Imaging modalities such as confocal reflectance microscopy, fluorescence by single- or two-photon excitation, second harmonic generation and coherent anti-Stokes Raman spectroscopy (CARS) provide different contrast mechanisms that can be combined to give structural, functional and molecular information of living tissue. Our group has developed a multimodal microscope in which up to three of these imaging modalities can be realized simultaneously. Images are acquired at video rate, which allows real-time monitoring of fast events in the living tissue.
In Vivo Flow Cytometry
Our group has developed an in vivo flow cytometer, based on confocal design. Contrary to the conventional flow cytometer, the in vivo flow cytometer can provide real-time detection and quantitative information on fluorescently labeled cells while in circulation in a live animal model. As the labeled cells pass through a slit of light focused across a blood vessel, fluorescence is excited. Its confocal detection makes it possible to observe the cell population of interest without the need to extract a blood sample. Furthermore, the same cell population can be tracked continuously and over long periods of time to examine the dynamic changes in the circulation of different types of cells in the same animal. The in vivo flow cytometer has been used to measure the circulation lifetime of different tumor cells and leukocyte populations in the peripheral circulation in response to immunological stress or therapeutic manipulation.
|In vivo flow cytometer.Vasculature of mouse ear with cartoons exemplifying cells flowing through the slit of excitation light. Example of fluorescence trace from the photodetector. Each spike represents one fluorescently labeled cell. Comparison of circulation time of two tumor cells with different metastasic potential. The population that is highly metastasic (MLL) depletes within hours from circulation. (Georgakoudi et al. Cancer Research 64, 5044-5047 (2004))|
In Vivo Imaging of Molecular Expression
While our microscope can image tissue structure by means of intrinsic contrast mechanisms, tracking of a specific cell population usually requires selective labeling of those cells via exogenous markers. While we use commercially available probes, we also develop novel labeling techniques. Examples include engineering of fluorescent antibody fragments and near-infrared quantum dot conjugates.
|Double labeling of blood vessel endothelial receptor molecules: PECAM-1 is a ubiquitous endothelial molecular marker that is required for neutrophils to extravasate from the vasculature. E-selecting is constitutively expressed in a subset of vessels and participates in the arrest of leukocytes prior to extravasation. (Runnels et al. Molecular Imaging, in press)|
Therapeutic Applications of Lasers