Laser Microdissection

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Laser Microdissection

• Introduction to LMD Technology
• Patrick Wojtkiewicz, Ph.D.
• North Louisiana Crime Lab
• [phone]
• pwojtkie_at_NLCL.org

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Microscope
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Basic Microscopy

• Terminology
• Magnification - apparent increase in size of the
  object, based upon microscopes objective,
  ocular, and body tube.
• Refraction change in direction (velocity) of
  light as it passes from one medium to another
  (refractive index)
• Illumination method of applying light to the
  sample from the light source.
• Köhler (Brightfield)
• Vertical illuminator (Fluorescence)

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Basic Microscopy

• Brightfield
• Light source is focused through sample
  collected by objective lens.
• Specimen refracts light, which bends it out of
  the collection angle aperture of the objective
  lens(dark).
• Objective lenses most important part of microscope

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Basic Microscopy

• Fluorescence
• Illuminate sample with UV (365 nm) light source
  (excitation)
• Fluorochromes are energized and electron moves to
  higher energy state.
• The energetic electron then drops back to its
  normal state releasing energy as a photon
  (emission).
• Bypass filters block excitation ?, allowing
  emission ? (gt420 nm)) to pass.

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Basic Microscopy

• Terminology
• Resolution the ability to distinguish two
  points as distinct and separate (1000NA).
• Empty magnification is when magnification exceeds
  useful resolution

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Basic Microscopy

• Differential Interference Contrast
• Optical method of creating relief (3-D) effects
  from a sample which has little inherent contrast.
• Interference within the recombined beams causes
  effect.
• Image formed by differences in the optical path
  caused by the specimens refractive index
  difference from the medium.
• Note Effect may be caused by a discontinuity in
  the film therefore, interpretations should be
  made with care.

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Concept of LMD
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Mechanism of Lasers
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Pulse Lasers and Ablation

• Ultrafast pulse lasers work by a multi-photon
  nonlinear process.
• Photons of the ultrafast pulse are bunched at a
  very high density and material absorbs several
  photons at once.       
• Multi-photon absorption works in transparent
  media, such as plastics.

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Pulse Lasers and ablation
A nanosecond laser pulse first heats the target
then melts it, then vaporizes it. The
surroundings are heated as well leading HAZ
problems.
A picosecond laser pulse heats up material much
faster. Heat has no time to spread. The material
has no time to melt it becomes plasma.
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Advantages of Fast Pulse Lasers

• The fast laser pulse (1.0 4.0 ns) is greater
  the heat diffusion time (0.5 ns).
• Material briefly melts then energy strips
  electrons from molecules (plasma).
• Nearly all of the energy of the pulse is used in
  plasma formation, not heating surrounding
  material.
• Little debris or recast material.
• Little collateral damage to surrounding area.

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Laser Microdissection

• History
• Relatively brief history for forensics but well
  established in the medical field
• First occurrence of laser microdissection was in
  1996
• Primarily used to excise individual cells or
  groups of cells embedded in other tissue
• First report on its forensic applications was at
  the AAFS meeting in 2004 but was initially used
  in 2003.

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Types of LMD Microscopes

• Leica
• Automated microscope
• Brightfield, fluorescence, alternative optics
• Slides have film coating
• Laser cuts plastic film, which allows the cut
  sample to drop into a tube. Up to four different
  samples can be placed on stage.
• UV laser does cutting (?355nm diode, 80 hz
  ?337nm gas, 30 hz).
• Laser beam is directed though objective by prisms.

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LCD Microscopes

• Molecular Machines Industries CellCut
• Samples collected on a sticky cap
• Samples can be easily inspected after cutting
• Pulse laser cutting by moving stage
• Contamination free