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In the last 20 years, research focused on developing retinal imaging as a source of potential biomarkers for Alzheimer's disease and other neurodegenerative diseases, has increased significantly. The Alzheimer's Association and the Alzheimer's & Dementia: Diagnosis, Assessment, Disease Monitoring editorial team (companion journal to Alzheimer's & Dementia) convened an interdisciplinary discussion in 2019 to identify a path to expedite the development of retinal biomarkers capable of identifying biological changes associated with AD, and for tracking progression of disease severity over time. As different retinal imaging modalities provide different types of structural and/or functional information, the discussion reflected on these modalities and their respective strengths and weaknesses. Discussion further focused on the importance of defining the context of use to help guide the development of retinal biomarkers. Moving from research to context of use, and ultimately to clinical evaluation, this article outlines ongoing retinal imaging research today in Alzheimer's and other brain diseases, including a discussion of future directions for this area of study.

Watertight closure of perforating corneoscleral lacerations is necessary to prevent epithelial ingrowth, infection, and potential loss of the eye. Complex lacerations can be difficult to treat, and repair with sutures alone is often inadequate. In this study, we evaluated a potentially sutureless technology for sealing complex corneal and scleral lacerations that bonds the amniotic membrane (AM) to the wound using only green light and rose bengal dye.The AM was impregnated with rose bengal and then sealed over lacerations using green light to bond the AM to the deepithelialized corneal surface. This process was compared with suture repair of 3 laceration configurations in New Zealand White rabbits in 3 arms of the study. A fourth study arm assessed the side effect profile including viability of cells in the iris, damage to the blood-retinal barrier, retinal photoreceptors, retinal pigment epithelium, and choriocapillaris in Dutch Belted rabbits.Analyses of the first 3 arms revealed a clinically insignificant increase in polymorphonuclear inflammation. In the fourth arm, iris cells appeared unaffected and no evidence of breakdown of the blood-retinal barrier was detected. The retina from green light laser-treated eyes showed normal retinal pigment epithelium, intact outer segments, and normal outer nuclear layer thickness.The results of these studies established that a light-activated method to cross-link AM to the cornea can be used for sealing complex penetrating wounds in the cornea and sclera with minimal inflammation or secondary effects.

Commercial microscopy systems make use of tandem scanning i.e. either slow or fast scanning. We constructed, for the first time, an advanced control system capable of delivering a dynamic line scanning speed ranging from 2.7 kHz to 27 kHz and achieve variable frame rates from 5 Hz to 50 Hz (512 × 512). The dynamic scanning ability is digitally controlled by a new customized open-source software named PScan1.0. This permits manipulation of scanning rates either to gain higher fluorescence signal at slow frame rate without increasing laser power or increase frame rates to capture high speed events. By adjusting imaging speed from 40 Hz to 160 Hz, we capture a range of calcium waves and transient peaks from soma and dendrite of single fluorescence neuron (CAL-520AM). Motion artifacts arising from respiratory and cardiac motion in small animal imaging reduce quality of real-time images of single cells in-vivo. An image registration algorithm, integrated with PScan1.0, was shown to perform both real time and post-processed motion correction. The improvement is verified by quantification of blood flow rates. This work describes all the steps necessary to develop a high performance and flexible polygon-mirror based multiphoton microscope system for in-vivo biological imaging.

To evaluate crosslinking of cornea in vivo using green light activation of Rose Bengal (RGX) and assess potential damaging effects of the green light on retina and iris.Corneas of Dutch belted rabbits were de-epithelialized, then stained with Rose Bengal and exposed to green light, or not further treated. Corneal stiffness was measured by uniaxial tensiometry. Re-epithelialization was assessed by fluorescein fluorescence. Keratocytes were counted on hematoxylin and eosin (HE)-stained sections, and iris cell damage was assessed by lactate dehydrogenase staining. Thermal effects on the blood-retinal barrier (BRB) were assessed by fluorescein angiography and those on photoreceptors, retinal pigment epithelium (RPE), and choriocapillaris by light microscopy and transmission electron microscopy.RGX (10-min irradiation; 150 J/cm) increased corneal stiffness 1.9-fold on day 1 (1.25 ± 0.21 vs. 2.38 ± 0.59 N/mm; P = 0.036) and 2.8-fold compared with controls on day 28 (1.70 ± 0.74 vs. 4.95 ± 1.86 N/mm; P = 0.003). Keratocytes decreased only in the anterior stroma on day 1 (24.0 ± 3.0 vs. 3.67 ± 4.73, P = 0.003) and recovered by day 28 (37.7 ± 8.9 vs. 34.5 ± 2.4, P = 0.51). Iris cells were not thermally damaged. No evidence of BRB breakdown was detected on days 1 or 28. Retina from RGX-treated eyes seemed normal with RPE cells showing intact nuclei shielded apically by melanosomes, morphologically intact photoreceptor outer segments, normal outer nuclear layer thickness, and choriocapillaris containing intact erythrocytes.The substantial corneal stiffening produced by RGX together with the lack of significant effects on keratocytes and no evidence for retina or iris damage suggest that RGX-initiated corneal crosslinking may be a safe, rapid, and effective treatment.

Differential interference contrast (DIC) microscopy is a powerful technique for imaging phase objects in transparent samples but does not work with scattering samples. This Letter, to the best of our knowledge, describes a new technique for obtaining DIC-like phase-gradient images in scattering media based on differential detection of forward-scattered light, using detectors arranged in a ring configuration around the microscope objective pupil or its conjugate pupil plane. This method, called pupil plane differential detection (P2D2) microscopy, does not need polarization optics or a confocal pinhole, yet produces images that are free of speckles and interference noises. We compared the P2D2 imaging technique with reflectance confocal microscopy and demonstrated P2D2 as a simple add-on to conventional laser scanning microscopes.

The bone marrow is a unique microenvironment where blood cells are produced and released into the circulation. At the top of the blood cell lineage are the hematopoietic stem cells (HSC), which are thought to reside in close association with the bone marrow vascular endothelial cells (Morrison and Scadden, Nature 505:327-334, 2014). Recent efforts at characterizing the HSC niche have prompted us to make close examinations of two distinct types of blood vessel in the bone marrow, the arteriolar vessels originating from arteries and sinusoidal vessels connected to veins. We found the two vessel types to exhibit different vascular permeabilites, hemodynamics, cell trafficking behaviors, and oxygen content (Itkin et al., Nature 532:323-328, 2016; Spencer et al., Nature 508:269-273, 2014). Here, we describe a method to quantitatively measure the permeability and hemodynamics of arterioles and sinusoids in murine calvarial bone marrow using intravital microscopy.

The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient’s blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertz's oblique back-illumination microscopy (OBM) (Ford et al, Nat. Meth. 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.

The bone marrow is a unique microenvironment where blood cells are produced and released into the circulation. At the top of the blood cell lineage are the hematopoietic stem cells (HSC), which are thought to reside in close association with the bone marrow vascular endothelial cells (Morrison and Scadden, Nature 505:327-334, 2014). Recent efforts at characterizing the HSC niche have prompted us to make close examinations of two distinct types of blood vessel in the bone marrow, the arteriolar vessels originating from arteries and sinusoidal vessels connected to veins. We found the two vessel types to exhibit different vascular permeabilites, hemodynamics, cell trafficking behaviors, and oxygen content (Itkin et al., Nature 532:323-328, 2016; Spencer et al., Nature 508:269-273, 2014). Here, we describe a method to quantitatively measure the permeability and hemodynamics of arterioles and sinusoids in murine calvarial bone marrow using intravital microscopy.

Transplantation of a single hematopoietic stem cell is an important method for its functional characterization, but the standard transplantation protocol relies on cell homing to the bone marrow after intravenous injection. Here, we present a method to transplant single cells directly into the bone marrow of live mice. We developed an optical platform that integrates a multiphoton microscope with a laser ablation unit for microsurgery and an optical tweezer for cell micromanipulation. These tools allow image-guided single cell transplantation with high spatial control. The platform was used to deliver single hematopoietic stem cells. The engraftment of transplants was tracked over time, illustrating that the technique can be useful for studying both normal and malignant stem cells in vivo.

Clonal heterogeneity and selection underpin many biological processes including development and tumor progression. Combinatorial fluorescent protein expression in germline cells has proven its utility for tracking the formation and regeneration of different organ systems. Such cell populations encoded by combinatorial fluorescent proteins are also attractive tools for understanding clonal expansion and clonal competition in cancer. However, the assignment of clonal identity requires an analytical framework in which clonal markings can be parameterized and validated. Here we present a systematic and quantitative method for RGB analysis of fluorescent melanoma cancer clones. We then demonstrate refined clonal trackability of melanoma cells using this scheme.

A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2+ HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2+ HSCs. Activation of the PPAR (peroxisome proliferator–activated receptor)–fatty acid oxidation pathway promotes expansion of Tie2+ HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2+ HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways.

We examine the effect of bone marrow transplantation (BMT) on retinal cell turnover by performing simultaneous cell tracking of native microglia and engrafting donor bone marrow-derived cell (BMDC) populations in the retinae of live mice using a custom-built multi-color confocal scanning laser ophthalmoscope (SLO) specifically developed for murine retinal imaging. CX3CR1GFP/+ mice whose retinal microglia express the green fluorescent protein (GFP) were exposed to a lethal dose of gamma radiation and subsequently rescued with bone marrow cells from universal DsRed donor mice. Over a time course of four months after the irradiation and BMT, progressive loss of GFP+ microglia was accompanied by delayed engraftment of DsRed+ BMDC. Morphologic examination revealed that the remaining GFP+ microglia were ramified, while engrafting DsRed+ cells exhibited both ramification and dendriform shape. Leukocyte endothelial interaction, normally absent in healthy retinal vasculature, was observed even after three months, ...

The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) provides a protective environment and resistance to therapeutic agents. We hypothesized that disruption of the interaction of MM cells with their BM milieu would lead to their sensitization to therapeutic agents such as bortezomib, melphalan, doxorubicin, and dexamethasone. We report that the CXCR4 inhibitor AMD3100 induces disruption of the interaction of MM cells with the BM reflected by mobilization of MM cells into the circulation in vivo, with kinetics that differed from that of hematopoietic stem cells. AMD3100 enhanced sensitivity of MM cell to multiple therapeutic agents in vitro by disrupting adhesion of MM cells to bone marrow stromal cells (BMSCs). Moreover, AMD3100 increased mobilization of MM cells to the circulation in vivo, increased the ratio of apoptotic circulating MM cells, and enhanced the tumor reduction induced by bortezomib. Mechanistically, AMD3100 significantly inhibited Akt phosphorylation and enhanced poly(ADP-ribose) polymerase (PARP) cleavage as a result of bortezomib, in the presence of BMSCs in coculture. These experiments provide a proof of concept for the use of agents that disrupt interaction with the microenvironment for enhancement of efficacy of cytotoxic agents in cancer therapy.

Spectral domain optical coherence tomography (SD-OCT) in patients can deliver retinal cross-sectional images with high resolution. This may allow the evaluation of the extent of damage to the retinal pigment epithelium (RPE) and the neurosensory retina after laser treatment. This article aims to investigate the value of SD-OCT in comparing laser lesions produced by conventional laser photocoagulation and selective retina treatment (SRT).In a retrospective study, conventional retinal laser (CRL) lesions and SRT laser lesions were evaluated with SD-OCT. One hundred seventy-five CRL lesions were investigated in 10 patients with diabetic maculopathy at timepoints between 1 hr and 4 years after treatment. Ninety-one SRT lesions were examined in 9 patients with central serous retinopathy, geographic atrophy, and diabetic maculopathy at timepoints between 1 hr and 2 years. CRL lesions were applied with an ophthalmoscopically slightly grayish-white appearance (Nd:YAG laser at 532-nm wavelength; power 100-200 mW; retinal spot diameter 100 microm; pulse duration 100 ms). SRT lesions were applied with a Nd:YLF (527 nm; pulse duration 200 ns [30 pulses at 100 Hz]; energy 100-200 microJ/pulse; retinal spot diameter 200 microm) and were visible only angiographically.All CRL lesions were characterized by high reflectivity in OCT images throughout the full thickness of the neurosensory tissue 1 hr after irradiation, suggesting complete neurosensory coagulation. Strong contraction through the full thickness of the neurosensory layers was observed within 7 days after treatment. In contrast, the neural retina appeared unaffected after SRT. For both lesion types, the RPE layer appeared to be regular or thinner immediately after treatment, whereas within a period of 4 weeks, a RPE thickening indicating RPE proliferation was observable. One year and later after treatment, CRL lesions were characterized by RPE atrophy combined with significant damage of the neurosensory tissue. SRT lesions aged one year and older revealed unaffected neurosensory structures and an intact RPE layer.Spectral domain OCT can be used clinically to follow the development of laser-induced lesions over time. Postoperative RPE proliferation was observed in both CRL and SRT laser lesions. RPE atrophy appeared subsequently only in CRL lesions, whereas the neurosensory retina appeared unaffected following SRT. These results suggest the selective effect of SRT in humans without causing adverse effects to the neurosensory retina.

Purpose: Selective retina therapy (SRT), the confined laser heating and destruction of retinal pigment epithelial cells, has been shown to treat acute types of central serous chorioretinopathy (CSC) successfully without damaging the photoreceptors and thus avoiding laser-induced scotoma. However, a benefit of laser treatment for chronic forms of CSC is questionable. In this study, the efficacy of SRT by means of the previously used 1.7-µs and shorter 300-ns pulse duration was evaluated for both types of CSC, also considering re-treatment for nonresponders. Material and Methods: In a two-center trial, 26 patients were treated with SRT for acute (n = 10) and chronic-recurrent CSC (n = 16). All patients presented with subretinal fluid (SRF) in OCT and leakage in fluorescein angiography (FA). SRT was performed using a prototype SRT laser system (frequency-doubled Q-switched Nd:YLF-laser, wavelength 527 nm) with adjustable pulse duration. The following irradiation settings were used: a train of 30 laser pulses with a repetition rate of 100 Hz and pulse durations of 300 ns and 1.7 µs, pulse energy 120-200 µJ, retinal spot size 200 µm. Because SRT lesions are invisible, FA was always performed 1 h after treatment to demonstrate laser outcome (5-8 single spots in the area of leakage). In cases where energy was too low, as indicated by missing FA leakage, energy was adjusted and the patient re-treated immediately. Observation intervals were after 4 weeks and 3 months. In case of nonimprovement of the disease after 3 months, re-treatment was considered. Results: Of 10 patients with active CSC that presents focal leakage in FA, 5 had completely resolved fluid after 4 weeks and all 10 after 3 months. Mean visual acuity increased from 76.6 ETDRS letters to 85.0 ETDRS letters 3 months after SRT. Chronic-recurrent CSC was characterized by less severe SRF at baseline in OCT and weaker leakage in FA than in acute types. Visual acuity changed from baseline 71.6 to 72.8 ETDRS letters after 3 months. At this time, SRF was absent in 3 out of 16 patients (19%), FA leakage had come to a complete stop in 6 out of 16 patients (38%). In 6 of the remaining chronic CSC patients, repeated SRT with higher pulse energy was considered because of persistent leakage activity. After the re-treatment, SRF resolved completely in 5 patients (83.3%) after only 25 days. Conclusion: SRT showed promising results in treating acute CSC, but was less effective in chronic cases. Interestingly, re-treatment resulted in enhanced fluid resolution and dry conditions after a considerably shorter time in most patients. Therefore, SRT including re-treatment if necessary might be a valuable CSC treatment alternative even in chronic-recurrent cases.

Hintergrund Selektive RPE-Lasertherapie ist durch repetitive Laserbestrahlung mit Mikrosekundenpulsen unter Schonung der Photorezeptorschicht moglich. Damit konnen zentrale Netzhautpathologien wie diabetische Makulopathie, weiche konfluierende Drusen im Rahmen der altersbedingten Makuladegeneration oder die Chorioretinopathia centralis serosa ohne konsekutive Laserskotome behandelt werden. Es war Ziel dieser Studie zu zeigen, ob ein selektiver RPE-Schaden auch mit einem konventionellen grunen cw-Laserstrahl erreicht werden kann, wenn dieser zur Bestrahlung uber die Netzhaut gescannt wird.

Background and Objectives Selective treatment of the retinal pigment epithelium (RPE) by repetitively applying green μs-laser pulses is a new method for retinal diseases associated with a degradation of the RPE, which spares the neural retina. We investigated an alternative approach to realize repetitive μs-laser exposure by rapidly scanning a continuous wave (CW)-laser beam across the RPE. Study Design/Materials and Methods An Ar+ laser beam (514 nm) with a diameter of 18.75 μm was repetitively scanned across porcine RPE samples in vitro providing an irradiation time of 1.6 μs per point on the central scan axis. RPE cell damage was investigated by means of the fluorescence viability assay Calcein-AM. Results The ED50 cell damage is 305 mJ/cm2 when applying 10 scans with a repetition rate of 500 Hz. The threshold decreases with the number of scans, a saturation was found at 135 mJ/cm2 with more than 500 exposures applied. The depth of focus in beam direction is 350 μm, defined by an increase of the threshold radiant exposure by 20%. Conclusions Targeting of pigmented cells with high local resolution has been proved with a laser-scanning device. Looking ahead selective RPE-treatment, the adaptation of a laser-scanning device on a slit-lamp or into a modified retina angiograph seems to be an attractive alternative to the pulsed μs laser device. Lasers Surg. Med. 32:252–264, 2003. © 2003 Wiley-Liss, Inc.

The bone marrow is an important site where all blood cells are formed from hematopoietic stem cells and where hematologic malignancies such as leukemia emerge. It is also a frequent site for metastasis of solid tumors such as breast cancer and prostate cancer. Intravital microscopy is a powerful tool for studying the bone marrow with single cell and sub-cellular resolution. To improve optical access to this rich biological environment, plasma-mediated laser ablation with sub-microjoule femtosecond pulses was used to thin cortical bone. By locally removing a superficial layer of bone (local laser osteotomy), significant improvements in multiphoton imaging were observed in individual bone marrow compartments in vivo. This work demonstrates the utility of scanning laser ablation of hard tissue with sub-microjoule pulses as a preparatory step to imaging.

Femtosecond laser pulses can be used to perform very precise cutting of material, including biological samples from subcellular organelles to large areas of bone, through plasma-mediated ablation. The use of a kilohertz regenerative amplifier is usually needed to obtain the pulse energy required for ablation. This work investigates a 5 megahertz compact fiber laser for near-video rate imaging and ablation in bone. After optimization of ablation efficiency and reduction in autofluorescence, the system is demonstrated for the in vivo study of bone regeneration. Image-guided creation of a bone defect and longitudinal evaluation of cellular injury response in the defect provides insight into the bone regeneration process.

Purpose Gamma irradiation and bone marrow transplantation (BMT) are established clinical procedures for the treatment of hematologic malignancies. The radiation targets cells in the bone marrow, but injury to other tissues, including the central nervous system (CNS), have been reported. Here, we examine if anti-inflammatory treatment can mitigate the radiation-induced turnover of retinal microglia and the replacement by bone marrow-derived cells (BMDCs). Methods Two-color chimeric mice were generated by lethal irradiation of heterozygous CX3CR1-GFP mice that express GFP in microglial cells and bone marrow transplantation from universal DsRed donor mice. Mice were treated with the corticosteroid dexamethasone; a control group received no dexamethasone treatment. The populations of resident microglia (GFP+) and BMDCs (DsRed+) were quantified by serial in vivo imaging for 10 weeks after irradiation with a confocal scanning laser ophthalmoscope that we custom-built specifically for multicolor imaging of the murine retina. Results Ionizing radiation resulted in loss of 75% of the resident retinal microglia population after 70 days. Recruitment of BMDCs was delayed with respect to the microglia loss, resulting in a transient depletion of the total immune cell number in the retina. With dexamethasone treatment, both the loss of the resident microglia and the infiltration of BMDCs were suppressed by at least 50%. Conclusions Anti-inflammatory treatment with the corticosteroidal agent dexamethasone preserves resident microglia and minimizes recruitment of BMDCs after ionizing radiation exposure and BMT.

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs)1–3, and LSC deficiency is a major cause of blindness worldwide4. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts5, a gene allowing for prospective LSC enrichment has not been identified so far5. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5)6,7 marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs2 in mice and p63α-positive LSCs8 in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

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.

Adult zebrafish are being increasingly used as a model in cancer and stem cell research. Here we describe an integrated optical system that combines a laser scanning confocal microscope (LSCM) and an in vivo flow cytometer (IVFC) for simultaneous visualization and cell quantification. The system is set up specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish (casper) that have been engineered to be optically transparent. Confocal imaging in this instrument serves the dual purpose of visualizing fish tissue microstructure and an imaging-based guide to locate a suitable vessel for quantitative analysis of circulating cells by IVFC. We demonstrate initial testing of this novel instrument by imaging the transparent adult zebrafish casper vasculature and tracking circulating cells in CD41-GFP/Gata1-DsRed transgenic fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. In vivo measurements allow cells to be tracked under physiological conditions in the same fish over time, without drawing blood samples or sacrificing animals. We also discuss the potential applications of this instrument in biomedical research.

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.

Purpose: An in vivo flow cytometer was developed recently, providing quantification of fluorescently labeled cells in live animals without extracting blood samples. This non-invasive procedure allows continuously tracking a cell population of interest over long periods of time to examine its dynamic changes in the circulation. However, it has not been shown that counting signals arise from individual cells. Furthermore, cell morphology and cell-cell interaction in the blood stream (e.g. aggregation) are not visualized. Here we describe an imaging in vivo flow cytometer. Material and Methods: Fluorescence images are obtained simultaneously with quantitative information on a DiD-labeled cell population. As fluorescent cells pass through the slit of light focused across a blood vessel, the excited fluorescence is detected confocally. This cell counting signal triggers a strobe beam and an intensified CCD camera to capture a snapshot image of the cell as it moves down-stream from the slit. Results: Nearly all peaks counted as circulating T-cells originate from individual cells, while cell aggregates were rarely observed (

Selective laser targeting of the retinal pigment epithelium (RPE) is an attractive method for treating RPE-associated disorders. We are developing a method for optically detecting intracellular microcavitation that can potentially serve as an immediate feedback of the treatment outcome. Thermal denaturation or intracellular cavitation can kill RPE cells during selective targeting. We examined the cell damage mechanism for laser pulse durations from 1 to 40 micros ex vivo. Intracellular cavitation was detected as a transient increase in the backscattered treatment beam. Cavitation and cell death were correlated for individual cells after single-pulse irradiation. The threshold radiant exposures for cell death (ED(50,d)) and cavitation (ED(50,c)) increased with pulse duration and were approximately equal for pulses of up to 10 micros. For 20 micros, the ED(50,d) was about 10% lower than the ED(50,c); the difference increased with 40-micros pulses. Cells were killed predominantly by cavitation (up to 10-micros pulses); probability of thermally induced cell death without cavitation gradually increases with pulse duration. Threshold measurements are discussed by modeling the temperature distribution around laser-heated melanosomes and the scattering function from the resulting cavitation. Detection of intracellular cavitation is a highly sensitive method that can potentially provide real-time assessment of RPE damage during selective laser targeting.

PURPOSE. Selective targeting of the retinal pigment epithelium (RPE) with repetitive laser pulses that minimize thermal damage to the adjacent photoreceptors is a promising new therapeutic modality for RPE-related retinal diseases. The selectivity of an alternative, more versatile scanning approach was examined in vivo by using a broad range of scanning parameters. METHODS. Acousto-optic deflectors repeatedly scanned the focus of a continuous wave (cw)-laser across the retina of Dutch belted rabbits, producing microsecond irradiation at each RPE cell. Two irradiation patterns forming separated lines (SEP) or interlaced lines (INT), different dwell times (2.5‐75 s), and repetition numbers (10 and 100 scans with 100-Hz repetition rate) were tested. Thresholds were evaluated by fundus imaging and angiography. Histology was performed for selected parameters. RESULTS. Selective RPE cell damage was obtained with moderate laser power. The angiographic threshold power decreased with pulse duration, number of exposures, and applying the INT pattern. Ophthalmoscopic thresholds, indicating onset of thermal coagulation, were higher than twice the angiographic threshold for most tested parameters. Histology confirmed selective RPE cell damage for SEP irradiation with 7.5 and 15 s; slower scan speeds or closed lines caused photoreceptor damage. CONCLUSIONS. A cw-laser scanner can be set up as a highly compact and versatile device. Selective RPE damage is feasible with dwell times up to 15 s. Greatest selectivity is achieved with short exposure times and separated scan lines. Interlaced lines and long exposure times facilitate heat conduction into photoreceptors. A scanner is an attractive alternative for pulsed selective targeting, because both selective targeting and thermal photocoagulation can be realized. (Invest Ophthalmol Vis Sci. 2007;48:1782‐1792) DOI:10.1167/iovs.06-0797

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.

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α1 subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α1-deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α1 and β1 subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.

Selective targeting of the retinal pigment epithelium (RPE) is a new strategy for treating certain retinal disorders while preserving adjacent photoreceptors. The treatment currently relies on a complex laser system to produce the required microsecond pulse structure. In our new approach, we scan the focus of a continuous-wave (cw) laser beam with acousto-optic deflectors to produce microsecond-long exposures at each RPE cell. Experiments were performed in vitro with a bench-top scanner on samples of young bovine RPE and in vivo on Dutch belted rabbits with a slit-lamp adapted scanner. Effective dose 50% (ED50) for RPE damage was determined in vitro by fluorescence cell viability assay and in vivo by fluorescein angiography. Damage to individual RPE cells was achieved with laser power on the order of 100 mW. Using separated scan lines, we demonstrate selectivity in the form of alternating lines of dead and surviving cells that resemble the scan pattern. Selectivity is also shown by the absence of retinal thermal coagulation in vivo. Selective RPE damage is feasible by rapidly scanning a cw laser beam. The scanning device is an attractive alternative to conventional laser coagulation and pulsed laser targeting of the RPE.