Introduction to Fluorescence Microscopy. Introduction to fluorescence microscopy Fluorescence Widefield Fluorescence microscopes Filters and Dichroics

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Text of Introduction to Fluorescence Microscopy. Introduction to fluorescence microscopy Fluorescence...

  • Slide 1
  • Introduction to Fluorescence Microscopy
  • Slide 2
  • Introduction to fluorescence microscopy Fluorescence Widefield Fluorescence microscopes Filters and Dichroics Objectives and abberation CCD cameras
  • Slide 3
  • Natural Fluorescence
  • Slide 4
  • Transmittance is subtractive while fluorescence is additive
  • Slide 5
  • excitation emission Fluorescence Microscopy: basics of theory Absorbance spectrum limits excitation. Energy states limit excitation Molecule returns to lowest vibrational state emitting heat Light is emitted on return to ground state
  • Slide 6
  • Fluorescence spectra correlated to sise of conjugated ring
  • Slide 7
  • Multichannel fluorescence labelling Direct coupling to macromolecules Fluorescent dyes and substrates Fluorescent fusion proteins Fluorescent Antibodies Ch2(Red) Texas Red anti-rabbit & Rabbit anti- Gal Ch1(Green) UBI-GFP Arterial edothelial cell Ch1(Green) FITC Tubulin Ch2(Red) mitotracker Ch3(Blue) DAPI Ch1 Ch2
  • Slide 8
  • Light path of a Epi-fluorescent Microscope http://microscopy.fsu.edu/primer/techniques/fluorescence/anatomy/ fluoromicroanatomy.html
  • Slide 9
  • Upright Scope Brightfield Source Epi- illumination Source Image from Nikon promotional materials
  • Slide 10
  • Inverted Microscope Brightfield Source Epi- illumination Source Image from Nikon promotional materials
  • Slide 11
  • Microscope for widefield epifluorescence Mercury Lamp Dichroic Reflector Objective Sample Stage DIC analyser Dichroic Reflector CCD camera
  • Slide 12
  • Mercury lamp spectra http://www.mcb.arizona.edu/ipc/fret/
  • Slide 13
  • Standard Band Pass Filters Transmitted LightWhite Light Source 625/50 nm BandPass Filter Texas Red 600 -650 nm Light
  • Slide 14
  • Standard Long Pass Filters Transmitted Light Light Source 500 nm Long Pass Filter >520 nm Light Transmitted Light Light Source 575 nm Short Pass Filter