Workgroup5

From GermanBioImaging
Jump to: navigation, search
Workgroup 5 aims at building up a consistent training scheme for facility users. An inventory of existing activities should be compiled and training activities from the different sites should be coordinated with each other and within the EuroBioImaging training workpackage.
Work Plan for WG5
  • Define a range of teaching modules for general use
  • Put together a list of recommendations for hands-on user training (individual or course-based)
  • Coordinate activities with EuBI training workpackage
  • Produce a list with current microscopy training activities around Germany (Europe?)


Overview

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.

Recommendations for Teaching Modules

See work in progress page for module proposals still in development.
  • 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

go to work in progress page
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)