Abstract
The vast majority of flow cytometers in use since the inception of this technology operate by making cellular measurements derived from a pulse of fluorescence or laser-light scatter generated as a particle intercepts a focused laser in a flow system. In the case of fluorescence, these "zero resolution" systems accurately quantify the amount of light without knowledge of the location or distribution of the source of this light within the cell, since the laser focal point typically is larger than the cell being measured.
There are situations where the spatial distribution of fluorescence in a cell can provide important additional information, but this information is not available in most contemporary zero resolution flow cytometers.
In the early 70s, there was interest in developing rapid automated diagnostic systems for the fields of hematology and cancer diagnostics based on either flow cytometry or microscopic image analysis. In the lab of Leon Wheeless at University of Rochester, scientists and engineers set forth to utilize Stanley Patten's diagnostic criteria in cytopathology towards the development of an automated pre-screening system for detection of atypical or malignant cells in cytologic samples derived from cervical sampling or urine.
The principle of concept of slit-scanning, or restricting the detection of light from a cell by sampling through a slit mechanically moved over the image of the cell, was initially tested with a custom fluorescence microscope. This proof of concept led to the development of a slit-scan flow cytometer, where the laser illumination field was narrowed to a plane of laser light, oriented orthogonal to the direction of cell flow, with a thickness much smaller than that of the cell. By evaluating the fluorescence intensity pulse shape in real time, this technique proved highly effective at detecting atypical or malignant cells in a flow cytometer. It was noted with this system, however, that there was an unacceptable false positive rate, although only slightly higher than that obtained using microscopic screening by trained cytotechnologists.
In order to evaluate cells in flow and their correlated real-time classification as normal or abnormal, a correlation system was built which provided images in flow of cells correlated with their pulse shape analysis and classification. This presentation will describe the characteristics and operation of this original imaging flow cytometer.