A modular detector for flow cytometric multicolor fluorescence measurements

Abstract

A modular detector for measuring multicolor fluorescence from cells illuminated by single or multiple lasers has been developed for flow cytometers. Motion picture projector, camera, and CCTVlvideo lenses were evaluated for use in the detector by comparing their physical characteristics, image quality, and light collection efficiencies. A 25-mm focal length F/0.95 CCTV lens was selected, based on our critera and test results. The detector was constructed out of square aluminum extrusion channels. A CCTV lens mounted on the outside of the first channel collected light emitted from cells and collimated it towards filters and secondary CCTV lenses located in each channel. The secondary lenses functioned as relay optics for directing and focusSeveral optical systems have been designed for measuring light scatter and fluorescence in flow cytometers (FCM). Early methods employed bright-fieladark-field microscopic on-axis measurements for quantifying absorption, light scatter (12), and fluorescence (11). In these detection systems, high numerical aperture (NA) objective lenses were used to magnify and image the cell stream onto apertures in front of photomultiplier tubes (PMTs). Dichroic color-separating filters were used to divide the absorption, light scatter, and fluorescence. A similar dark-field illumination instrument for quantifying fluorescence and light scatter was constructed around a 10 x low-power microscope (27).Epiilluminators (15) have been used to measure fluorescence (5,241 and light scatter (23) and they have been used with flow chambers that have electronic particlevolume sensing (9) and cell-sorting (13) capability. High-NA objectives serve as both the excitation and emission collection lens. The illuminating light is reflected by a n excitation dichroic mirror and imaged onto the cell stream. The longer-wavelength fluorescence emission is then collected by the same lens and transmitted by the dichroic mirror for imaging onto PMT detectors. Multibeam, sequential excitation also has been used to illuminate cells for measuring absorption, scatter, and ing light onto pinhole spatial filters for measurement by photomultipliers. The detector design allowed any number of channels to be connected together and the versatility for making simultaneous or sequential measurements. Measurements on lymphocytes labeled with four monoclonal antibodies conjugated to fluorescent dyes and measurements on viable tumor cells stained for DNA content and with three fluorescent-labeled antibodies were used to demonstrate the detector's capabilities.Key terms: Fluorescence detector, immunofluorescence, lymphocytes, tumor-cell analysis multicolor fluorescence (3). Recently, the fluorescence emission spectra of cells have been recorded using a monochromator connected to a vertical-illuminator flow cytometer (25).The orthogonal dark-field method uses a lens placed perpendicular to the laser-beadcell-stream axes to collect the emitted fluorescence and light scatter. Two projector lenses were originally used as relay optics (1:l...

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