Imaging leukocyte trafficking in vivo with two-photon-excited endogenous tryptophan fluorescence

We describe a new method for imaging leukocytes in vivo by exciting the endogenous protein fluorescence in the ultraviolet (UV) spectral region where tryptophan is the major fluorophore. Two-photon excitation near 590 nm allows noninvasive optical sectioning through the epidermal cell layers into the dermis of mouse skin, where leukocytes can be observed by video-rate microscopy to interact dynamically with the dermal vascular endothelium. Inflammation significantly enhances leukocyte rolling, adhesion, and tissue infiltration. After exiting the vasculature, leukocytes continue to move actively in tissue as observed by time-lapse microscopy, and are distinguishable from resident autofluorescent cells that are not motile. Because the new method alleviates the need to introduce exogenous labels, it is potentially applicable for tracking leukocytes and monitoring inflammatory cellular reactions in humans

An inflammatory checkpoint regulates recruitment of graft-versus-host reactive T cells to peripheral tissues

Transfer of T cells to freshly irradiated allogeneic recipients leads to their rapid recruitment to nonlymphoid tissues, where they induce graft-versus-host disease (GVHD). In contrast, when donor T cells are transferred to established mixed chimeras (MCs), GVHD is not induced despite a robust graft-versus-host (GVH) reaction that eliminates normal and malignant host hematopoietic cells. We demonstrate here that donor GVH-reactive T cells transferred to MCs or freshly irradiated mice undergo similar expansion and activation, with similar up-regulation of homing molecules required for entry to nonlymphoid tissues. Using dynamic two-photon in vivo microscopy, we show that these activated T cells do not enter GVHD target tissues in established MCs, contrary to the dogma that activated T cells inevitably traffic to nonlymphoid tissues. Instead, we show that the presence of inflammation within a nonlymphoid tissue is a prerequisite for the trafficking of activated T cells to that site. Our studies help to explain the paradox whereby GVH-reactive T cells can mediate graft-versus-leukemia responses without inducing GVHD in established MCs

Monitoring intracellular cavitation during selective laser targeting of the retinal pigment epithelium

Thesis (Mech. E.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2002.Includes bibliographical references (leaves 39-45).by Costas M. Pitsillides.Mech.E

Selective cell targeting with light-absorbing particles

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2000.Includes bibliographical references (p. 41-43).by Costas M. Pitsillides.S.M

Monitoring tumor burden by multicolor in vivo flow cytometry

In vivo measurement of tumor burden, both in cancer research models and in patients, is an important parameter for the accurate assessment of disease progression and the response to therapeutic intervention [1]. Several in vivo imaging modalities have been utilized in the assessment of tumor burden, including functional magnetic resonance imaging, computer tomography and positron emission tomography [2, 3], fluorescence imaging [4, 5], intravital microscopy [6] and bioluminescence imaging [7]. More recently, the detection/quantification of circulating cancer cells has been explored as a method to evaluate tumor burden in the context of assessing disease stage, prognosis as well as monitoring disease progression following therapeutic intervention in cancer patients [8, 9]. Clinically, various ex vivo assays have been developed to detect cancer cells shed in circulation by primary tumors, including breast cancer, prostate cancer and small-cell lung cancer [10, 11]. In vivo flow cytometry has been developed as a method for real-time detection of circulating cancer cells injected into the circulation of experimental animals. The method does not require extraction of blood samples and is therefore well suited for long-term monitoring of circulating tumor cells. In this report, we report on the development of a multichannel in vivo flow cytometer to detect and quantify circulating cancer cells as a means of assessing the tumor burden in animal models

Facilitating the development of novel therapeutic strategies via in vivo optical imaging techniques

The BioLISYS Laboratory at CUT is developing novel fluorescence-based techniques for in vivo imaging of small animals with applications in cardiovascular and cancer therapeutics. These include the development of an in vivo flow cytometer in order to monitor fluorescently labeled cells in circulation as well as a whole body reflectance imaging system for detection of fluorescence and bioluminescence signal from cells and tissues in murine models of disease. The in vivo flow cytometer has been designed as a minimally invasive optical tool for the real time detection/quantification of fluorescent cells in circulation of living animals without the need to sequentially extract blood samples or sacrifice animals. Thus the system allows for the continuous monitoring of a cell population of interest over long time periods in order to assess dynamic changes in circulation. The optical reflectance imaging system combines fluorescence and bioluminescence imaging capabilities with a large field of view in order to enable imaging over a wide area of the animal. The noninvasive, quantitative method enables longitudinal studies of physiological changes in disease and allows for continuous monitoring in the same mouse over an extended time period, in order to evaluate biodistribution and therapeutic response of experimental therapeutic agents. The imaging systems have been employed in the in vivo analysis of cardiovascular implants and novel biomaterials in order to evaluate the inflammatory response of vascular tissue to stent implantation and stent biocorrosion via the in vivo monitoring of the degree of inflammation, macrophage infiltration and cytokine expression in tissue surrounding stents deployed in mice abdominal aortas. In cancer therapeutics, the in vivo imaging systems have been used to develop a novel therapeutic system for targeted miRNA delivery to tumors, via microparticles that are derived from mesenchymal stem cells. Fluorescently labeled miRNA-loaded microparticles injected into the tail vein of tumor bearing mouse were monitored in circulation via the in vivo flow cytometer while their biodistribution and targeting specificity was detected in tumor sites via the fluorescence based whole body reflectance imaging system. Furthermore, tumor progression and therapeutic response to miRNA therapy delivered via local and systemic administration of the MSC-derived microparticles was monitored in real time via the imaging of fluorescence and bioluminescence expressing tumors by whole body reflectance imaging

Monitoring intracellular cavitation during selective targeting of the retinal pigment epithelium

PURPOSE Selective targeting of the Retinal Pigment Epithelium (RPE), by either applying trains of microsecond laser pulses or, in our approach, by repetitively scanning a tightly focused spot across the retina, achieves destruction of RPE cells while avoiding damage to the overlying photoreceptors. Both techniques have been demonstrated as attractive methods for the treatment of retinal diseases that are caused by a dysfunction of the RPE. Because the lesions are ophthalmoscopically invisible, an online control system that monitors cell death during irradiation is essential to ensure efficient and selective treatment in a clinical application. MATERIALS AND METHODS Bubble formation inside the RPE cells has been shown to be the cell damage mechanism for nano- and picosecond pulses. We built an optical system to investigate whether the same mechanism extends into the microsecond regime. The system detects changes in backscattered light of the irradiating beam during exposure. Samples of young bovine eyes were exposed in vitro using single pulses ranging from 3 μs to 50 μs. Using the viability assay calcein-AM the ED50 threshold for cell death was determined and compared to the threshold for bubble formation. We also set up a detection system on our slit lamp adapted scanning system in order to determine the feasibility of monitoring threshold RPE damage during selective laser treatment in vivo. RESULTS AND DISCUSSION Intracellular cavitation was detected as a transient increase in backscattering signal, either of an external probe beam or of the irradiation beam itself. Monitoring with the irradiation beam is both simpler and more sensitive. We found the threshold for bubble formation to coincide with the threshold for cell damage for pulse durations up to 20 μs, suggesting that cavitation is the main mechanism of cell damage. For pulse widths longer than 20 μs, the cell damage mechanism appears to be increasingly thermal as the two thresholds diverge. We conclude that bubble detection can be used to monitor therapeutic endpoint for pulse durations up to 20 μs (or equivalent dwell time in a scanning approach). We have integrated a detection module into our slit lamp adapted laser scanner in order to determine threshold RPE damage during selective laser treatment in viv

Facilitating the development of novel therapeutic strategies via in vivo optical imaging techniques

The BioLISYS Laboratory at CUT is developing novel fluorescence-based techniques for in vivo imaging of small animals with applications in cardiovascular and cancer therapeutics. These include the development of an in vivo flow cytometer in order to monitor fluorescently labeled cells in circulation as well as a whole body reflectance imaging system for detection of fluorescence and bioluminescence signal from cells and tissues in murine models of disease. The in vivo flow cytometer has been designed as a minimally invasive optical tool for the real time detection/quantification of fluorescent cells in circulation of living animals without the need to sequentially extract blood samples or sacrifice animals. Thus the system allows for the continuous monitoring of a cell population of interest over long time periods in order to assess dynamic changes in circulation. The optical reflectance imaging system combines fluorescence and bioluminescence imaging capabilities with a large field of view in order to enable imaging over a wide area of the animal. The noninvasive, quantitative method enables longitudinal studies of physiological changes in disease and allows for continuous monitoring in the same mouse over an extended time period, in order to evaluate biodistribution and therapeutic response of experimental therapeutic agents. The imaging systems have been employed in the in vivo analysis of cardiovascular implants and novel biomaterials in order to evaluate the inflammatory response of vascular tissue to stent implantation and stent biocorrosion via the in vivo monitoring of the degree of inflammation, macrophage infiltration and cytokine expression in tissue surrounding stents deployed in mice abdominal aortas. In cancer therapeutics, the in vivo imaging systems have been used to develop a novel therapeutic system for targeted miRNA delivery to tumors, via microparticles that are derived from mesenchymal stem cells. Fluorescently labeled miRNA-loaded microparticles injected into the tail vein of tumor bearing mouse were monitored in circulation via the in vivo flow cytometer while their biodistribution and targeting specificity was detected in tumor sites via the fluorescence based whole body reflectance imaging system. Furthermore, tumor progression and therapeutic response to miRNA therapy delivered via local and systemic administration of the MSC-derived microparticles was monitored in real time via the imaging of fluorescence and bioluminescence expressing tumors by whole body reflectance imaging