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Microglia are the resident immune cells of the central nervous system and play a crucial role in maintaining neuronal health and function. Their dynamic behavior, that is, the constant extension and retraction of microglia processes, is thought to be critical for communication between microglia and their cellular neighbors, such as neurons, astrocytes and vascular endothelial cells. Here, we investigated the morphology and dynamics of retinal microglia in vivo under normal conditions and in response to focal laser injury of blood vessel endothelial wall, using a scanning laser ophthalmoscope (SLO) designed specifically for imaging the retina of live mice. The multichannel confocal imaging system allows retinal microstructure, such as the processes of microglia and retinal vasculature, to be visualized simultaneously. In order to generate focal laser injury, a photocoagulator based on a continuous wave (cw) laser was coupled into the SLO. An acousto-optic modulator chopped pulses from the cw laser. A tip-tilt-scanner was used to direct the laser beam into a blood vessel of interest under SLO image guidance. Mild coagulation was produced using millisecond-long pulses. Microglia react dynamically to focal laser injury of blood vessel endothelial walls. Under normal conditions, microglia somas remain stationary and the processes probe a territory of their immediate environment. In response to local injury, process movement velocity approximately doubles within minutes after injury. Moreover, the previously unpolarized process movement assumes a distinct directionality towards the injury site, indicating signaling between the injured tissue and the microglia. In vivo retinal imaging is a powerful tool for understanding the dynamic behavior of retinal cells.

Zebrafish have become a powerful vertebrate model organism for drug discovery, cancer and stem cell research. A recently developed transparent adult zebrafish using double pigmentation mutant, called casper, provide unparalleled imaging power in in vivo longitudinal analysis of biological processes at an anatomic resolution not readily achievable in murine or other systems. In this paper we introduce an optical method for simultaneous visualization and cell quantification, which combines the laser scanning confocal microscopy (LSCM) and the in vivo flow cytometry (IVFC). The system is designed specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish casper, under physiological conditions in the same fish over time. The confocal imaging part in this system serves the dual purposes of imaging fish tissue microstructure and a 3D navigation tool to locate a suitable vessel for circulating cell counting. The multi-color, multi-channel instrument allows the detection of multiple cell populations or different tissues or organs simultaneously. We demonstrate initial testing of this novel instrument by imaging vasculature and tracking circulating cells in CD41: GFP/Gata1: DsRed transgenic casper fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. Circulating fluorescent cell incidents were recorded and counted repeatedly over time and in different types of vessels. Great application opportunities in cancer and stem cell researches are discussed.

In vivo retinal imaging is an outstanding tool to observe biological processes unfold in real-time. The ability to image microstructure in vivo can greatly enhance our understanding of function in retinal microanatomy under normal conditions and in disease. Transgenic mice are frequently used for mouse models of retinal diseases. However, commercially available retinal imaging instruments lack the optical resolution and spectral flexibility necessary to visualize detail comprehensively. We developed an adaptive optics scanning laser ophthalmoscope (AO-SLO) specifically for mouse eyes. Our SLO is a sensor-less adaptive optics system (no Shack Hartmann sensor) that employs a stochastic parallel gradient descent algorithm to modulate a deformable mirror, ultimately aiming to correct wavefront aberrations by optimizing confocal image sharpness. The resulting resolution allows detailed observation of retinal microstructure. The AO-SLO can resolve retinal microglia and their moving processes, demonstrating that microglia processes are highly motile, constantly probing their immediate environment. Similarly, retinal ganglion cells are imaged along with their axons and sprouting dendrites. Retinal blood vessels are imaged both using evans blue fluorescence and backscattering contrast.

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. 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. 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 vivo.

Selective targeting the retinal pigment epithelium (RPE) while sparing adjacent tissue such as the photoreceptors hasbeen demonstrated by repetitively irradiating the fundus with a train of green µs-laser pulses. The aim of this study wasto investigate selective RPE effects alternatively by means of rapidly scanning a cw-laser beam across the RPE to obtainthe required µs-illumination times.The radiation of an Ar + laser (514 nm) was transmitted through a 25 µm core diameter fiber to a scanner unit. The fibertip was imaged onto the object plane with a magnification of 0.75. The beam was repetitively scanned across porcineRPE samples in vitro providing an irradiation time of 1.6 µs. Cell damage was investigated with a fluorescence viabilityassay.The ED 50 cell damage power was determined to 580 mW when applying 10 exposures with a repetition rate of 500 Hz.It decreases to 250 mW for 500 exposures and more. The threshold keeps constant for an axial focus displacement ofabout 250 µm.In conclusion, selective cell targeting has been proved with a laser scanning device. The technique can be adapted forclinical RPE treatment to a slit-lamp or by modifying a retina angiograph.KEY WORDS : fluorescence microscopy, melanosome, laser scanner, RPE, selective photocoagulation, viability assay

Laser photocoagulation is a well-established treatment modality for a variety of retinal disorders, but is difficult to use near the fovea due to thermal retinal destruction. Certain diseases, such as drusen maculopathy, are thought to be caused by a dysfunction of the Retinal Pigment Epithelium. For those diseases selective targeting of the RPE, sparing the adjoining photoreceptors, might be the appropriate treatment to avoid laser scotoma, as it has been shown with application of a train of ms laser pulses by Birngruber and Roider. Our new approach is to use a conventional green cw laser and rapidly scan a small laser spot over the retina so as to produce microsecond(s) -illumination at each RPE cell. Two scanning devices were developed using acousto-optic deflectors. For the in vitro experiments the ED50 value RPE cell damage was 170 mW with 100 exposures, scanning with a speed of 1 spot diameter/3 microsecond(s) . In vivo experiments demonstrated an angiographic ED50 threshold of 66 mW for 100 exposures while scanning with an effective illumination time of 5 microsecond(s) . The ophthalmoscopic threshold was higher than a factor of 2 times the angiographic ED50. Using separated scan lines we show selectivity in the form of surviving cells in between irradiated lines. Selective destruction of RPE cells is possible using laser-scanning devices.© (2002) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.