Comparison and Implications of Three Optical Microscopy Data Acquisition Modalities

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Comparison and Implications of Three Optical
Microscopy Data Acquisition Modalities
Widefield Fluorescence
Laser Scanning Cytometry (LSC)
Confocal
LSC Images Courtesy CompuCyte Corporation
James Butler Ph.D. Nikon Instruments, Inc.
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Widefield Fluorescence
Widefield Point Spread Function (PSF)
Tube Lens
Dichromatic Mirror
CCD
0.23µm

• Light from the Focal Plane --------

• Light from outside the Focal Plane --------

1.0µm using
60x Plan Apochromat 1.4NA Oil Objective
Objective Lens
Focal Plane
Depth 2-20µm
Specimen
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Confocal
Confocal PSF
Pinhole
PMT
Scanning Mirrors

• Confocal or in Focus Fluorescence

0.4µm using

• Fluorescence from Below Focus

• Fluorescence from Above Focus

60x Plan Apochromat 1.4NA Oil Objective

• Laser Point Scanning, e.g. Nikon C1 Confocal
• Fast Scanning, e.g Nikon Livescan SFC Confocal
• Other techniques deconvolution, structured
  illumination, TIRF

Depth lt5µm
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Laser Scanning Cytometry (LSC)

• Including CompuCyte iCyte, iCys

PMT

• Scan line width 2.5 to 10µm possibly smaller
  depending on objective used

Scanning Mirror (1 direction line scan)

• Light from extended depth of field above and
  below objective focal plane gt 20µm

Depth 20-100µm
Stage Translation (gt0.25µm steps) Perpendicular
to Scan line
Laser Scan Image
Confocal Image
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Observations

• Data collected is very much dependent on modality
  particularly in z
• There are benefits/tradeoffs for each of the
  modalities i.e. resolution, speed, quantity of
  data collected, etc.
• Application specific implications - required data
  may be present regardless of resolution