Fluorescence Microscopy

1
Fluorescence Microscopy

• http//micro.magnet.fsu.edu/primer/

2
Microscopy Basics

• The microscope must accomplish three tasks
• produce a magnified image of the specimen,
• separate the details in the image,
• render the details visible to the human eye or
  camera.
• multiple-lens (compound microscopes) designs with
  objectives and condensers
• also very simple single lens instruments that are
  often hand-held, such as a loupe or magnifying
  glass.

3

• The objective
• Gathers the light coming from each of the various
  parts or points of the specimen.
• Has the capacity to reconstitute the light coming
  from the various points of the specimen into the
  various corresponding points in the image
  (Sometimes called anti-points).
• Constructed so that it will be focused close
  enough to the specimen so that it will project a
  magnified, real image up into the body tube.
• The eyepiece serves to further magnify the real
  image projected by the objective.
• In visual observation, the eyepiece produces a
  secondarily enlarged virtual image.
• In photomicrography, it produces a secondarily
  enlarged real image projected by the objective.
• The image can be projected on the photographic
  film in a camera or upon a screen held above the
  eyepiece.

4
(No Transcript)
5

• Optical Tricks to increase contrast and provide
  color variations in specimens
• Polarized light- Anisotropic samples. After
  exiting the specimen, the light components become
  out of phase, but are recombined with
  constructive and destructive interference when
  they pass through the analyzer.
• Phase contrast imaging- An optical method devised
  by F. Zernike for converting the focused image of
  a phase object (one with differences in
  refractive index or optical path but not in
  absorbance), which ordinarily is not visible in
  focus, into an image with good contrast.
• Differential interference contrast - A mode of
  contrast generation in microscopy that yields an
  image with a shadow relief. The relief reflects
  the gradient of optical path difference. DIC,
  which is a form of interference microscopy that
  uses polarizing beam splitters, can be of the
  Smith or Nomarski type.
• Fluorescence illumination
• Darkfield illumination - Any method of
  illumination which illuminates the specimen but
  does not admit light directly to the objective.
  It may be by substage (dark field, q.v.)
  condensers.

6
Microscope Optical Train

• Illuminator light source and collecting lens
• Light Conditioner modify image contrast
  (spatial frequency, phase, polarization,
  absorption, fluorescence, others)
• Condenser - the lens mounted before the
  microscope stage, which transmits light to the
  object.
• Specimen
• Objective - the primary magnifying system of a
  microscope. A system, generally of lenses, less
  frequently of mirrors, forming a real, inverted,
  and magnified image of the object
• Image filter alter the light going to the
  eyepiece or detector
• Eyepiece magnifies image slightly and allows
  detection by eye
• Detector Video or digital camera

7
Fluorescence Microscopy

• Irradiate the specimen with a desired and
  specific band of wavelengths separate the
  weaker emitted fluorescence from exciting light
• Exciting light much more intense than emitted
  fluorescence

8
(No Transcript)
9
Epi-fluorescence Microscope

• Illuminator between the observation viewing
  tubes and nosepiece housing,
• Light is directed onto specimen by passing
  through objective (acts as a condenser), then
  emitted light is collected through the same
  objective
• Since these two are single components alignment
  perfect

10
Filter Cube

• Excitation light filter used to collect
  wavelength band of light, block unwanted light
• Dichromatic beamsplitting mirror interference
  filter passes longer wavelength light (45 degree
  tilt reflects light 90 to focus on specimen)
• Fluorescence emission collected through
  objective serving an image forming function
• Light can pass through dichromatic mirror because
  fluorescence occurs at longer wavelengths
• Emission Filter collects light from fluorescing
  sample
• Filter cubes rotating cubes turret can
  accommodate 4-6 cubes
• Lamphouse contains - Infrared light suppression
  filter and shutter, nuetral density filter
  holders to reduce exciting light

11
(No Transcript)
12
Filter Selection

• Excitation/emission filters wavelength band
  chosen that includes peak
• Microscope optics may vary with different
  wavelength selections
• Absorption properties and quantum yields of
  fluorophores

13
Photobleaching

• Irreversible decomposition of fluorescent
  molecules because of their interaction with
  molecular oxygen in excited state
• FRAP fluorescence recovery after
  photobleaching. Rates and recovery in
  photobleached area (new fluorophores are
  diffusing into bleached area)
• FLIP fluorescence loss in photobleaching.
  Decrease in fluorescence in a defined region
  lying adjacent to photobleached area.

14
(No Transcript)
15
Detection of single molecules

• TIRFM- total internal fluorescence microscopy
• Evanescent wave that is developed when light is
  totally internally reflected at the interface
  between two media having dissimilar refractive
  indices
• Light directed onto specimen at the critical
  angle TIR occurs at the interface
• Light permeates 200 nm or less into the lower
  refractive index space
• Extremely low background because very small area
  is exposed to exciting light

16
(No Transcript)