Laser-assisted targeted gene delivery to degenerated retina improves retinal function

Delivery of therapeutic genes into retina is proving to reverse degeneration and restore vision, however, viral vector-based gene delivery is prone to immunorejection, inflammatory/immune-response and nontargeted. Here, we report nonviral gene delivery and expression of opsin encoding genes in mouse retina in-vitro and in-vivo by use of pulsed femtosecond laser microbeam. In-vitro patch-clamp recording of the opsin-sensitized retinal cells and visually evoked in-vivo electrical recording from laser-transfected eye of mouse with degenerated retina showed functional response. The ultrafast laser-based naked gene delivery showed minimal damage and reliable expression of therapeutic opsin in cell membrane of the selected cells and in targeted retinal region. Laser-based “naked DNA gene therapy” in a spatially targeted manner will pave the way for treatment of inherited retinal diseases.     

Cytoprotection by endogenous zinc in the vertebrate retina

Our recent studies have shown that endogenous zinc, co‐released with glutamate from the synaptic terminals of vertebrate retinal photoreceptors, provides a feedback mechanism that reduces calcium entry and the concomitant vesicular release of glutamate. We hypothesized that zinc feedback may serve to protect the retina from glutamate excitotoxicity, and conducted in vivo experiments on the retina of the skate (Raja erinacea) to determine the effects of removing endogenous zinc by chelation. These studies showed that removal of zinc by injecting the zinc chelator histidine results in inner retinal damage similar to that induced by the glutamate receptor agonist kainic acid. In contrast, when an equimolar quantity of zinc followed the injection of histidine, the retinal cells were unaffected. Our results are a good indication that zinc, co‐released with glutamate by photoreceptors, provides an auto‐feedback system that plays an important cytoprotective role in the retina.

     

In vivo optoretinography of phototransduction activation and energy metabolism in retinal photoreceptors

The objective of this study is to verify the anatomic correlate of the second (2nd) outer retina band in optical coherence tomography (OCT), and to demonstrate the potential of using intrinsic optical signal (IOS) imaging for concurrent optoretinography (ORG) of phototransduction activation and energy metabolism in stimulus activated retinal photoreceptors. A custom-designed OCT was employed for depth-resolved IOS imaging in mouse retina activated by a visible light flicker stimulation. The spatiotemporal properties of the IOS changes at the photoreceptor outer segment (OS) and inner segment (IS) were quantitatively evaluated. Rapid IOS change was observed at the OS almost right away, and the IOS at the IS was relatively slow. Comparative analysis indicates that the OS-IOS reflects transient OS deformation caused by the phototransduction activation, and IS-IOS might reflect the energy metabolism caused by mitochondria activation in retinal photoreceptors. The consistency of the distribution of the IS-IOS and the 2nd OCT band supports the IS ellipsoid (ISe), which has abundant mitochondria, as the signal source of the 2nd OCT band of the outer retina.     

Characterization of blood coagulation dynamics and oxygenation in ex‐vivo retinal vessels by fluorescence hyperspectral imaging

Blood coagulation mechanisms forming a blood clot and preventing hemorrhage have been extensively studied in the last decades. Knowing the mechanisms behind becomes very important particularly in the case of blood vessel diseases. Real‐time and accurate diagnostics accompanied by the therapy are particularly needed, for example, in diseases related to retinal vasculature. In our study, we employ for the first time fluorescence hyperspectral imaging (fHSI) combined with the spectral analysis algorithm concept to assess physical as well as functional information of blood coagulation in real‐time. By laser‐induced local disruption of retinal vessels to mimic blood leaking and subsequent coagulation and a proper fitting algorithm, we were able to reveal and quantify the extent of local blood coagulation through direct identification of the change of oxyhemoglobin concentration within few minutes. We confirmed and illuminated the spatio‐temporal evolution of the essential role of erythrocytes in the coagulation cascade as the suppliers of oxygenated hemoglobin. By additional optical tweezers force manipulation, we showed immediate aggregation of erythrocytes at the coagulation site. The presented fluorescence‐based imaging concept could become a valuable tool in various blood coagulation diagnostics as well as theranostic systems if coupled with the laser therapy.     

Osteoporosis evaluation through full developed speckle imaging

Osteoporosis is a disease characterized by bone mineral density reduction, weakening the bone structure. Its diagnosis is performed using ionizing radiation, increasing health risk. Optical techniques are safer, due to non‐ionizing radiation use, but limited to the analyses of bone tissue. This limitation may be circumvented in the oral cavity. In this work we explored the use of laser speckle imaging (LSI) to differentiate the sound and osteoporotic maxilla and mandible bones in an in vitro model. Osteoporosis lesions were simulated with acid attack. The samples were evaluated by optical profilometry and LSI, using a custom software. Two image parameters were evaluated, speckle contrast ration and patches ratio. With the speckle contrast ratio, it was possible to differentiate sound from osteoporotic tissue. From speckle patches ratio it was observed a negative correlation with the roughness parameter. LSI is a promissory technique for assessment of osteoporosis lesions on alveolar bone.     

LASER SAFETY THRESHOLDS FOR CETACEANS AND PINNIPEDS

An increase in the use of oceanographic lidar has raised concern over laser safety for marine mammals. We were able to address some of these concerns by combining information about current laser safety standards, retinal damage mechanisms for humans, and research on eye anatomy for humans, cetaceans, and pinnipeds. To estimate the irradiance at the retina, the image size at the retina and pupil diameter must be known. We estimate the smallest spot size using retinal resolution or visual acuity for six species of cetaceans and five species of pinnipeds. A sensitivity ratio was calculated for each species using the ratio of the irradiance at the retina of the marine mammal to the irradiance at the retina of humans. The sensitivity ratio was used to suggest exposure limits for the various species. Because the human eye is more sensitive than either the cetacean or pinniped eye, we conclude that laser energies that are eye-safe for humans will also be safe for marine mammals, and higher laser irradiances may be permissible if illumination of humans is avoided.     

A new model for laser-induced thermal damage in the retina

We describe a new model for laser-induced retinal damage. Our treatment is prompted by the failure of the traditional approach to accurately describe the image size dependence of laser-induced retinal injuries and by a recently reported study which demonstrated that laser injuries to the retina might not appear for up to 48 h post exposure. We propose that at threshold a short-duration, laser-induced, temperature rise melts the membrane of the melanosomes found in the pigmented retinal epithelial cells. This results in the generation of free radicals which initiate a slow chain reaction. If more than a critical number of radicals are generated then cell death may occur at a time much later than the return of the retina to body temperature. We show that the equations consequent upon this mechanism result in a good fit to the recent image size data although more detailed experimental data for rate constants of elementary reactions is still required. This paper contributes to the current understanding of damage mechanisms in the retina and may facilitate the development of new treatments to mitigate laser injuries to the eye. The work will also help minimize the need for further animal experimentation to set laser eye safety standards. Unemployed; no address available.     

Is oblique scanning laser ophthalmoscope applicable to human ocular optics? A feasibility study using an eye model for volumetric imaging

Oblique scanning laser ophthalmoscopy (oSLO) is a novel imaging modality to provide volumetric retinal imaging without depth sectioning over a large field of view (FOV). It has been successfully demonstrated in vivo in rodent eyes for volumetric fluorescein angiography (vFA). However, engineering oSLO for human retinal imaging is challenging because of the low numerical aperture (NA) of human ocular optics. To overcome this challenge, we implement optical designs to (a) increase the angle of the intermediate image under Scheimpflug condition, and (b) expand the magnification in the depth dimension with cylindrical lens to enable sufficient sampling density. In addition, we adopt a scanning‐and‐descaning strategy, resulting in a compact oSLO system. We experimentally show that the current setup can achieve a FOV of ~3 × 6 × 0.8 mm³, and the transverse and axial resolutions of 7 and 41 μm, respectively. This feasibility study serves an important step for future in vivo human retinal imaging.     

Pseudo‐real‐time retinal layer segmentation for high‐resolution adaptive optics optical coherence tomography

We present a pseudo‐real‐time retinal layer segmentation for high‐resolution Sensorless Adaptive Optics‐Optical Coherence Tomography (SAO‐OCT). Our pseudo‐real‐time segmentation method is based on Dijkstra's algorithm that uses the intensity of pixels and the vertical gradient of the image to find the minimum cost in a geometric graph formulation within a limited search region. It segments six retinal layer boundaries in an iterative process according to their order of prominence. The segmentation time is strongly correlated to the number of retinal layers to be segmented. Our program permits en face images to be extracted during data acquisition to guide the depth specific focus control and depth dependent aberration correction for high‐resolution SAO‐OCT systems. The average processing times for our entire pipeline for segmenting six layers in a retinal B‐scan of 496 × 400 and 240 × 400 pixels are around 25.60 and 13.76 ms, respectively. When reducing the number of layers segmented to only two layers, the time required for a 240 × 400 pixel image is 8.26 ms.     

The saltatory effect in vision.

Events in vision triggered by brief flashes in train prove to be strictly analogous to those generated in the skin by a succession of taps. A requisite condition for their appearance in vision is projection of the flashes into the peripheral retinal field. Experiments are described that establish the general relation between extent of saltatory leaping and degree of retinal eccentricity and between leaping and retinal subtense of stimulus patches. Additionally, a curious "dip" phenomenon and several qualitative experiments in which color has been used to "tag" the saltatory image are reported.     

Evaluation of a 345 nm Femtosecond Laser for Corneal Surgery with Respect to Intraocular Radiation Hazard

Purpose

We report our findings from a preclinical safety study designed to assess potential side effects of corneal ultraviolet femtosecond laser treatment on lens and retina.

Methods

Refractive lenticules (-5 dpt) with a diameter of 6 mm were created in the right cornea of eight Dutch Belted rabbits. Radiant exposure was 0.5 J/cm² in two animals and 18 J/cm² in six animals. The presence of lens opacities was assessed prior to and up to six months following laser application using Scheimpflug images (Pentacam, Oculus) and backscatter analysis (Opacity Lensmeter 702, Interzeag). Ganzfeld flash and flicker electroretinogram (ERG) recordings were obtained from both eyes prior to and up to six weeks following laser application. At the study endpoint, retinas were examined by light microscopy.

Results

Independent of energy dose applied, no cataract formation could be observed clinically or with either of the two objective methods used. No changes in ERG recordings over time and no difference between treated and untreated eye were detected. Histologically, retinal morphology was preserved and retinal pigment epithelium as well as photoreceptor inner and outer segments appeared undamaged. Quantitative digital image analysis did not reveal cell loss in inner or outer nuclear layers.

Conclusions

Our analysis confirms theoretical considerations suggesting that ultraviolet femtosecond laser treatment of the cornea is safe for intraocular tissues. Transmitted light including stray light induces no photochemical effects in lens or retina at energy levels much higher than required for the clinical purpose. These conclusions cannot be applied to eyes with pre-existing retinal damage, as these may be more vulnerable to light.     

Front Cover

This work studied two-photon collagen crosslinking (CXL) in human corneal lenticules induced by near infrared femtosecond laser, possessing the advantages of multiphoton absorption and near-infrared light. The corneal stiffness can be enhanced by up to 296% without significantly reducing corneal transparency (<3%), paving the way to treat ophthalmic disorders such as keratoconus by 3D CXL of in vivo human cornea with higher safety, precision and efficacy. Further details can be found in the article by Zhenzhou Cheng, Nan Zhang, Le Chang, Pengfei Qi, Lin Zhang, Lie Lin, Yan Wang, and Weiwei Liu ( e202200160 ).

     

High-speed volumetric imaging in vivo based on structured illumination microscopy with interleaved reconstruction

Wide-field fluorescence microscopy (WFFM) is widely adopted in biomedical studies, due to its high imaging speed over large field-of-views. However, WFFM is susceptible to out-of-focus background. To overcome this problem, structured illumination microscopy (SIM) was proposed as a wide-field, optical-sectioning technique, which needs multiple raw images for image reconstruction and thus has a lower imaging speed. Here we propose SIM with interleaved reconstruction, to make SIM of lossless speed. We apply this method in volumetric imaging of neural network dynamics in brains of zebrafish larva in vivo.     

Comparative study on the inactivation of MS2 and M13 bacteriophages using energetic femtosecond lasers

Femtosecond (fs) laser irradiation techniques are emerging tools for inactivating viruses that do not involve ionizing radiation. In this work, the inactivation of two bacteriophages representing protective capsids with different geometric constraints, that is, the near‐spherical MS2 (with a diameter of 27 nm) and the filamentous M13 (with a length of 880 nm) is compared using energetic visible and near‐infrared fs laser pulses with various energies, pulse durations, and exposure times. Intriguingly, the results show that inactivation using 400 nm lasers is substantially more efficient for MS2 compared to M13. In contrast, using 800 nm lasers, M13 was slightly more efficiently inactivated. For both viruses, the genome was exposed to a harmful environment upon fs‐laser irradiation. However, in addition to the protection of the genome, the metastable capsids differ in many properties required for stepwise cell entry that may explain their dissimilar behavior after (partial) disassembly. For MS2, the dominant mechanism of fs‐laser inactivation was the aggregation of the viral capsid proteins, whereas aggregation did not affect M13 inactivation, suggesting that the dominant mechanism of M13 inactivation was related to breaking of secondary protein links.     

A novel in vivo Cerenkov luminescence image‐guided surgery on primary and metastatic colorectal cancer

Intraoperative Cerenkov luminescence imaging (CLI) can effectively improve the performance of tumor surgery. Nevertheless, the existing approaches are still unsatisfying to the clinical demands of open surgery. This study develops a novel intraoperative in vivo CLI approach to investigate the potential and value of Cerenkov luminescence (CL) image‐guided surgery. A system characterized with high sensitivity (19.61 kBq mL^{?1 18}F‐FDG) and desirable spatial resolution (88.34 μm) is developed. CL image‐guided surgery is performed on colorectal cancer (CRC) models of mice and swine. Tumor surgery is guided by the static CL images, and the resection quality is evaluated quantitatively and contrasted with other imaging modalities exemplified by bioluminescence imaging (BLI). The in vivo results demonstrated the effectiveness of the proposed intraoperative CLI approach for removing primary and metastatic CRC. Safety of performing in vivo CL image‐guided surgery is verified as well through radiation measurements of related staffs. Overall, the developed intraoperative in vivo CLI approach can efficiently improve the cancer treatment.     

Issue Information

Photographs of Kulikovskiyia triundulata gen. et sp. nov. along with holotypic locality. Upper left: Photograph of the holotypic locality (lateritic rocky plateau of Maharashtra, India). Upper middle: Live cell showing chloroplast structure in valve view. Upper right: DIC image of a frustule in valve view. Lower: SEM image of the external valve showing peripheral and apical spines, siliceous slats and tri‐undulate valve shape. See Roy et al. in this issue. Cover picture from: Article link here

     

Optical radiation and the eyes with special emphasis on children

The Sun is the most abundant source of optical radiation for the child eye. New hand-held visible lasers are a threat to the child eye. Some scientific data suggest that near infrared radiation may cause cumulative damage in the ocular lens. The child eye usually is exposed to ambient solar radiation, gazing at the horizon. Ambient Sun ultraviolet radiation (UVR) exposure to the child is complex due to atmospheric scattering and strong dependence on background reflection. Solar exposure causes biological damage, only by photochemical mechanisms. UVR exposure to a child eye is mainly a threat to the anterior segment of the eye, but also age dependently to the retina. Above threshold exposure to UVR, for short delay onset of damage, causes a toxic reaction on the surface of the eye, snow blindness, and cataract. Sub-threshold daily exposure to UVR over decades is associated with several ocular surface pathologies and eye lid cancer. Visible radiation is a threat to the retina. A single above threshold exposure, for short delay onset of damage to the retina causes immediate photochemical Type II retinal damage, Sun blindness. A single exposure of the retina to a very high intensity laser beam may cause thermal or thermo-mechanical damage in the retina. In environments with high irradiance of optical radiation, the child eye should be protected. Legislation and public information is required for avoidance of damage from high intensity laser systems. More research is urgently needed to exclude the potential hazard of near infrared radiation.     

The effects of transcranial laser photobiomodulation and neuromuscular electrical stimulation in the treatment of post-stroke dysfunctions

Post-stroke sequelae includes loss functions, such as cognitive and sensory-motor which lead to emotional and social problems, reducing quality of life and well-being. The main aim of our study was to investigate the effects of transcranial laser photobiomodulation together with neuromuscular electrical stimulation (NMES) in post-stroke patients. We performed a clinical trial and an ex vivo study. For the clinical trial, hemiplegic patients were separated into two groups: Treated Group (TG): Hemiplegics treated with transcranial laser (on) associated with NMES (on) and; Placebo Group (PG): Hemiplegics treated with placebo transcranial laser (off) associated with NMES (on). The cluster prototype includes 12 diode laser beams (4 × 660 nm, 4 × 808 nm and 4 × 980 nm) with average power of 720 mW per cluster applied during one minute, leading to 43.2 J energy per cluster. Fifteen regions for all head were irradiated by cluster, leading to 648 J energy per session. The parameters of NMES of the paretic limbs to generate extension wrist and ankle dorsiflexion were symmetrical biphasic rectangular waveforms, 50 Hz frequency, 250 μs pulse duration, and adjustable intensity to maintain the maximum range of motion (amplitude between 0 and 150 mA). Our clinical trial showed improvement of cognitive function, pain relief, greater manual dexterity, enhancement of physical and social–emotional health which lead to better quality of life and well-being. There was also increased temperature in the treated regions with laser and NMES. For the ex vivo study, the distribution of infrared and red radiation after penetration through the cranium and hemihead of cadavers were showed. Therefore, transcranial laser photobiomodulation associated with NMES can be an important therapeutic resource for rehabilitation after stroke.     

Lightsheet fluorescence lifetime imaging microscopy with wide‐field time‐correlated single photon counting

We report on wide‐field time‐correlated single photon counting (TCSPC)‐based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single‐photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide‐field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.