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.

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

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

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

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

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

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

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

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)