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C. Alt, I. Veilleux, H. Lee, C. M. Pitsillides, D. Côté, C. P. Lin; Retinal Flow Cytometer. Invest. Ophthalmol. Vis. Sci. 2007;48(13):142.
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The in vivo flow cytometer is a valuable tool for studying the circulation kinetics of fluorescently labeled cells in a live animal model. However, probing a single artery in the mouse ear results in a sampled volume smaller than 1 microliter/minute, limiting the sensitivity of the technique. Furthermore, the precision of experimental results can suffer, especially when rare cells (e.g. GFP-expressing progenitor or stem cells), or cells with short circulation times (e.g. cancer cells with high metastasic potential) are investigated. We have developed a novel retinal flow cytometer to address these limitations.
The retinal flow cytometer is a line-scanning confocal microscope that scans a small laser spot rapidly (5 kHz) in a circle around the optic nerve head in the mouse eye. Since the circular scan intersects with all major retinal vessels that diverge from the optic nerve head, the retinal flow cytometer probes five blood vessel pairs simultaneously. We injected 106 fluorescently labeled lymphocytes (freshly isolated from extracted lymph nodes) into a BALB/c mouse. Cell counts acquired with the retinal flow cytometer were compared with those of the original in vivo flow cytometer that were acquired in the ear of the same mouse.
Counting circulating cells, both manually and by using software algorithms, we demonstrate that the retinal flow cytometer detects about five times more cells per minute than the original in vivo flow cytometer does in the ear of the same mouse.
Improved cell count of the retinal flow cytometer indicates larger sample volume and can help to increase statistical confidence in performing flow cytometry in vivo. Furthermore, taking advantage of the transparent media of the eye, tissue effects, such as scattering and autofluorescence, can be minimized and, thus, lead to increased signal-to-noise ratio.
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