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

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