The main research focus of our group is to develop minimally invasive optical techniques for In-vivo imaging and monitoring of cells and tissues as well as therapeutic applications of lasers. The diagnostic techniques will help to answer important biological questions, such as:
- In-vivo monitoring of cell trafficking in circulation
- Imaging of vasculature and microenvironment in tissue
- Interaction of cells with microenvironment
The therapeutic techniques allow us to target cellular and subcellular structures by means of selective absorption of endogenous or exogenous chromophores. Selective targeting may be helpful for treating various pathologies, such as retinal diseases or destruction of tumors, without causing adverse side effects to healthy tissue.
ADVANCED MICROSOCOPY
Multimodal Microscopy
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 fluorescence imaging of the bone marrow environment is used for real-time observation of tumor cell metastasis into the bone marrow. This image illustrates that the extravasation of human lymphoblastic leukemic cells (NALM-6, shown in green) into the marrow in the skull of immunocompromised mice is limited to discrete microdomains of the marrow vasculature (shown in red).
(Sipkins et al. Nature 435 (16): 969 - 973 (2005)) |
Confocal reflectance (a) and single fluorescence image (b) of the optic disk of a mouse eye. In the reflectance image the tissue structure is visible. In the fluorescence image the deep capillary bed can be distinguished.
(C.P. Lin et al. Invest. Ophthalmol. Vis. Sci. 2005 46: E-Abstract 3464.) |
This image shows sebaceous glands of hair follicles (red) surrounded by vasculature (green) as imaged In-vivo by combined CARS and two-photon fluorescence. Green is fluorescence from FITC-labeled dextran in bloodstream. Red is CARS signal from intrinsic CH2 stretch vibration. (Conors et al. PNAS, in press) |
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| The movie shows the trafficking of GFP-labeled T cells (green) in the mouse ear after drug-induced inflammation. Lymphatic ducts in the skin are stained red using Cy5.5- conjugated anti-LYVE-1 antibody. Images were acquired every 30 sec for about 20 min using the video-rate laser scanning confocal microscope. |
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. A) Vasculature of mouse ear with cartoons exemplifying cells flowing through the slit of excitation light. B) Example of fluorescence trace from the photodetector. Each spike represents one fluorescently labeled cell. C) 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
A major focus is to investigate the cellular effects of localized energy deposition into endogenous or exogenous chromophores.
Micro- and nano-particle assisted selective cell targeting
By labeling cell population of interest with highly absorbing micro- or nano-particles, spatial confinement of laser energy deposition has been demonstrated. This technique allows us to selectively destroy cells and transiently permeabilize cell membrane to allow delivery of molecules (drugs, genes, etc.)
Selective cell targeting for the treatment of retinal diseases
The presence of strongly absorbing microparticles (melanosomes) in the retinal pigment epithelium (RPE) behind the photoreceptors, enables us to develop new therapeutic strategies to selectively treat the RPE cells in certain retinal diseases (such as diabetic macular edema) without damaging the photoreceptors. We have developed a laser scanner that rapidly scans the small spot of a continuous wave laser across the retina so as to produce microsecond-short irradiation at each exposed RPE cell. We have shown successfully, that individual RPE cells can be damaged.
Calcein stained explants of bovine RPE, before (left) and after irradiation (right) with a pre-defined scan pattern of cw green laser (middle). After irradiation damaged cells loose the fluorescence and turn dark. Alternating lines of dead and surviving cells that resemble the scan pattern suggest selectivity. (Alt et al. J. Biomedical Optics, in press) |
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