Lasers and Confocal

							Lasers and Confocal
                    Laser
• Acronym: Light Amplification by Stimulated
  Emission of Radiation
• Ordinary light emission: Comes from
  spontaneous decay of excited state to
  ground levels
• Stimulated emission: molecule remains in
  excited state until stimulated to emit by
  incoming light that is insufficient to raise it
  to the next higher excited state
               Simulation
• http://micro.magnet.fsu.edu/primer/java/las
  ers/electroncycle/index.html
              Design of a laser
• Medium (such as ruby crystal) that has reflective mirrors
  at both ends
• Mechanism to pump energy (stimulated absorption) in
  (flashtube, accelerating coils, pump laser) so that we get
  a population inversion: circumstyance in which there are
  more (atoms, molecules) in the excited state than the
  ground state
• Under these circumstances, additional light is more likely
  to generate stimulated emission than stimulated
  absorption
• At that point, further pulses give stimulated emission.
        Design of a laser (cont’d)
• This phenomenon of stimulated emission gives
  rise to a standing wave
• That standing wave can generate constructive
  interference to escape from the end of the
  crystal

 Different lasers with different
 pumps
                Ruby laser
• Ruby laser
• Length of cavity, index of refraction of
  material determines wavelength


Note that emission is:
      Phase coherent
      Nearly
      monochromatic
Cavity resonance modes and gain
            bandwidth
• Multimode lases are not monochromatic
• Wavelengths of light are extremely small
  compared to size of cavity
• Laser modes are distibuted over a narrow
  range of frequencies, termed gain
  bandwidth
  Varying cavity modes can affect
          gain bandwidth
• http://micro.magnet.fsu.edu/primer/java/las
  ers/gainbandwidth/index.html
               Types of lasers
• Argon ion laser – ionize argon gas to produce excited
  state
• Continuous wave emission
• http://micro.magnet.fsu.edu/primer/java/lasers/gainband
  width/index.html
• Argon ion lasers can produce approximately 10
  wavelengths in the ultraviolet region and up to 25 in the
  visible region, ranging from 275 to 363.8 nanometers
  and 408.9 to 686.1 nanometers, respectively. In the
  visible light spectral region
• Typically most power at 458, 488, 514 are in visible
  range
Ion laser spectra
   Semiconductor diode laser
• Electrical pumping
• Wide variety of wavelengths
 Beam shaping in diode lasers
• http://micro.magnet.fsu.edu/primer/java/las
  ers/diodelasers/index.html

 Ti-sapphire
 mode-locked
 lasers
         Ti-sapphire lasers
• Wavelength adjustable by
  changing cavity length
• Modelocking ensures
  better monochromacity
• Tunable over a broad
  range using prism to
  spread spectrum and slit
  to select wavelength
   Laser illuminators for widefield
             fluorescence
• Because lasers are phase coherent, you
  set up standing wavers between optical
  components
• Results in fringes when you try to image
• Solution: optical fiber mode scramblers
           Optical fibers total internal
                    reflection
                            Scramblers work by curving optical
                            fibers to remove phase coherence:



Advantages of laser sources for
widefield fluorescence:
        - Monochromacity
         - Intense illumination in a
        small spot
Confocal laser scan microscopy
• Instead of defocussing source over the
  image plane, focus it to a point
• Scan that point over the specimen to buld
  up an image
Advantage: Out of focus loght may
 be rejected by a paired emission
             aperture
Result: Optical sections
Pollen grain optical sections
Reconstruction of optical stacks
       Confocal technologie
• Specimen scan confocal
  – Use a Piezo device to scan specimen as you
    build up images
  – Advantage: can be used in transmission

  – Major disadvantages:
    • specimen size limitation
    • Shear on specimen
Laser scan confocal microscope

                    Advantages:
                           Flexibility
                           Ease of use


                    Disadvantages
                           Speed
                           Monochromacity
                           Cannot be used for
                           transmitted-light
                           confocal
Spinning disc confocal

                 Advantages:
                        White light
                        Speed


                 Disadvantages
                        Lack of
                        sensitivity
      Intermediate techniques
• Slit scan confocal – Use a cylindrical lens
  to spread beam into a fan bean
• Scan that beam across specimen
• Instead of pinhole, use a slit to reject out-
  of-focus information, and use a line
  detector
  – Real time speed
  – However, resolution, contrast, and optical
    sectioning are nonisotropic
          Confocal caveats
• The meaning of optical sections: no
  sharply defined boundaries; Gaussian
  intensity distribution
• Means that very bright objects can “spill
  over”
• Importance of setting black level and gain
• In X and Y, maximum resolution is ~0.1
  µm; in Z, approximately 0.8 µm. Problems
  for colocalization
       The problem of chromatic
              aberration
• Lenses that have chromatic abberration
  bring different wavelengths to focus at
  different points




• Even apchromats are only corrected at blue, green and
red; we often use purple (DAPI) or near infrared (Cy5) dyes
           Problems (continued)
• Spherical aberration
   – As we focus into a specimen, we are focusing though aqueous
     medium.
   – If we are using an oil immersion lens, we will get spherical
     aberration, because η is wrong
   – One solution: High NA water-immersion objectives
• Signal-to-noise: much worse for confocal than
  deconvolved widefield
• Fluorophore overlap: rhodamine, for example, is excited
  by 488, as well as 514
   – Detection: turn of 514 excitation
   – Fix
       • 1. Use other dyes
       • 2. Sequential scanning
       • Multispectral analysis to deconvolve overlapping fluorophores