss the essential parts to understand them
• Point out that there are modalities and buttons to switch to them (Eye-observation, Epifluorescence with camera, Spinning-disc modalities).
• Point out that we were using the Eye-observation mode all this time
• Switch to Epifluorescence with camera mode
• Choose a pre-set optical configuration that should reveal a fluorescent color that is in the sample
• Point out that the two settings that matter now are a light intensity slider (lower left) and a camera exposure time (top left), set them both to low values
• Click Capture image and probably see nothing (black image, but it may just be a low amount of signal!)
• Switch on auto-scale (above the picture, a graph symbol with a Play button on top of it) and explain that this will always show what the camera sees, even if it is weak or strong signal. Now you will see something.
• Click Live to live-view with the camera for making further adjustments
• Raise the light source and exposure times and see the signal becoming higher than the noise, explain why/that it should be higher than the noise
• Then lower the both down and see the signal disappearing in the noise (undesirable in a real experiment)
• Save the light and exposure time adjustments using right-click on the Optical Configuration and choosing "Assign" - explain that this is necessary upon changing something
• Select another pre-set (e.g. mCherry) and point out that another fluorescent color will be activated now and why
• Move on from Epifluorescence-by-eye mode as this is not worth spending the users' brain power on too much.

Confocal Spinning Disc mode (the main thing you do with this microscope)

• Switch to the Spinning Disc modality
• Activate a pre-set, e.g. "488"
• Point out that it now looks different but we will also go step-by-step
• Point out that there are now 5 lasers available
• Capture Image and inspect the result
• Live view and adjust laser and exposure time to get decent signal
• Point out that we are now in confocal mode and the optical section is thinner than before
• Let the user move the focus up/down and see clearly different layers of the sample, especially noticable because it's in confocal mode
• Point out the lack of out-of-focus blur to highlight the confocality again.
• Adjust the light intensity and exposure times to show that the same principles apply, you need signals stronger than noise.
• Repeat this for another pre-set Optical Configuration.
  

Camera settings in more detail (worth spending time on this)

• Binning: You can choose to sacrifice resolution to gain sensitivity to low signals by increasing binning
• Bit-depth: Always 16-bit, because it has the most accurate intensity registration. 12-bit doesn't make sense to use for any obvious reason.
• Exposure time: Adjust in multiples of 5. You can show what happens with an exposure time of 2 ms (stripes!)
• Scan mode: Always on "Fast" (the other ones are just doing on-chip averaging to get more signal-to-noise, whereas you can just increase exposure time to achieve the same).
• Full-sensor or smaller sensor area (custom): Start with full-sensor always. The only application for a smaller sensor size is when you need extremely rapid imaging, as those images will then be smaller in data-size to transfer from the camera to the computer.

SoRa Superresolution, how to switch on/off (link)

• This should theoretically be able to reach resolutions of around 150 nm (at 510 nm)

ND Acquisition

This is used to do automated multidimensional experiments

Start with a Z-stack, because the full SoRa pipeline to get maximum resolution needs this.

Perfect Focus System for keeping/using exact focus levels in live view and during ND Acquisition experiments

FRAP (optional)

Dual camera mode (optional)

Data saving (nd2 files) and transfering (SURF file sender)

User account creation

Booking system