Leica SP8

Introduction protocol

Based on Hänsch, Sebastian 27.11.2018

Start

Safety

• Genetic safety - ask for "Excerpt/wrapper of Formblatt Z" for each new project - to describe GMOs, send to CAi
• Chemical safety - corrosion, toxic substances
• Laser safety - no mirrors, opening boxes, etc. - and complete the laser safety course!
• Log-book writing and error reporting, how to do.

Booking

• Booking policies (show website: 3 h slots, 4-wk in advance max)
• Don't book and then just before, remove a massive session
• We use the booking system to generate bills

Incubator

• We keep it at 21.5°C, all the time (do not leave it open!)
• Other cooling: right-side of the table is at 17°C, all the time, for the objective turret
• Leave coolings on!

Switch-on

• Buttons in numbered sequence
• Pause between buttons, do you need the fluorescent lamp?

Log-in

• TCS user - machine - DMI8 stage
• Generally non-active (but optionally on):
• Resonant scanner (greatly increase scan speed), STED (high resolution), AFC (adaptive focus control - good for keeping time course in focus)
• Initialize table (for multiple locations imaging; raise microscope arm first, objectives go down automatically)

Software - lasers

• Laser config: STED vs. non-STED
• Which lasers do we have? What do they do? WLL (pulsed, pick wavelength, gating), Argon (more powerful, fan cooling)
• How to switch them off (especially Argon)
• The fluorescent lamp needs at least 30 minutes between off-on

Touch panel

• Also has a rotary knob with forward = toward sample
• Objective switching, fluorecent light / cubes on-off, pick, transmitted light
• Immersion of objectives: which one to choose?

• Save positions using touch pad: working/safe positions. Careful not to crash automatically...

Table inlet

• Is for fine z-adjustments, eg. active when xz-scanning
• Do not lean on, or push down hard on!

Sample placement/preparation

• Table sample holder: move holders to accomodate sample - leave open to allow raising sample
• Objective selection + apply immersion oil
• Focus finding: see droplet become wider, use fluorescence/DIC

Software - overview

• Left: acquisition settings, pixel size, speed etc.
• Middle: beam path, filter settings, detectors etc.
• Right: images already taken, current scan displayed

Beam path

• Go through beam path on-screen, what it schematically represents
• Lasers: switch on check boxes, switch between classic/spectrum-picker views. Start with 2-5% power
• Notch filters: picking laser lines at notch filter wavelengths is preferable over exactly matching the dye's excitation spectrum.
• Detectors: PMT (robust, noisy) vs. HyD (sensitive, advanced functions: gating, photon counting, low noise)

Laser/filter settings

• Picking emission spectrum: Keep laser out of range, at least 10 nm
• Transmitted light on: in parallel to laser scanning -can just activate in any sequential scan
• Show dye spectrum and "dye assistant" (help with setting up scans)

Acquisition

• Rotary knobs on touch-pad, configurable too!
• Autofocus can find focus automatically, but isn't used often (by "hand", rather)
• Live mode (quick and dirty), doesn't take into account detailed settings

Live

• We see nothing at first - why? PMT used, needs gain (600-800), adjust contrast in histogram to keep up
• Motor correction collar: What does it do and why?
• Motor correction can set up on control panel, and can adjust until the signal is brightest (eg. use XZ scanning)
• Use "range indicator" to view live, and to optimize motorized correction - in steps, judge when signal is going down again

Fine tuning acquisition

• Pixel size is important - talk about the extremes, and what's wrong with those
• Optimally, pixel size should be 3-4 times smaller than the expected resolution (which is roughly 1/2 the emission wavelength)
• Pinhole: can change values to increase fluorescent signal intensity, at the cost of resolution
• Averaging is employed (not in live) to counteract noise, such as PMT detector noise
• Accumulation is employed (also in live) in situations with low signal intensity, but also low noise

• Optimise the signal for each channel using laser intensity, detector gain, scanning speed, averaging/accumulation, zoom, and display range
• Optional: smooth rendering (leica display option) can be disabled, r/clicking the channel image

Multi-channel experiments

• Activate T-PMT → immediately have a multi-channel experiment
• Mention sequential scanning (line, frame)
• Add channel ("Seq")
• Cannot change detector order, physically arranged that way
• Add a laser line + detector window, point out you need to switch them on/off in each sequential scan
• Dye Assistant does these things automatically, but needs scrutiny
• Can use Dye Assistant to point out potential cross-talk and bleedthrough issues (spectra are visualised)
• The real way to address bleedthrough/crosstalk problems: USE CONTROL SAMPLES! With eg. only one colour of fluorophore expressed.
• Then, suspicious signal can be evaluated; does it occur in the sample itself? or because of the other fluorophore? at all?

Z-stacks

• Use gentle imaging conditions, and the control-panel Z-controls, define top and bottom of the stack
• The knobs on the microscope control the height of the whole table, the knob on the control panel controls the smaller Z-inlet
• Change step-size, best is system optimized, but can reduce them a bit

Time series/focus control

• Define total experiment time, and intervals between acquisitions
• The series will use the currently active acquisition settings (speed, format, etc.)
• Now the Automated Focus Control (AFC) becomes relevant, which uses IR laser reflection to hold-current-position over time
• "Show autofocus channel" can show what's going on there

HyVolution

• PSF always messes up your image, but can be partially countered using deconvolution
• HyVolution slider drags between best speed and best resolution, for deconvolution
• Enter which mounting medium was used
• Change from aggressive-standard-weak setting; test same acquisition region for the differences
• Try to overdo it, get artifacts, to show what it looks like when going wrong

Saving

• Can save data locally during the experiment,
• Then transfer to the web drive (Daten) at the end of the session
• We do not take responsibility for data locally saved!
• Can make "collections" out of images to create separate LIF files
• Keep LIF files, use FIJI to analyze them, export things from there

Switch-off

• Depends on next user: if same-day, can keep system on
• Clean the objectives with a few wipes using a Kimtech-wipe
• Lasers off in software, wait for Argon laser to cool (can hear)
• Then, switch off PC, and reverse-order the power buttons on the panel
• Close the incubator and write session details in log-book