Imaging cytometry by multiparameter fluorescence

Abstract

A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections. Key terms: Image processing, quantitation, lymphocytesMulticolor fluorescence image analysis of cells present in tissue sections, smears, and imprints by microscopy offers a potentially powerful approach for correlating the locations of cells as well as their quantity in the two-or three-dimensional space of the sample. Intracellular and surface antigens, nucleic acid sequences from RNA and DNA, DNA content, and chromo/fluorogenic substrate precipitates are examples of parameters that could be related to sample morphology. In order for image cytometry to realize this potential, instrumentation with optimal light sources, filter sets, and detectors must be combined with software that permits identification of stained structures, accurate quantitation of the fluorescence or absorption signals, morphological analysis, and data reduction and presentation.While single-color fluorescence image cytometry of cells has been carried out previously in a number of laboratories (8,9,12,14,19), the optics, probes, hardware, and computers have recently advanced significantly, and now multicolor quantitative analysis offers enormous potential €or analysis of fixed and living cells and tissues. For example, the availability of high-resolution cooled CCD cameras with a broad spectral range, minimal spatial distortion, excellent linearity, and a wide dynamic range have made quantitative fluorescence analysis more simple than before (7,10,18). Laser scanning microscopes equipped with multiple excitation lines and detectors provide an alternative approach for multicolor immunofluorescence (2). Also, improvements in interference filters have made it possible to image multiple fluorophores that emit with narrow Stokes shifts in the visible region of the spectrum (6). Furthermore, additional fluorophores have become available at new wavelengths, such as CY5 (171, which emits in the deep red region of the spectrum where CCD cameras are very sensitive. And finally, imaging computers continue to be more powerful and cost effective year by year.Here we have developed software and methods for the four-color analysis of lymphocyte subsets on microscope slides. This work repre...

“Virtual flow cytometry” of immunostained lymphocytes on microscopic tissue slides: iHCFlow™ tissue cytometry

“…Also a few hybrid microscope-FC were developed to obtain COBs information to mimic FC (29). Immunofluorescent techniques were applied on slide substrate (30) to obtain cell populations using laser scanning instrumentation. We believe our approach does not need the complex, fluorescent, and expensive instrumentation not readily available to a regular pathologist or oncologist.…”

High-Precision Pinpointing of Luminescent Targets in Encoder-Assisted Scanning Microscopy Allowing High-Speed Quantitative Analysis

“…The existing techniques based on digital microscopy [13][14][15][16] , however, are time-consuming and resource-demanding, as images are typically captured for the entire sample area, or even through three-dimensional space [17][18][19][20] , followed by stitching and processing to identify and quantitate targets of interest. Their quantification is also less accurate, because different types of noise and background emission interfere in the measurement of absolute intensities, and targets that are randomly located at the periphery of the field-of-view (FOV) have large variation in excitation and detection efficiencies [21][22][23] .…”

Automated multichannel fluorescent whole slide imaging and its application for cytometry

“…The development of computer and charge coupled device (CCD) camera technology about 20 years ago made it already possible to have imaging cytometer systems with acceptable throughput and data handling capability (7)(8)(9)(10)(11). Since that time different systems were developed to push the boundaries of image cytometry.…”