Lasers and Confocal

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Lasers and Confocal
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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

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Simulation

• http//micro.magnet.fsu.edu/primer/java/lasers/ele
  ctroncycle/index.html

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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.

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Design of a laser (contd)

• 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
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Ruby laser

• Ruby laser
• Length of cavity, index of refraction of material
  determines wavelength

Note that emission is Phase coherent Nearly
monochromatic
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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

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Varying cavity modes can affect gain bandwidth

• http//micro.magnet.fsu.edu/primer/java/lasers/gai
  nbandwidth/index.html

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Types of lasers

• Argon ion laser ionize argon gas to produce
  excited state
• Continuous wave emission
• http//micro.magnet.fsu.edu/primer/java/lasers/gai
  nbandwidth/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

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Ion laser spectra
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Semiconductor diode laser

• Electrical pumping
• Wide variety of wavelengths

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Beam shaping in diode lasers

• http//micro.magnet.fsu.edu/primer/java/lasers/dio
  delasers/index.html

Ti-sapphire mode-locked lasers
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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

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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

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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
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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

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Advantage Out of focus loght may be rejected by
a paired emission aperture
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Result Optical sections
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Pollen grain optical sections
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Reconstruction of optical stacks
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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

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Laser scan confocal microscope
Advantages Flexibility Ease of
use Disadvantages Speed Monochromacity Cannot
be used for transmitted-light confocal
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Spinning disc confocal
Advantages White light Speed Disadvantages La
ck of sensitivity
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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

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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

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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

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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 ? 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