Conférencier: Mark Niedre
Sensing and enumeration of rare circulating cells in the bloodstream is important in many areas of pre-clinical biomedical research including study of cancer metastasis and the immune system. Normally this is achieved by drawing small blood samples which are later analyzed using a number of analytical techniques, or more recently non-invasively using microscopy-based in vivo fluorescence flow cytometry. In either case, the blood sampling volume is relatively small so that very rare circulating cells – on the order of 103 cells/mL or less - may escape detection entirely. This is insufficient for a number of important applications, such as the study of minimal residual disease or early stage circulating tumor cells. Therefore, a major research focus in our lab is developing new methods for sensing, enumerating and tracking very rare circulating cells non-invasively in vivo. In this presentation, two such methods are discussed. First, we describe a new instrument – termed the “diffuse fluorescence flow cytometer” - that utilizes multi-spectral detection of diffuse fluorescence light with a miniaturized optical ring placed around a mouse limb, thus allowing interrogation of relatively large blood vessels and blood volumes. The system is capable of detecting circulating cells at average concentrations less than 1 cell per mL. Second, a computer vision approach to rare cell in vivo flow cytometry is described. The instrument utilizes widefield laser illumination and a high-sensitivity electron multiplied CCD to sample large blood volumes from blood vessels in the mouse ear. We developed an automated computer vision algorithm allowed removal of this background and tracking and enumeration of moving fluorescent cells through the image sequences with substantial noise and autofluorescence content. Overall, this instrument had sensitivity of less than 10 cells per mL. We anticipate these methods will have many uses in the preclinical study of rare circulating cell types in vivo.