
- 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.
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- 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
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Theory (S/T: 12 (20)):
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Refraction at the lens surface and chromatic aberration.
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The compound microscope: beam path and properties of objectives, oculars and condensers
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The stereo microscope: beam paths for oculars and camera
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Finite and infinite optics
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Spherical aberration and coverslip thickness
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Diffraction (Airy disks)
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Resolution (Rayleigh, Sparrow, Abbe, FWHM)
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Aperture angle, NA and influence on resolution
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Immersion
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Köhler alignment
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How to keep microscope clean
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Practice (S/T: 2 (4)) (S/I: 2 (4)):
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Building a 'compound microscope' with simple magnifying lenses
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Setting up Köhler alignment
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Working with immersion
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Contrast Enhancement in Transmitted Light (CE)
- Modules required to start this module: BMO
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Theory (S/T: 12 (20)):
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Brightfield
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Amplitude objects and phase objects
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Phase contrast
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Differential interference contrast (DIC, Nomarski)
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Polarization microscopy
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Darkfield and Rheinberg
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Oblique illumination and Hoffman modulation contrast
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Practice (S/T: 2 (4)) (S/I: 2 (4))
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Setting up Köhler alignment
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Influence of the condenser aperture on contrast and resolution (e.g. with Pleurosigma angulatum).
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Demonstration of the various techniques
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Principles of Fluorescence and Fluorescence Microscopy (PoF)
- Modules required to start this module: BMO
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Theory (S/T: 12 (20)):
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Physico-chemical basics:
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Jablonski diagram
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S0, S1, S2 states
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Fluorochrome characteristics:
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Extinction coefficient, quantum yield, brightness
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Spectral properties
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Stoke's shift
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Combining several fluorochromes, bleed through and how to avoid it
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The principle of spectral unmixing
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Bleaching and counter measures
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Fluorescence lifetime
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Different kinds of fluorochromes:
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Organic dyes
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Fluorescent proteins
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Quantum dots
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Autofluorescence
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The fluorescence microscope:
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Filters, beam splitters and their positions
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Light sources:
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Spectral properties and handling (including safety issues)
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Lamps: halogen, metal halide, LED, mercury vapor
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Lasers
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Practice (S/T: 4 (6)) (S/I: 2 (4)):
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Fluorescence microscopy with test samples
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Measurement of bleaching (time series)
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Demonstration of bleed through
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Demonstration of diffraction rings by focusing through point emitters (beads, quantum dots...)
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Measurement of chromatic aberration in 2D (also in 3D if automated z-drive is available)
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Basics of Digital Imaging (BDI)
- Modules required to start this module: BMO
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Theory (S/T: 12 (20)):
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Pixels and voxels
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Nyquist
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Poisson noise, photons and contrast
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Signal-to-noise
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Dynamic range/ saturation/ bitdepth
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File formats
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Caveats of image manipulation
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Practice (S/T: 6 (10)) (S/I: 2 (4)):
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Capture an image
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Estimate SNR and background level
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Put in a scalebar
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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
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Theory (S/T: 12(20)):
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The confocal principle
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Beampath in a confocal laser scanning microscope, scanning, zooming
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Confocal resolution and optical sectioning; pinhole size
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Confocal detection of fluorescence and reflection; laser transmission detection
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Equipment:
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Point detectors: PMTs, GaAsPs and Hybrid detectors
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Scousto-optical devices: AOTF, AOM, AOBS and the like
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Spectral detection: Prisms, Meta detector, spectrometer, interferometer
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Spectral sensitivity of detectors
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Sources of noise
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Practice (S/T: 2(4)) (S/I: 2(4)):
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Confocal microscopy with test samples: zoom and resolution
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Demonstration of bleed through
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Recording of a PSF, FWHM measurement
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Measurement of chromatic aberration in 3D
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Reflection confocal microscopy and transmission images
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Hyperspectral Imaging (HI)
- Modules required to start this module: BMO, BDI, PoF, LSCM
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Theory (S/T: 12 (20)):
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Applications in
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Fluorescence microscopy
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(Patho-)histology
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Remote sensing
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Different acquisition methods for hyperspectral datasets
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Lambda scanning
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Spectral array detector
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Interferometer
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Different methods to analyse spectral datasets (for each also mentioning the required control/ reference samples)
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Spectral unmixing
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Linear unmixing
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Blind unmixing (e.g. PoissonNMF tool in ImageJ)
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Spectral mapping
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Practice (S/T: 2 (4)) (S/I: 2 (4))
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Acquisition of a multichannel image of a sample with bleedthrough and unmix it/ analyse it with different spectral tools
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Acquisition of a hyperspectral image (spectral information on each pixel of a sample) with bleedthrough and unmix it/ analyse it with different spectral tools
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Image processing and presentation (IPP)
- Modules required to start this module: BMO, BDI
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Theory (S/T: 12(20)):
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Grayscale, RGB, false colors, lookup tables (LUTs)
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How to measure: resolution, chromatic aberration, size (micrometer)
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How does human vision work?
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Colour Blindness
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(Colour) sensitivity
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Image enhancement vs. data manipulation: limits, pitfalls, do's&don't's
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Practice (S/T: 6 (10)) (S/I: 2 (4)):
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Create a RGB image and an image with 4+ channels
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Experiment with visualising the data
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Adjust images for colour blind vision with vischeck plugins
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Brightness and contrast adjustment
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Measure pixel size and image size with an object micrometer
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Make a scalebar
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Measure the FWHM of the PSF in x,y and z.
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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
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- 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
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- 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
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- 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)