Leica SP8 Liachroic intro

Introduction

1. Welcome, outline of what the introduction will be (takes a few hours)
2. The General Instrumentation department where we are
3. The room and the general layout (sink, microscope, cupboard, chairs)
4. The microscope conceptually (Confocal Laser Scanning Microscope)
   1. Confocal = observing only a small horizontal layer of the sample, not seeing blurry higher or lower objects.
   2. Laser = specific wavelengths, focused spot, to only see specifically what we want
   3. Scanning = doesn't take a picture in one shot, but scans every pixel by pixel until the whole picture of a square field of view is built.
5. The microscope physically (components, roughly what parts do we see? -> microscope, scanner (left, attached to microscope on air table), laser box (left next to air table))

Switch-on

1. Quick inspection (everything looks ok visually?)
2. Switch-on procedure of components (buttons in numbered order)
3. Log-in in Windows (use yours, user will get an account) and software starting (LAS X)

Stage and objective movement controls (XY stage joystick, Z objectives, Switch Objectives on the touch screen - start with the 10X lens. You normally start low, then go higher)

Sample placement (sample types, coverslip towards the bottom, adjusting holder, put demo or user sample there, don't clamp it but let it rest (otherwise breaks when objective hits))

Brightfield (where does light come from, what do we expect to see)

Focusing (sample finding, and then moving around, warning: hitting lens to sample possible)

Epifluorescence (see visually light shining, see returning light, discuss different filters, user responsible for knowing their dyes and wavelengths, Searchlight spectra viewer. This method is used to see where is the signal, is there any signal, do I already see something suitable for confocal imaging?)

Confocal scanning (gonna be a big part, mostly on computer now, switch in mood/user to focus on screen not on microscope - instructor sits on the far-right side of the desk to control the mouse, attendees sit right in front of the screen to see everything properly)

- Launch the software LAS X which controls the microscope, and choose the right startup settings (microscope = DMi8, settings = standard) and wait for it to appear completely. The software is going to ignore any wavelength/imaging modality (BF, EPI) choices you have made using the touch screen of the microscope and do confocal anyway.

- Overview of software areas:

1. We start in "Acquire" (top) and will be spending most time there
2. The big vertical line in the middle divides the view into settings (left) and image display (right)
3. Making any adjustments to settings in the image display (right) is not going to change any raw data values or any microscope settings - just how the image looks on the screen
4. Making adjustments on the left will change actual microscope settings, such as wavelengths used, scanning behavior etc.
5. At the bottom, there are the buttons that make the microscope actually do something (Live, Capture Image, Start - etc.) - to prevent confusion when clicking "open laser shutter" in the software, or any other buttons.
6. We make 1 small excursion to "Configuration" (left of "Acquire") and then "Laser Config" in order to switch on the lasers we know we will need.

- Laser setup

1. choose the right wavelength
2. give it a tiny bit of power (philosophy: start low, go higher if needed - instead of bleaching everything straight away)
3. click round button under the laser to open the shutter
4. Confirm yellow line appears in the schematic light path below -> laser ready.

- Detector setup

1. Acknowledge we have a few detectors available, one is switched on.
2. Prefer to always choose HyD (hybrid detectors), they are better. Typically they are in standard mode with 100 gain (digital gain) and you can use them like that. You can destroy the detector by setting its detection window to include the current laser wavelength used (dotted line in detector area spectrum reminds you which lasers are used).
3. PMT detectors are less sensitive = good for strong signals (usually you have weak signals), to use one you have to give it some gain (couple hundred Volts, until image looks good to you)
4. Move the detection window and resize it to capture the desired fluorescence emission wavelengths. Avoid including the laser wavelength at all cost!! This will visualise the laser, which we do not want to see - and it can damage the detector.

- First image

1. Click "Capture Image"
2. Probably, nothing will show up!
3. Is there something there, but just weak? -> Set the Image Display (right) to "auto-scale" (button called "M" because it starts on Manual scale) -> have a look if something shows up.
4. Is there nothing there anyway, maybe you need more laser -> Increase laser power to 20-30%, capture another image -> have a look if something shows up.
5. Is there still nothing, maybe the focus is slightly wrong (we're in confocal mode after all...) -> use the Z-adjustment knob to focus up and down (either on the microscope, on the joystick, or on the USB panel) -> have a look if something shows up, now it really should.
6. If there's still nothing, visually check if the sample is still alright, and then use Brightfield or Epifluorescence through the binoculars to find focus again. Then try confocal again, using the software.
7. If there's still nothing, there is a problem with the microscope or the sample. Change samples to a known working sample - a piece of paper responds to 405 nm, a stroke of Staedler Yellow Highlighter responds to 488 nm.

- Evaluating the first image, boundary conditions

1. It could be too weak (barely jumping out of the noise), so we need more laser until it's visible (improving the scan will help too, but this comes later) at the cost of possibly bleaching
2. It could be too strong (huge areas look intense, and same intensity - mouse-over the pixel and see the maximum value 255 show up under the picture = too bright!) you want to avoid saturation.

- Optimizing the scan (better picture, takes longer, make it as good as you need - and not better. Every improvement you make can be compared to the previous situation by flipping through the last couple of acquired images)

1. This part is about losing acquisition speed but gaining image quality.
2. There are three main settings that most influence the image quality, which you can use to maximize the details and signals that the current objective is able to see. (if not enough, you need a higher mag. objective) as follows:
3. Format (number of pixels that are scanned -> note that the square area of the sample that you are scanning does not change in size, just gets divided into smaller steps i.e. more measurement points i.e. pixels). This influences the resolution of the image (lower = worse). Use the "Optimize" button on the left of Format to always get the right number of pixels to describe the details that are available through this objective.
4. Speed (lower values = slower, takes longer but results in higher signal-to-noise). This means the laser spends more time on each pixel, and thus illuminates that place for longer, and thus gets more signals if there are signals. Noise always stays the same.
5. Averaging (higher values = longer, also more signal-to-noise). This means one horizontal line will be acquired that number of times and the values averaged. Noise randomly appears everywhere. Signal always appears, on every scan consistently, when you look at a location that has signal.

- Adding a second channel and using Sequential Scanning

1. Set the settings to "bad" again (512x512 pixels, 400 speed, no averaging) to save some time.
2. Set up another laser (e.g. 561, if you used 488) to activate the other fluorophores
3. Set up another detector (e.g. HyD3 if you used HyD1) to detect the other fluorophores' signals
4. Do another "Capture Image" and make optimizations where necessary (use the Evaluating the second image just like in the first image section above).
5. Sequential Scanning: both molecule populations and both detectors are active at the same time, so bleedthrough might happen.
6. Switch on "SEQ" at the top left, a sequential scanning menu will appear.
7. Add another sequence using "+" and switch off one of the detectors in sequence 2
8. Flip back and forth between seq 1 and 2 so that you can see the difference in settings.
9. Now set up sequence 1 for one imaging channel, and sequence 2 for the other: Lasers should be "switched off" by putting their power on 0 where necessary, in each sequence.

- Switching to a higher magnification lens (use navigator to register 1 pic, switch to immersion lens, go back to original location).

1. Higher magnification does not automatically mean higher detail on a confocal scanning microscope.
2. Higher NA does mean higher detail on a confocal. Provided that the scan settings are also optimized (just done that, can repeat it).

- Z-stack (look at the menu: set start height, go to a different focus and set end height, and that's it - would always recommend to leave the rest of the Z-stack settings as-is for the introduction)

- Time course (switch modes using dropdown menu, then set (1) interval (none, or amount of time) and (2) duration (unlimited, or amount of time, or amount of pictures)

- Tilescan (Use the Navigator for this, do one small one, several of them, focus map, stitching)

1. Make sure some objects are in the view in normal viewer
2. Switch to Navigator and do "Spiral"
3. After an area is revealed click "Stop"
4. Use a drawing tool (bottom, e.g. the rectangle) to designate a small tilescan area
5. Execute ("Start") the tilescan
6. In the left menu "Stage", confirm that "Merge images during acquisition" is on, and "blend" is on "Smooth"
7. Inspect the result, be skeptical of overlap areas, they should be stitched well

Rounding off

- Saving data (save the projects as LIF files locally, then before ending your session move the raw data to a safe location e.g. through SURF or network drive, our policy is that the computer is not a safe storage space and we can't count on it)

- Saving settings (sequential scan settings in the sequential scan menu, single settings above the laser set-up area, or you can reload settings from an image in a LIF file: right-click on one and "apply".

- Closing software (is someone coming after us? do we need to keep the whole system on just logging out, or switch the whole system off?)

- Cleaning oil objective, then put back at 10X

- Shutting down microscope (if necessary, otherwise just logging out)

- Leaving room and system tidy for next user!

Optional functions

- Autofocus

- Emission wavelength scanning (check whether the signal behaves like the fluorophore should)

- Closing the pinhole (more resolution, lower signal)

- Changing Z-stack settings like step size (speed thigns up, but you may skip layers and thus lose information)