Flexible solid immersion meniscus lens (SIMlens) approach for enhancing biological imaging of cleared samples.

Tissue-clearing techniques have revolutionized the field of biological imaging by rendering biological specimens transparent and enabling inside optical detection. Light-sheet fluorescence microscopy (LSFM) is a powerful tool for three-dimensional imaging of large biological samples. Combining tissue-clearing techniques with LSFM has advanced the efficient 3D visualization of these samples. A crucial challenge with LSFM is the requirement for the objective to operate within the clearing reagent, which can cause aberrations. To address this issue, we introduce a novel, to our knowledge, approach for the flexible design of the solid immersion refractive meniscus lens (SIMlens), facilitating the use of air objectives with cleared samples. Compared to the previous SIMlens, this method not only eliminates aberrations but also offers customized options for enhancing the numerical aperture and working distance of the objective lens, achieving at least a 10% improvement. We have demonstrated the feasibility of this new method using mouse brain samples.

Efficient 3D imaging and pathological analysis of the human lymphoma tumor microenvironment using light-sheet immunofluorescence microscopy.

Rationale: The composition and spatial structure of the lymphoma tumor microenvironment (TME) provide key pathological insights for tumor survival and growth, invasion and metastasis, and resistance to immunotherapy. However, the 3D lymphoma TME has not been well studied owing to the limitations of current imaging techniques. In this work, we take full advantage of a series of new techniques to enable the first 3D TME study in intact lymphoma tissue. Methods: Diverse cell subtypes in lymphoma tissues were tagged using a multiplex immunofluorescence labeling technique. To optically clarify the entire tissue, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+), clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) and stabilization to harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD) were comprehensively compared with the ultimate dimensional imaging of solvent-cleared organs (uDISCO) approach selected for clearing lymphoma tissues. A Bessel-beam light-sheet fluorescence microscope (B-LSFM) was developed to three-dimensionally image the clarified tissues at high speed and high resolution. A customized MATLAB program was used to quantify the number and colocalization of the cell subtypes based on the acquired multichannel 3D images. By combining these cutting-edge methods, we successfully carried out high-efficiency 3D visualization and high-content cellular analyses of the lymphoma TME. Results: Several antibodies, including CD3, CD8, CD20, CD68, CD163, CD14, CD15, FOXP3 and Ki67, were screened for labeling the TME in lymphoma tumors. The 3D imaging results of the TME from three types of lymphoma, reactive lymphocytic hyperplasia (RLN), diffuse large B-cell lymphoma (DLBCL), and angioimmunoblastic T-cell lymphoma (AITL), were quantitatively analyzed, and their cell number, localization, and spatial correlation were comprehensively revealed. Conclusion: We present an advanced imaging-based method for efficient 3D visualization and high-content cellular analysis of the lymphoma TME, rendering it a valuable tool for tumor pathological diagnosis and other clinical research.

Detection of Choroidal Neovascularization Using Optical Tissue Transparency.

Purpose: Optical tissue transparency (OTT) provides a tool for visualizing the entire tissue block. This study provides insights into the potential value of OTT with light-sheet fluorescence microscopy (LSFM) in detecting choroidal neovascularization (CNV) lesions. Methods: OTT with LSFM, hematoxylin and eosin (H&E) staining of paraffin sections, choroidal flatmount immunofluorescence, and optical coherence tomography angiography (OCTA) were used to obtain images of CNV. We determined the rate of change as (Data of week 1 - Data of week 2)/Data of week 1 × 100%. Finally, we compared the rate of change acquired from OTT with LSFM and the other methodologies. Results: We found that OTT with LSFM can realize three-dimensional (3D) visualizations of the entire CNV. The results showed that the decline in the rate of change from week 1 to week 2 after laser photocoagulation was 33.05% with OTT, 53.01% with H&E staining, 48.11% with choroidal flatmount, 24.06% with OCTA (B-scan), 18.08% with OCTA (en face), 10.98% with OCTA (3D reconstruction), and 7.74% with OCTA (vessel diameter index). Conclusions: OTT with LSFM will continue to be an invaluable resource for investigators to detect more visualized and quantified information regarding CNV. Translational Relevance: OTT with LSFM now serves as a tool for detecting CNV in mice, and it may undergo human clinical trials in the future.

TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability.

For comprehensive studies of the brain structure and function, fluorescence imaging of the whole brain is essential. It requires large-scale volumetric imaging in cellular or molecular resolution, which could be quite challenging. Recent advances in tissue clearing technology (e.g. CLARITY, PACT) provide new solutions by homogenizing the refractive index of the samples to create transparency. However, it has been difficult to acquire high quality results through immunofluorescence (IF) staining on the cleared samples. To address this issue, we developed TSA-PACT, a method combining tyramide signal amplification (TSA) and PACT, to transform samples into hydrogel polymerization frameworks with covalent fluorescent biomarkers assembled. We show that TSA-PACT is able to reduce the opacity of the zebrafish brain by more than 90% with well-preserved structure. Compared to traditional method, TSA-PACT achieves approximately tenfold signal amplification and twofold improvement in signal-to-noise ratio (SNR). Moreover, both the structure and the fluorescent signal persist for at least 16 months with excellent signal retention ratio. Overall, this method improves immunofluorescence signal sensitivity, specificity and stability in the whole brain of juvenile and adult zebrafish, which is applicable for fine structural analysis, neural circuit mapping and three-dimensional cell counting.

The effects of platform motion and target orientation on the performance of trackball manipulation.

The trackball has been widely employed as a control/command input device on moving vehicles, but few studies have explored the effects of platform motion on its manipulation. Fewer still have considered this issue in designing the user interface and the arrangement of console location and orientation simultaneously. This work describes an experiment carried out to investigate the performance of trackball users on a simple point-and-click task in a motion simulator. By varying the orientation of onscreen targets, the effect of cursor movement direction on performance is investigated. The results indicate that the platform motion and target orientation both significantly affect the time required to point and click, but not the accuracy of target selection. The movement times were considerably longer under rolling and pitching motions and for targets located along the diagonal axes of the interface. Subjective evaluations carried out by the participants agree with these objective results. These findings could be used to optimise console and graphical menu design for use on maritime vessels. STATEMENT OF RELEVANCE: In military situations, matters of life or death may be decided in milliseconds. Any delay or error in classification and identification will thus affect the safety of the ship and its crew. This study demonstrates that performance of manipulating a trackball is affected by the platform motion and target orientation. The results of the present study can guide the arrangement of consoles and the design of trackball-based graphical user interfaces on maritime vessels.

Minutes-timescale 3D isotropic imaging of entire organs at subcellular resolution by content-aware compressed-sensing light-sheet microscopy.

Rapid 3D imaging of entire organs and organisms at cellular resolution is a recurring challenge in life science. Here we report on a computational light-sheet microscopy able to achieve minute-timescale high-resolution mapping of entire macro-scale organs. Through combining a dual-side confocally-scanned Bessel light-sheet illumination which provides thinner-and-wider optical sectioning of deep tissues, with a content-aware compressed sensing (CACS) computation pipeline which further improves the contrast and resolution based on a single acquisition, our approach yields 3D images with high, isotropic spatial resolution and rapid acquisition over two-order-of-magnitude faster than conventional 3D microscopy implementations. We demonstrate the imaging of whole brain (~400 mm3), entire gastrocnemius and tibialis muscles (~200 mm3) of mouse at ultra-high throughput of 5~10 min per sample and post-improved subcellular resolution of ~ 1.5 µm (0.5-µm iso-voxel size). Various system-level cellular analyses, such as mapping cell populations at different brain sub-regions, tracing long-distance projection neurons over the entire brain, and calculating neuromuscular junction occupancy across whole muscle, are also readily accomplished by our method.

Effects of translational and rotational motions and display polarity on visual performance.

This study investigated effects of both translational and rotational motion and display polarity on a visual identification task. Three different motion types--heave, roll, and pitch--were compared with the static (no motion) condition. The visual task was presented on two display polarities, black-on-white and white-on-black. The experiment was a 4 (motion conditions) x 2 (display polarities) within-subjects design with eight subjects (six men and two women; M age = 25.6 yr., SD = 3.2). The dependent variables used to assess the performance on the visual task were accuracy and reaction time. Motion environments, especially the roll condition, had statistically significant effects on the decrement of accuracy and reaction time. The display polarity was significant only in the static condition.

Assessment of human color discrimination based on illuminant color, ambient illumination and screen background color for visual display terminal workers.

Human performance on color discrimination in visual display terminals may be affected by illuminant colors, the level of ambient illumination and background colors of the monitor. Few studies have focused on this topic. This study investigated human color discrimination ability in a simulated control room. Ten subjects were recruited as participants to perform a series of experimental tasks. A complete factorial (2 x 3 x 3) within-subject design was used. The independent variables were three illuminant colors (red, blue, and white), two ambient illumination levels (50 lux and 300 lux), and three background colors (black, blue and brown); the three dependent variables were the color discrimination ability (error scores), completion time and subject preference. The results showed that the illuminant colors and the screen background colors both significantly influenced human color discrimination ability (p<0.01). The result of this research can be used in control room design when considering the effect of color.

Effects of VDT workstation lighting conditions on operator visual workload.

Industrial lighting covers a wide range of different characteristics of working interiors and work tasks. This study investigated the effects of illumination on visual workload in visual display terminal (VDT) workstation. Ten college students (5 males and 5 females) were recruited as participants to perform VDT signal detection tasks. A randomized block design was utilized with four light colors (red, blue, green and white), two ambient illumination levels (20 lux and 340 lux), with the subject as the block. The dependent variables were the change of critical fusion frequency (CFF), visual acuity, reaction time of targets detection, error rates, and rating scores in a subjective questionnaire. The study results showed that both visual acuity and the subjective visual fatigue were significantly affected by the color of light. The illumination had significant effect on CFF threshold change and reaction time. Subjects prefer to perform VDT task under blue and white lights than green and red. Based on these findings, the study discusses and suggests ways of color lighting and ambient illumination to promote operators' visual performance and prevent visual fatigue effectively.

A study for safety and health management problem of semiconductor industry in Taiwan.

The main purpose of this study is to discuss and explore the safety and health management in semiconductor industry. The researcher practically investigates and interviews the input, process and output of the safety and health management of semiconductor industry by using the questionnaires and the interview method which is developed according to the framework of the OHSAS 18001. The result shows that there are six important factors for the safety and health management in Taiwan semiconductor industry. 1. The company should make employee clearly understand the safety and health laws and standards. 2. The company should make the safety and health management policy known to the public. 3. The company should put emphasis on the pursuance of the safety and health management laws. 4. The company should prevent the accidents. 5. The safety and health message should be communicated sufficiently. 6. The company should consider safety and health norm completely.