comparison and implications of three optical microscopy data acquisition modalities james butler...
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Comparison and Implications of Three Optical Microscopy Data
Acquisition Modalities
James Butler Ph.D.Nikon Instruments, Inc.
Widefield Fluorescence
Confocal Laser Scanning Cytometry (LSC)
LSC Images Courtesy CompuCyte Corporation
Widefield Fluorescence
CCD
Tube Lens
Objective Lens
Specimen
Focal Plane
Dichromatic Mirror
Light from the Focal Plane --------
Light from outside the Focal Plane --------
Depth ~2-20μm
Widefield Point Spread Function (PSF)
y
x
z
x
~0.23μm
60x Plan Apochromat 1.4NA Oil Objective
~1.0μm
using
Confocal
Confocal PSF
PMT
x y
Confocal or “in Focus” Fluorescence
Pinhole
Scanning Mirrors
Fluorescence from Below Focus
Fluorescence from Above Focus
Depth <5μm
60x Plan Apochromat 1.4NA Oil Objective
~0.4μm
using
• Laser Point Scanning, e.g. Nikon C1 Confocal
• Fast Scanning, e.g Nikon Livescan SFC Confocal
• Other techniques – deconvolution, structured illumination, TIRF
Laser Scanning Cytometry (LSC)
PMT
yScanning Mirror
(1 direction = line scan)
Stage Translation (>=0.25µm steps)
Perpendicular to Scan line
Light from extended depth of field above and below objective focal plane > 20µm
Collimated laser light for
extended depth
excitation
Depth ~20-100μm
• Scan line width 2.5 to 10µm – possibly smaller depending on objective used
• Including: CompuCyte iCyte, iCys
Laser Scan Image Confocal Image