In vivo flow cytometer for real-time detection and quantification of circulating cells

Abstract

An in vivo flow cytometer is developed that allows the real-time detection and quantification of circulating fluorescently labeled cells in live animals. A signal from a cell population of interest is recorded as the cells pass through a slit of light focused across a blood vessel. Confocal detection of the excited fluorescence allows continuous monitoring of labeled cells in the upper layers of scattering tissue, such as the skin. The device is used to characterize the in vivo kinetics of red and white blood cells circulating in the vasculatúre of the mouse ear. Potential applications in biology and medicine are discussed.Current methods to detect and quantify various types of cells within the blood stream involve extraction of blood from the patient or animal followed by ex vivo labeling and detection. For example, standard flow cytometry involves taking blood samples, fluorescently labeling specific cell populations, and passing these cells in a single file through a flow stream. 1 The cells are interrogated by a light source (usually a laser) to determine the types and number of cells based on their fluorescence and light-scattering signals. Another example is a hemocytometer, which involves manual counting of cells against a grid while viewing them with a microscope. Although both methods are useful, they provide only a single time sample. Consequently, if the cell population of interest varies unpredictably or rapidly with time, it is difficult to obtain a valid temporal population profile, since it is difficult to know when to sample. In addition, with both methods, blood must be withdrawn for each time point, and there is a significant time delay between blood withdrawal and analysis. The development of confocal and two-photon imaging techniques has allowed the detection of static and circulating fluorescently labeled cells in vivo. 2 However, extraction of quantitative information about the number and flow characteristics of a specific cell population can be extremely tedious. In addition, the high velocity of flowing cells, especially in the arterial circulation, makes it difficult and sometimes impossible to track the cells, even when images are captured at video rates. To remedy these problems, we have constructed a flow cytometer with the capability of detecting and quantifying the number and flow characteristics of fluorescently labeled cells in vivo and over a continuous time period.The underlying principle of operation of the in vivo flow cytometer is confocal excitation and detection of fluorescently labeled cells in circulation. A schematic of the experimental setup is shown in Fig. 1. The animal to be studied is anesthetized and placed on the stage with its ear adhered to a microscope slide with glycerine. A blood vessel of appropriate diameter is identified (see below). Light from a He-Ne laser is then focused into a slit by a cylindrical lens and imaged across the selected blood vessel with a microscope objective lens (40×, 0.6 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA...

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