GerBI-GMB microscopy teaching modules

The basic teaching unit is called a module, covering a defined set of topics, e.g. 'basic microscope optics' or 'fluorescence'. While modules generally cover topics exclusively, some overlap may occur, such as PMTs covered in 'confocal' and 'multi-photon' modules. Several modules can be combined to a course to include all topics which are required to master a specific microscopic technique. For example, a course for confocal microscopy will include a module on confocality but also modules on fluorescence, basic microscopy, etc.
  • The recommended student:teacher ratio is given as S/T: [optimal]([maximum])
  • For the practical parts there is also a student:instrument ratio given as S/I: [optimal]([maximum]); instruments can be microscopes, analysis workstations, laboratory benches, etc.

Basic Microscope Optics (BMO)

Modules required to start this module: none
  1. Theory (S/T: 12 (20)):

    • Refraction at the lens surface and chromatic aberration.

    • The compound microscope: beam path and properties of objectives, oculars and condensers

    • The stereo microscope: beam paths for oculars and camera

    • Finite and infinite optics

    • Spherical aberration and coverslip thickness

    • Diffraction (Airy disks)

    • Resolution (Rayleigh, Sparrow, Abbe, FWHM)

    • Aperture angle, NA and influence on resolution

    • Immersion

    • Köhler alignment

    • How to keep microscope clean

  2. Practice (S/T: 2 (4)) (S/I: 2 (4)):

    • Building a 'compound microscope' with simple magnifying lenses

    • Setting up Köhler alignment

    • Working with immersion

  3. material

Contrast Enhancement in Transmitted Light (CE)

Modules required to start this module: BMO
  1. Theory (S/T: 12 (20)):

    • Brightfield

    • Amplitude objects and phase objects

    • Phase contrast

    • Differential interference contrast (DIC, Nomarski)

    • Polarization microscopy

    • Darkfield and Rheinberg

    • Oblique illumination and Hoffman modulation contrast

  2. Practice (S/T: 2 (4)) (S/I: 2 (4))

    • Setting up Köhler alignment

    • Influence of the condenser aperture on contrast and resolution (e.g. with Pleurosigma angulatum).

    • Demonstration of the various techniques

  3. Materials

Principles of Fluorescence and Fluorescence Microscopy (PoF)

Modules required to start this module: BMO
  1. Theory (S/T: 12 (20)):

    • Physico-chemical basics:

      • Jablonski diagram

      • S0, S1, S2 states

    • Fluorochrome characteristics:

      • Extinction coefficient, quantum yield, brightness

      • Spectral properties

        • Stoke's shift

        • Combining several fluorochromes, bleed through and how to avoid it

        • The principle of spectral unmixing

      • Bleaching and counter measures

      • Fluorescence lifetime

    • Different kinds of fluorochromes:

      • Organic dyes

      • Fluorescent proteins

      • Quantum dots

      • Autofluorescence

    • The fluorescence microscope:

      • Filters, beam splitters and their positions

      • Light sources:

        • Spectral properties and handling (including safety issues)

        • Lamps: halogen, metal halide, LED, mercury vapor

        • Lasers

  2. Practice (S/T: 4 (6)) (S/I: 2 (4)):

    • Fluorescence microscopy with test samples

    • Measurement of bleaching (time series)

    • Demonstration of bleed through

    • Demonstration of diffraction rings by focusing through point emitters (beads, quantum dots...)

    • Measurement of chromatic aberration in 2D (also in 3D if automated z-drive is available)

  3. material

Basics of Digital Imaging (BDI)

Modules required to start this module: BMO
  1. Theory (S/T: 12 (20)):

    • Pixels and voxels

    • Nyquist

    • Poisson noise, photons and contrast

    • Signal-to-noise

    • Dynamic range/ saturation/ bitdepth

    • File formats

    • Caveats of image manipulation

  2. Practice (S/T: 6 (10)) (S/I: 2 (4)):

    • Capture an image

    • Estimate SNR and background level

    • Put in a scalebar

  3. material

Basic Laser Scanning Confocal Microscopy (LSCM)

This module covers point scanners ('normal' confocals) but not spinning disk systems.
Modules required to start this module: BMO, BDI, PoF
  1. Theory (S/T: 12(20)):

    • The confocal principle

    • Beampath in a confocal laser scanning microscope, scanning, zooming

    • Confocal resolution and optical sectioning; pinhole size

    • Confocal detection of fluorescence and reflection; laser transmission detection

    • Equipment:

      • Point detectors: PMTs, GaAsPs and Hybrid detectors

      • Scousto-optical devices: AOTF, AOM, AOBS and the like

      • Spectral detection: Prisms, Meta detector, spectrometer, interferometer

      • Spectral sensitivity of detectors

    • Sources of noise

  2. Practice (S/T: 2(4)) (S/I: 2(4)):

    • Confocal microscopy with test samples: zoom and resolution

    • Demonstration of bleed through

    • Recording of a PSF, FWHM measurement

    • Measurement of chromatic aberration in 3D

    • Reflection confocal microscopy and transmission images

  3. Materials

Hyperspectral Imaging (HI)

Modules required to start this module: BMO, BDI, PoF, LSCM
  1. Theory (S/T: 12 (20)):

    • Applications in

      • Fluorescence microscopy

      • (Patho-)histology

      • Remote sensing

    • Different acquisition methods for hyperspectral datasets

      • Lambda scanning

      • Spectral array detector

      • Interferometer

    • Different methods to analyse spectral datasets (for each also mentioning the required control/ reference samples)

      • Spectral unmixing

        • Linear unmixing

        • Blind unmixing (e.g. PoissonNMF tool in ImageJ)

      • Spectral mapping

  2. Practice (S/T: 2 (4)) (S/I: 2 (4))

    • Acquisition of a multichannel image of a sample with bleedthrough and unmix it/ analyse it with different spectral tools

    • Acquisition of a hyperspectral image (spectral information on each pixel of a sample) with bleedthrough and unmix it/ analyse it with different spectral tools

  3. Materials

Image processing and presentation (IPP)

Modules required to start this module: BMO, BDI
  1. Theory (S/T: 12(20)):

    • Grayscale, RGB, false colors, lookup tables (LUTs)

    • How to measure: resolution, chromatic aberration, size (micrometer)

    • How does human vision work?

      • Colour Blindness

      • (Colour) sensitivity

    • Image enhancement vs. data manipulation: limits, pitfalls, do's&don't's

  2. Practice (S/T: 6 (10)) (S/I: 2 (4)):

    • Create a RGB image and an image with 4+ channels

      • Experiment with visualising the data

      • Adjust images for colour blind vision with vischeck plugins

    • Brightness and contrast adjustment

    • Measure pixel size and image size with an object micrometer

    • Make a scalebar

    • Measure the FWHM of the PSF in x,y and z.

  3. material

Recommendations for User Trainings

The following is a recommendation and may have to be adapted to the circumstances of an imaging site depending on e.g. staff situation or complexity of instrumentation.

Before Going to the Microscope

  • Before the training, the general experimental design should be discussed with the user (and his/her supervisor if applicable).
    • What's the biological question and which is the ideal instrument for answering it
    • Sample preparation
    • How will the data be analysed
  • Introduce user to imaging resources at hand (staff, wiki, books, internet resources, etc.)
  • Admin stuff
    • Booking system
    • Facility rules
    • Usage costs
    • Registration
    • Safety issues (biosafety, lasers, waste, working in dark rooms etc.)

1st Training Session

  • Background to general light microscopy techniques, eg. contrasting techniques, fluorescence, beampath, pinhole, detector etc. (possible without microscope)
  • Introduction to hardware: how to switch on/off.
  • Introduction to software: how to properly acquire an image, z-stack, time series, etc.
  • Set up an imaging experiment from scratch (with a known sample)
  • Data handling (original and exported file formats, metadata, where to store data and backups) and data safety
  • Cleaning up of microscope and workplace

2nd Training Session

  • 2nd training session with the user involves applying the knowledge from the 1st training on own samples
  • After user introduction to a microscope, the user should receive guidance and support from facility staff when imaging their own samples for the first time

Full User Status

  • User has to sign facility user rules
  • If applicable, user needs to sign laser safety rules
  • If applicable (e.g. external user), the users group leader has to sign a declaration for cost assumption (should ideally happen before the introductions already)