Optical architectures for microvolume laser-scanning cytometers

Microvolume laser scanning cytometry (MLSC) is a method for analyzing the expression of biological markers in a biological fluid. See, e.g., U.S. Pat. Nos. 5,547,849 and 5,556,764; Dietz etal., Cytometry 23:177-186 (1996); U.S. Provisional Application No. 60/144,798, filed Jul. 21, 1999, each of which is incorporated herein by reference. A sample, such as blood, is incubated in a capillary with one or more fluorescently-labeled probes that specifically binds to particular biological markers, such as membrane proteins displayed on the surface of a blood cell. The sample is then analyzed by a MLSC instrument, which scans excitation light from a laser over the sample along one axis of the capillary, while the capillary itself is moved in an orthogonal axis by an automated stage. Fluorescent probes in the sample emit Stokes-shifted light in response to the excitation light, and this light is collected by the cytometer and used to form an image of the sample. In such images, the cells or particles that bind to the fluorescent probes can be identified and quantitated by image analysis algorithms. The resulting information on the expression of biological markers in the sample can be used for diagnostic and prognostic medical purposes. Methods and instrumentation for performing charge coupled device (CCD)-based confocal spectroscopy with a laser spot array are provided. The methods and instruments of the invention are useful in any spectroscopic application, including, but not limited to, microscopy and microvolume laser scanning cytometry (MLSC).