Zeiss LSM 710

Introduction protocol

Based on Hänsch, Sebastian and Weidtkamp-Peters, Stefanie , October 2018

The user's situation

• Do they have samples? What are they? What are they interested in?
• Do they know about the booking calendar/rules, what if another person comes after

Safety

• Objectives: you can destroy them (using the sample, the table, etc.)
• Lasers: don't put a mirror in the beam, don't open the laser boxes, do the laser safety course

Switch-on

• Buttons in ordered numbers, wait a couple seconds between each one
• Argon laser: button, turn key, hear fan turn on, later: switch on emission control, adjust intensity if necessary
• PC on, log in as user
• Start ZEN black
• RTC reset necessary? Have a brief look with the user at "common problems"

Hardware walk-through

• What is each part?
• Incubator, stage adjustment knobs, joystick (fine/coarse)
• Touchpad, load/work position, finding focus, take care with work position/slide

Objectives

• On pad, or in software
• Immersion: air, water, oil etc. - where to find the media
• Switch filter blocks for ocular fluorescence, can also do in software
• Sample
• Raise the arm (it is also a laser safety measure)
• Use 20x obj. to start with
• Table inlet: variable, place slide but do not clamp at first
• Joystick move to ROI on sample

ZEN software

Locate

• What is the rough workflow (locate, acquire)
• Beam path overview for TL, manual and quick-set-up
• Switch TL on, find focus manually through oculars
• Move objective very close, then go back (roll-button backwards)
• Can now activate fluorescence for ocular view, if lamp was switched on

Acquire

• Point out Live, Continuous, Snap, Start Experiment, and what they do
• Focus and Exposure, we don't need

Beam path

• Start with some manual set-up
• Laser source, MBS, detectors, colors, detection range
• Which lasers and detectors to use for which dyes?
• Smart Set Up: Software helps, based on your dye selection.
• Smart Set Up: Talk about sequential scanning, crosstalk etc.
• Apply quick-set-up, eg. 2-color
• Display different spectra (detection range), manually tune the range, show emission profile for fluorophore
• Laser not in detection range!
• Point out: Lasers used are on
• Point out: Dichroic, laser light can pass

Live

• What do we see? Nothing
• Show histogram
• Detectors on?

Channels window

• Laser intensity is low, raise from 0.2 (~default) to 2-5% at first
• Detector gain is low, raise from 0 (~default) to 600-700 at first, explain what it is
• Digital gain is fake news
• Pinhole size: does what? Compromise for 1 AU.
• Activate live mode again, sample should be visible now!
• Adjust parameters: laser intensity, detector gain, display settings (range)
• Get nice settings, using:

Continuous mode

Acquisition window

• Here, resolution / magnification need optimization
• Explain pixel size, dwell time, resolution, numerical aperture
• Aim for 2-3x smaller pixels than the expected resolution at this objective, NA and wavelength
• Click 'optimal' xy pixel numbers (stress: always!)
• Compare live with continuous - why is it slower?
• Why choose high scan speed? (bleach sensitive dyes, low pixel dwell time)
• Averaging, what does it do? Demonstrate with extreme gain (act against noise)
• Main variable parameters during user imaging
• Magnification, piel size, scan speed, averaging, gain, zoom, laser power, uni- vs. bidirectional scanning

Histogram

• Displays what?
• Hi-Lo (range indicator) lut, show saturation (avoid saturation)
• Adjust pinhole size, laser intensity, gain, then see Saturation occur!
• Auto, can use to always adapt

Zoom/tile/rotate

• Position imaging area at exactly the right spot of the sample
• Analog/digital zoom
• Crop, rotate (make sure to stay near the middle of the imaging area, see: obj. flat field/aberrations)

Z-stack window

1. Enable under 'live' button
2. Live: set top and bottom slices, explain schematic display of stack
3. Set step size based on Nyquist, or optimal
4. Acquire z-stack by Start Experiment
5. Complete, go through slices (gallery)

Re-use

• Mess up settings / re-use from existing image
• Recover from previous experiment in top-right
• Right-click/re-use
• Or, load saved settings from the top-left (please don't delete other users' settings)

Check point

• Need more detailed explanation of anything
• Need introduction to new functions (tile scanning, bleaching, time series)?
• Time for small practice run

Saving data

• Do not store locally, transfer to network drive (not USB: virus danger)

Closing the system

• Laser stand-by or all off, depending on user next
• Close argon laser emission key (let cool down until quiet)
• Shut down PC
• When laser quet, switch off switches in reverse-order

Log book

• Remind of laser safety course
• Remind of image analysis help too

Finish