MOLGENLAB A2: Fluorescence microscopy

• Fluorescence microscopy excites and observe fluorescent molecules.
• Fluorescence microscopy is the most commonly used microscopy with

  • high resolution
  • sensitive with low background
  • multi-channel…
• Fluorescence microscopy comes with variations (fancy names).

  • deconvolution,
  • OMX,
  • deltavision confocal,
  • spinning disc,
  • two photon
  • TIRF,
  • FRAP,
  • FRET,
  • FLIM,
  • iFRAP,
  • FCS
  • PALM,
  • STED,
  • STORM,
  • SIM,
  • (super-resolution)
• With fluorescence microscope, you can:

  • Determine the localisation of specific (multiple) proteins
  • Determine the shape of organs, cells, intracellular structures
  • Examine the dynamics of proteins
  • Study protein interactions or protein conformation
  • Examine the ion concentration etc.
  • can observe in live cells
• Fluorescence microscopy has an upright microscope light path.
• The main parts of a fluorescence microscope are the: camera, filter cube, lamp, objective lens, and sample (for brightfield microscopy.
• Fluorescence microscopy starts with the lamp (arc lamp).

  A) gas

  B) high voltage

  
• Parts of fluorescent microscope

  A) camera

  B) filter cube

  C) lamp

  D) objective lens

  E) sample

  F) for brightfield microscopy

  
• To obtain uniform illumination, centering or alignment of both lamp and mirror should be done. This is called as the Koeller illumination. The objective lens works as condenser.
• Koeller illumination

  A) mirror

  B) lamp house

  
• Koeller illumination

  A) sample plane

  B) illumination plane

  C) back focal plane

  D) focal plane

  
• LASER or Light Amplification by Stimulated Emission of Radiation is used for confocal microscopy or FRAP.
• Property of light from lasers

  • High intensity
  • uniform wavelength, phase, polarity
  • can be tightly focused
• Gases in laser diode can be Helium, Neon, Argon, and Krypton
• Gases in lamps can be mercury and xenon.
• Laser diodes can be gas or solid.

  A) pumping energy

  B) 100% mirror

  C) 99% mirror

  
• Filters – the heart of fluorescence microscopy
• Filter cube contains three filters

  A) dichroic mirror

  B) Emission filter

  C) Emission filter

  D) Excitation filter

  E) Excitation filter

  F) dichroic mirror

  
• Filter wheels are often used for speed

  A) Emission filter wheel

  B) Excitation filter wheel

  C) dichroic mirror

  
• One wheel + multiband pass filter. Selecting filter sets is critical for sensitivity, colour separation.
• How to tell the property of filters

  A) Long Pass (LP) Filter

  B) Band Pass (BP) Filter

  C) Short pass (SP) filter

  D) Multiband pass filter

  
• Objective lens – making it bigger

  A) Correction

  B) Magnification/NA

  C) Phase contrast or DIC

  D) Tube length / coverslip thickness

  E) Immersion media

  
• Magnification /numerical aperture (NA)

  Resolution: propotional to 1/NA

  Brightness: propotional to (NA)4 / (magnification)2
• Correction of optical aberration

  Better correction: Achromat>Fluorite>Apochromat

  Curveture of field: Plan

  Plan Apochromat is the best corrected (may not be the brightest)

  A) Ideal lense

  B) Spherical aberration

  C) Chromatic aberration

  D) Curveture of field

  
• Other considerations of correction are Thick sample and Lack of Registration.
• For thick samples,

  • Use a water-immersion lens (for live samples)
  • Use immersion oil with different reflactive index
  • Use a lens with a movable internal lens.

  A) Not corrected

  B) Corrected

  C) Immersion medium

  D) objective

  
• For Lack of Registration, Light with different wavelengths from the same point does not focus on the same place which Can be caused by:

  • objective lens
  • filters
  • mechanical
• Detectors – capturing data

  1. Eye
  2. Film
  3. PMT
  4. CCD
• PMT (photo multiplier tube)

  • no space information
  • very high time resolution
  • used for laser scanning confocal microscope
• CCD (charge coupled devise) camera

  • space information
  • low time resolution
  • very sensitive
  • (quantum efficiency: >70% vs 25% (PMT), 2% (film))
  • most commonly used
• CCD camera – how it works

  1. Generate and accumulate charge in response to photon - charge is propotional to the number of photon can achieve high sensitivity by longer exposure
  2. Readout by transferring charges by one pixel to the next - slow download

  
• Property of CCD camera

  Resolution: pixel size

  Field size: pixel number x size

  Time resolution: read-out rate (Hz)

  Dynamic range: bit (12,14 etc), full well capacity

  Sensitivity: quantum efficiency (wave-length dependent), "back-thinned" (QE >90%)

  Noise: cooling temperature
• Property of CCD camera

  Monochrome vs Colour

  Colour camera is, in general,

  • less sensitive
  • less resolution
  • more expensive
• Property of CCD camera

  A) Front illuminated

  B) Back illuminated, Back-thinned

  C) light

  D) electrode

  E) silicon

  
• Reducing noise: on-chip amplification

  • Dark noise: significant at a long exposure, can be reduced by cooling the chip (-50, -70oC)
  • Readout noise: significant at a low signal, can be reduced by slow readout, on-chip amplification
• Camera with on-chip amplification: EMCCD, EBCCD, iCCD

  (low readout noise, high readout rate)

  A) EMCCD (Electron multiplying CCD)

  B) On-chip amplifier

  
• Useful function of CCD camera

  A) Binning

  B) no binning

  C) 2x binning

  D) Subarray readout

  E) full

  F) subarray

  G) up

  
• Fluorescence in situ Hybridization (FISH)

  • a process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules
  • Opening picture - Human Mphase spread using DAPI stain

  
• Fluorescence in situ Hybridization (FISH)

  • Identifies chromosomal abnormalities
  • Aids in gene mapping, toxicological studies, analysis of chromosome structural aberrations, and ploidy determination
  • Used to identify the presence and location of a region of DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections
• Fluorescence in situ Hybridization (FISH)

  This means you can view a segment or entire chromosome with your own eyes

  Was often used during M phase but is now used on I phase chromosomes as well