Laser Scanning Research Articles

Gel-Gel Phase Separation in Clustered Poly(ethylene glycol) Hydrogel with Enhanced Hydrophobicity.

The development of hydrophobic poly(ethylene glycol) (PEG) hydrogels, which are typically hydrophilic, could significantly enhance their application as artificial extracellular matrices. In this study, we designed PEG hydrogels with enhanced hydrophobicity through gel-gel phase separation (GGPS), a phenomenon that uniquely enhances hydrophobicity under ambient conditions, and we elucidated the pivotal role of elasticity in this process. We hypothesized that increased elasticity would amplify GGPS, thereby improving the hydrophobicity and cell adhesion on PEG hydrogel surfaces, despite their inherent hydrophilicity. To test this hypothesis, we engineered dilute oligo-PEG gels via a two-step process, creating polymer networks from tetra-PEG clusters with multiple reaction points. These oligo-PEG gels exhibited significantly higher elasticity, turbidity, and shrinkage upon water immersion compared to dilute PEG gels. Detailed characterization through confocal laser scanning microscopy, rheological measurements, and cell adhesion assays revealed distinct biphasic structures, increased hydrophobicity, and enhanced cell attachability in the dilute oligo-PEG gels. Our findings confirm that elasticity is crucial for effective GGPS, providing a novel method for tailoring hydrogel properties without chemical modification. This research introduces a new paradigm for designing biomaterials with improved cell-scaffolding capabilities, offering significant potential for tissue engineering and regenerative medicine.

Static and dynamic open loop control of selective laser flash sintering conducted with direct current electric fields

AbstractSelective laser flash sintering utilizes a scanning laser as a heat source to locally initiate flash sintering in the regions scanned by the laser. A key challenge towards utilizing this process for additive manufacturing (AM) is the induced electrical current that arises during flash sintering. With traditional scan patterns conducted with static electric fields, electrical and thermal runaway and associated thermal shock cracking occur. In this study, several open‐loop control strategies with static and dynamic applied electric fields were utilized to control peak currents. These strategies were shown to be effective in reducing peak currents to below 1.0 µA and reducing the severity of, but not completely eliminating cracking. Alternative strategies are suggested that could lead to complete elimination of cracks.

Reducing Laser Exposure Time of PBF-LB/P by Providing High Energy Without Thermal Degradation and Its Effect on Part Strength and Process Time

Abstract One of the most promising additive manufacturing technologies for the production of end-use parts is powder bed fusion of polymer with laser beam (PBF-LB/P). This technology can reduce production costs by increasing process efficiency and production speed. As PBF-LB/P is a layer-wise additive manufacturing process, the production speed can be increased by reducing the layering time. Although some operations such as recoating are performed during the layering process, considerable time is spent on laser scanning. To reduce the laser exposure time while maintaining proper powder melting, a high-power beam should be irradiated to the powder layer to prevent energy shortage. However, as the laser beam power increases, the irradiance at the beam center increases significantly, causing powder degradation such as thermal decomposition or sublimation. In this study, an appropriate input energy range was determined by obtaining an input energy limit that does not cause powder deterioration via experimental observations and temperature estimations during the process. Furthermore, the influence of the scanning parameters on the mechanical properties of built specimens was investigated to reduce the layering time within an indicated range. Results show that the mechanical strength of the built parts decreases slightly as the scan spacing increases following the expansion of the beam diameter. This study also validated the effects of scanning parameters on layering time. As a result, by doubling the scan speed and spacing, the layering time can be reduced by up to 1/3.

A precise registration method for large-scale urban point clouds based on phased and spatial geometric features

Abstract The dense high-rise buildings and multipath effects in urban areas significantly reduce the positioning signal accuracy of laser scanning systems, leading to layering and offset issues in the collected point cloud data on the same road. In order to acquire comprehensive and consistent three-dimensional information on the objects, thereby providing field inspection data for large-scale road traffic network scenarios, in this paper, an improved point cloud registration method is proposed to divide the registration process into two stages: elevation registration and plane registration. Elevation registration takes the ground point cloud as the registration primitive, reduces the number of point clouds through curvature down-sampling, and constrains the feature point sequence with a fixed range to provide a good initial pose for fine registration. The plane registration first inherits the elevation registration parameters, combining the dynamic distance parameters of spherical region step based on the median, using robust multi-scale loss functions to address residual points, effective adjacent point pairs are selected to obtain the spatial transformation matrix, and realizes accurate registration. Experimental results with multiple sets of urban point cloud data show that the root mean square error of point cloud registration can be controlled within 0.06 m, achieving a relatively superior registration accuracy, it can provide detailed prior data for measurement information analysis.

Transdermal Drug Delivery Systems: Different Generations and Dermatokinetic Assessment of Drug Concentration in Skin.

Transdermal drug delivery systems (TDDS) are a highly appealing and innovative method of administering drugs through the skin, as it enables the drugs to achieve systemic effects. A TDDS offers patient convenience, avoids first-pass hepatic metabolism, enables local targeting, and reduces the toxic effect of drug. This review details several generations of TDDS and the advancements made in their development to address the constraints associated with skin delivery systems. Transdermal delivery methods of the first generation have been consistently growing in their clinical application for administering small, lipophilic, low-dose drugs. Second-generation TDDS, utilizing chemical enhancers and iontophoresis, have led to the development of clinical products. Third-generation delivery systems employ microneedles, thermal ablation, and electroporation to specifically target the stratum corneum, which is the skin's barrier layer. Dermatokinetics is the study of the movement of drugs and formulations applied to the skin over a period of time. It provides important information regarding the rate and extent to which drugs penetrate skin layers. Several dermatokinetic techniques, including tape stripping, microdialysis, and laser scanning microscopy, have been used to study the intricate barrier properties and clearance mechanisms of the skin. This understanding is essential for developing and improving effective TDDS.

Direct formation of pure copper layer on aluminum nitride by multibeam laser deposition with blue diode lasers

A demand of power module devices has been increasing for highly energy efficient social system. The insulated heat sink printed circuit board is the key component of power module devices, which is composed of copper and aluminum nitride (AlN) due to high thermal conductivity. The conventional method of the joining between copper and AlN is active metal brazing method, but there is an issue that the high heat treatment of this method generates thermal residual stress on the board and decrease the reliability. For that reason, the development of a direct bonding process between copper and AlN has been demanded. Therefore, we focused on the laser metal deposition (LMD) method, which is a selective heating and microfabrication process. The LMD method is a method in which a powder material is supplied to the processing point and irradiated with a laser to melt the powder and form layer on the surface of the substrate. Then, a blue diode laser with a wavelength of 450 nm, which has a high absorptivity of 60% for copper, is used as a heat source. In addition, the multibeam type LMD system has been installed for the uniform heating of powder. In this study, the laser power density, laser scanning speed, and powder feeding rate were changed to explore the conditions for forming a copper layer on the AlN substrate.

Filling the Holes on 3D Heritage Object Surface Based on Automatic Segmentation Algorithm

ABSTRACTReconstructing and processing 3D objects are activities in the research field of computer graphics, image processing and computer vision. The 3D objects are processed based on geometric modelling (a branch of applied mathematics and computational geometry) or machine learning algorithms based on image processing. The computation of geometrical objects includes processing the curves and surfaces, subdivision, simplification, meshing, holes filling or reconstructing the 3D surface's objects on both point cloud data and triangular mesh. While the machine learning methods are developed using deep learning models. With the support of 3D laser scan devices and LiDAR techniques, the obtained dataset is close to the original shape of the real objects. Besides, photography and its application based on modern techniques in recent years help us collect data and process the 3D models more precisely. This article proposes a new method for filling holes on the 3D object's surface based on automatic segmentation. Instead of filling the hole directly as the existing methods, we now subdivide the hole before filling it. The hole is first determined and segmented automatically based on the computation of its local curvature. It is then filled on each part of the hole to match its local curvature shape. The method can work on both 3D point cloud surfaces and triangular mesh surfaces. Compared to the state‐of‐the‐art (SOTA) methods, our proposed method obtained higher accuracy of the reconstructed 3D objects.

Morphological Analysis and Bond Strength to Root Canal Dentin of Endodontically Treated and Retreated Teeth: An Ex Vivo Study.

To assess the bond strength and the hybrid layer (HL) micro-morphological characteristics at the cement-dentin interface (CD-i) between root canal walls and two adhesive resin cements [self-etch (SERc) and self-adhesive (SARc)] in root-canal-treated (RCT) and naturally aged retreated teeth (RCR-T). Vital (n = 16) and RCT (n = 16) teeth were, respectively, endodontically treated or retreated. Fiber posts were luted either with SERc (Clearfil Universal Bond Quick + DC Core Plus) or SARc (iCEM). Samples were then sectioned into 1 mm thick slices perpendicular to the fiber post and submitted to push-out bond strength test. Vital (n = 4) and RCT (n = 4) first maxillary molars were also selected and prepared to evaluate CD-i morphology through confocal laser scanning microscopy (CLSM). Three-way analysis of variance (ANOVA) and Tukey post-hoc tests were assessed to statistically analyze the obtained data (p 0.05). Bond strength was significantly jeopardized in retreated teeth and in the root apical half, while the cements had no significant influence. Most failures occurred between dentin and resin cement. HL thickness was also hindered in retreated teeth. iCEM produced a thinner HL compared to SERc. Resin tag formation was significantly hampered in the root apical half. SARc performed as well as SERc on aged RCT radicular dentin. Clinicians can rely on simplified one-step luting systems when adhesion is required in unfavorable substrates such as the root canal post space of aged RCT teeth.

Design of Aerated Oleogel-Hydrogel Mixtures for 3D Printing of Personalized Cannabis Edibles.

Cannabis seed oil oleogel structured with Glycerol Monostearate (20% w/w) was mixed with xanthan gum hydrogel (2% w/w) at different ratios ranging from 0% w/w hydrogel to 75% w/w hydrogel, using a syringe-to-syringe apparatus, for the preparation of 3D-printable food inks. This process enabled the simultaneous blend of oleogel and hydrogel phases and the incorporation of air in a reproducible and accurate manner. The printability of bigel inks with different mass ratios was evaluated by using a conventional benchtop food 3D printer. The printability of the inks was found to be negatively affected by the presence of higher portions of the hydrogel phase, while the printing performance of pure cannabis seed oil oleogel was superior compared to the printing performance of the bigel inks. The physicochemical properties of hybrid gels were investigated with rheological studies, thermophysical studies (Differential Scanning Calorimetry), Polarized Light Microscopy, and Confocal Laser Scanning Microscopy. The microstructure of the aerated inks was affected by the presence of a higher oleogel fraction, in terms of air bubble shape and distribution. The addition of hydrogel at concentrations higher than 50% w/w had a strong negative effect on the mechanical properties of the inks leading to a partial collapse of the printed structures and subsequently to poor printing performance.

Optimization and Preparation of Ultrasound-Treated Whey Protein Isolate Pickering Emulsions.

This study aimed to create Pickering emulsions with varying oil fractions and assess the impact of ultrasonic treatment on the properties of Whey Protein Isolates (WPIs). At 640 W for 30 min, ultrasound reduced WPI aggregate size, raised zeta potential, and improved foaming, emulsifying, and water-holding capacities. FTIR analysis showed structural changes, while fluorescence and hydrophobicity increased, indicating tertiary structure alterations. This suggests that sonication efficiently modifies WPI functionality. Under ideal conditions, φ = 80 emulsions were most stable, with no foaming or phase separation. Laser scanning revealed well-organized emulsions at φ = 80. This study provides a reference for modifying and utilizing WPI.

Change in components by pretreatments alters the antimicrobial activity of Strobilanthes cusia biomass

The plant Strobilanthes cusia contains various antibacterial components, but the mechanisms behind its antimicrobial activity are not yet fully understood. This study demonstrates that alterations in components due to different pretreatments significantly impacted the antimicrobial activity of S. cusia biomass. Biomass dried using microwave pretreatment, which contained a high amount of indican, exhibited the lowest antibacterial activity, whereas heat-dried biomass without indican showed the highest antibacterial activity. High-performance liquid chromatography analysis indicated that pretreatments influenced the ratios of indican, indoxyl, indigo, and indirubin in S. cusia biomass, likely due to the effect of pretreatments on β-glucosidase activity. Extracts with higher levels of indoxyl, an intermediate product of indican hydrolysis, exhibited stronger inhibitory effects against Staphylococcus aureus. Indoxyl increased the NAD+/NADH ratio, which led to elevated hydrogen peroxide generation and subsequent cell damage. Furthermore, transmission electron microscopy, scanning electron microscopy, and confocal laser scanning microscopy revealed distinct morphological changes in cells exposed to indoxyl. Finally, leveraging the water-soluble properties of indoxyl, we propose a novel approach using microwave drying assisted by β-glucosidase to enhance the antimicrobial activity of S. cusia biomass. These findings provide a theoretical and methodological foundation for the evaluation and future application of S. cusia's bioactive substances.

Laser Powder Bed Fusion Parameters Optimization for Enhanced Mechanical Properties of EOS Co-Cr Dental Alloy.

This research explores how variations in laser powder bed fusion (LPBF) parameters-laser power (P), scanning speed (v), and base plate preheating temperature (ϑp)-affect the mechanical properties of the EOS Co-Cr SP2 dental alloy. A central composite design (CCD) was used to optimize the process parameters. Mechanical testing focused on crucial properties for dental applications, including yield strength (Rp0.2), elongation (ε), toughness (KVa), and flexural strength (Rms). Microstructural analysis was conducted using light and electron microscopy, while XRD identified microstructural phases. Statistical analysis (ANOVA, Scheffé post hoc test, α = 0.05) revealed significant effects of P, v, and ϑp on the mechanical properties. Response surface models (RSMs) were developed, and optimal parameters were determined to achieve maximum toughness and flexural strength. Maximum values were obtained with laser power above 205 W and base plate preheating at 310 °C. The mathematical model predicted toughness values with less than 5% deviation from experimental results, indicating high accuracy.

Photo-responsive Pickering emulsions triggered by in-situ pH modulation using a photoacid generator

HypothesisPickering emulsions that respond to changes in pH by the addition of acid or alkali have been extensively studied, but the development of photo-responsive Pickering emulsions has been more challenging. This study attempts to demonstrate a novel approach to achieve photo-responsiveness in Pickering emulsions by incorporating a photoacid generator (PAG) into the oil phase. Upon UV irradiation, the PAG is expected to release protons (H+), which can then regulate the pH of the emulsion system and control its stability. ExperimentsAmphiphilic colloidal particles obtained by modifying silica particles with poly (2-(dimethylamino)ethyl methacrylate) (SiO2-PDMAEMA) are used to stabilize the Pickering emulsions. The protonation and deprotonation of the SiO2-PDMAEMA particles at different pH values allow for the tuning of emulsion stability. By introducing the PAG into the stable Pickering emulsion system and applying UV irradiation to trigger the in-situ release of H+, the pH of the emulsion is systematically decreased, and the corresponding changes in emulsion stability are investigated. FindingsThe results show that UV irradiation alone cannot induce emulsion instability. However, when PAG is added to the oil phase, the Pickering emulsions exhibit a significant decrease in pH under UV irradiation, ultimately leading to emulsion destabilization and phase separation. At a UV intensity of 20 mW/cm2 for 2 min, the H+ release from the PAG significantly lower the emulsion’s pH, causing the SiO2-PDMAEMA particles to detach from the oil–water interface and resulting in emulsion instability. Higher concentrations of SiO2-PDMAEMA particles in the emulsion require more PAG to induce instability, as confirm by confocal laser scanning microscopy (CLSM) image. This study presents a versatile approach to develop photo-responsive Pickering emulsions which can have potential applications in areas such as drug delivery, cosmetics, and responsive materials.

Enhancing the Precision of Forest Growing Stock Volume in the Estonian National Forest Inventory with Different Predictive Techniques and Remote Sensing Data

Estimating forest growing stock volume (GSV) is crucial for forest growth and resource management, as it reflects forest productivity. National measurements are laborious and costly; however, integrating satellite data such as optical, Synthetic Aperture Radar (SAR), and airborne laser scanning (ALS) with National Forest Inventory (NFI) data and machine learning (ML) methods has transformed forest management. In this study, random forest (RF), support vector regression (SVR), and Extreme Gradient Boosting (XGBoost) were used to predict GSV using Estonian NFI data, Sentinel-2 imagery, and ALS point cloud data. Four variable combinations were tested: CO1 (vegetation indices and LiDAR), CO2 (vegetation indices and individual band reflectance), CO3 (LiDAR and individual band reflectance), and CO4 (a combination of vegetation indices, individual band reflectance, and LiDAR). Across Estonia’s geographical regions, RF consistently delivered the best performance. In the northwest (NW), the RF model achieved the best performance with the CO3 combination, having an R2 of 0.63 and an RMSE of 125.39 m3/plot. In the southwest (SW), the RF model also performed exceptionally well, achieving an R2 of 0.73 and an RMSE of 128.86 m3/plot with the CO4 variable combination. In the northeast (NE), the RF model outperformed other ML models, achieving an R2 of 0.64 and an RMSE of 133.77 m3/plot under the CO4 combination. Finally, in the southeast (SE) region, the best performance was achieved with the CO4 combination, yielding an R2 of 0.70 and an RMSE of 21,120.72 m3/plot. These results underscore RF’s precision in predicting GSV across diverse environments, though refining variable selection and improving tree species data could further enhance accuracy.

Stone instance segmentation of rubble masonry based on laser scanning point clouds

Laser scanning allows for objective area-based analysis of water dam structures to enable targeted interventions in case of displacements, requiring comparison of the same areas over different epochs. This comparison improves if identical areas, e.g. stones, from multiple epochs can be automatically derived in advance. We present an instance segmentation algorithm based on a 3D point cloud with reflected intensity information to identify individual stones in rubble masonry dams that can be used within deformation monitoring. The algorithm uses initial k-means classification followed by image-based processing steps, resulting in a segmented 3D point cloud with individual stones. It is evaluated against a manually labeled reference and analyzed on terrestrial laser scanning (TLS) and UAV-based data sets. Correctly identified stones are 89.92% for TLS and 90.82% for UAV data. Additionally, stone centroids and shapes were evaluated without significant deviation. A first outlook on deformation analysis was provided using ICP matching of stones from two simulated epochs.

Association of tear osmolarity and corneal nerves structure in dry eye disease: an in vivo study.

To evaluate the structural changes in corneal sub-basal nerves of dry eye disease (DED) patients with tear hyperosmolarity versus normosmolar tears. A prospective evaluation of the tear film (keratograph 5M), tear osmolarity, and sub-basal corneal nerves (laser scanning in-vivo confocal microscopy) was performed in a cohort of 53 DED patients (106 eyes) diagnosed as per DEWS II criteria. Patients with tear hyperosmolarity (Group 1, n = 48 eyes) were compared with DED patients without tear hyperosmolarity (Group 2, n = 58 eyes). Of 53 patients (27 females), 28 had Sjogren's syndrome, and the rest had meibomian gland dysfunction. There were more SS patients (21 vs 7) and females in Group 1. The two groups were similar in age, TMH, NIBUT, meibomian gland loss, bulbar redness, and corneal staining, except for Schirmer I (p < 0.001), and tear osmolarity (p < 0.001; worse in group 1). The groups did not differ in dendritic cell density, whether immature (53.8 vs. 38) or mature (2.7 vs. 0). The significantly different corneal nerve parameters were nerve fiber length (p = 0.005), density (p = 0.01), and branching density (p = 0.04), with lower values observed in group 1. Only tear osmolarity had a weak negative correlation with corneal nerve fiber length (r, -0.38), density (r, -0.32), and branching (r, -0.28). SS patients with hyperosmolar tears had reduced nerve fiber length and branching compared to SS patients with normosmolar tears. Tear hyperosmolarity is associated with reduced nerve branching, fiber density, and fiber length despite similar levels of conjunctival congestion, tear film stability, and meibomian gland loss in DED patients. What is known • Corneal nerves are reduced in density and length in dry eye disease patients. • Laboratory studies have shown fragmentation of corneal nerves on exposure to hyperosmolar solutions. What is new • Tear hyperosmolarity is associated with reduced nerve branching, fiber density, and fiber length in dry eyes compared to normosmolar tears. • The effect is independent of dendritic cell density.

Block copolymer micelles stabilized Pickering emulsions in templating ultralight conducting PEDOT: PSS elastomeric aerogels

Amphiphilic copolymers poly(N-(2-methacryloylxy) ethyl pyrrolidone)-block-poly(2-(perfluorooctyl) ethyl methacrylate) (PNMPm-b-PFMAn) with similar m/n of about 10 preferred to form spherical micelles in water regardless of the molecular weight. These PNMPm-b-PFMAn micelles as Pickering emulsifiers commonly favored the formation of O/W Pickering emulsions in the cyclohexane/water systems, and the corresponding interfacial structures were characterized by the confocal laser scanning microscopy (CLSM) method. Moreover, high internal phase Pickering emulsions (HIPPEs) were formed at high volume ratio of oil/water (VO/VW), which were excellent templates in generating structurally well-defined 3D porous PEDOT:PSS aerogels with low density and high conductivity. It was noticed that the HIPPE-templated PEDOT:PSS aerogels were mainly composed of hierarchically connected nano-/micro-level fibers, which were significantly different from the randomly intertangled lamellar micro-belts of PEDOT:PSS aerogels prepared by the hydrogel method under the same conditions. In addition, the HIPPE-templated stretchable and compressible PEDOT:PSS aerogels showed wide strain-invariant resistance (SIR) range and remarkable cycling stability, which were far superior to the hydrogel ones.

An Acoustic Sensor System to Measure Aeolian Ripple Morphology and Migration Rates.

Acoustic distance sensors have a long history of use to detect subaqueous bedforms. There have been few comparable applications for aeolian bedforms such as ripples. To address this, we developed a simple and reliable apparatus comprising a pair of distance sensors, a bracket upon which they are mounted, and a base upon which the bracket can slide. Our system relies on two Senix Corporation (Hinesburg, VT, USA), ToughSonic® model 14-TSPC-30S1-232 acoustic distance sensors: one to measure surface elevation changes (in this case, ripple morphology) and a second to measure horizontal location. The ToughSonic® vertical resolution was 0.22 mm and the horizontal scan distance was about 0.60 m with a locational accuracy of 0.22 mm. The measurement rate was 20 Hz, but we over-sampled at 1 KHz. Signal processing involves converting volts to meters, detrending the data, and removing noise. Analysis produces ripple morphologies and migration rates that conform with independent measurements. The advantages of this system relative to terrestrial laser scanning or structure from motion are described.

Nisin-loaded chitosan/sodium alginate microspheres enhance the antimicrobial efficacy of nisin against Staphylococcus aureus.

To develop and evaluate nisin-loaded chitosan/sodium alginate (CS/SA) microspheres as an improved antimicrobial delivery system targeting Staphylococcus aureus strains. The microspheres were prepared using a modified water-in-oil emulsion cross-linking method, resulting in spherical particles sized 1-8µm with a surface charge of -7.92±5.09mV, confirmed by scanning electron microscopy (SEM) and Zetasizer analysis. Encapsulation efficiency (EE) and loading capacity (LC) of nisin were 87.60±0.43% and 1.99±0.01%, respectively. In vitro release studies over 48 hours indicated a controlled release pattern of nisin, described by the Korsmeyer-Peppas model, with higher release rates at 37°C and alkaline pH. Antimicrobial assays showed an enhanced efficacy of nisin-loaded CS/SA microspheres compared to free nisin, with minimum inhibitory concentration (MIC) values reduced by 50%. Confocal laser scanning microscopy (CLSM), SEM and transmission electron microscopy (TEM) showed significant bacterial membrane damage and cellular disruption induced by the microspheres. This study highlights the potential of nisin-loaded CS/SA microspheres as an innovative antimicrobial delivery system with improved stability and antimicrobial efficacy against S. aureus, addressing limitations associated with nisin alone.

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy.

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and Müller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.