1. 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

2. 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

3. 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

4. Laser Scanning Cytometry (LSC) PMT y Scanning 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

5. 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