Increased Multiplexity in Optical Tissue Clearing-Based Three-Dimensional Immunofluorescence Microscopy of the Tumor Microenvironment by Light-Emitting Diode Photobleaching. | LitMetric
Department of Pharmaceutical Sciences, University of Illinois, Chicago, Chicago, Illinois; University of Illinois Cancer Center, University of Illinois Chicago, Chicago, Illinois. Electronic address:
Published: June 2024
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Article Abstract
Optical tissue clearing and three-dimensional (3D) immunofluorescence (IF) microscopy is transforming imaging of the complex tumor microenvironment (TME). However, current 3D IF microscopy has restricted multiplexity; only 3 or 4 cellular and noncellular TME components can be localized in cleared tumor tissue. Here we report a light-emitting diode (LED) photobleaching method and its application for 3D multiplexed optical mapping of the TME. We built a high-power LED light irradiation device and temperature-controlled chamber for completely bleaching fluorescent signals throughout optically cleared tumor tissues without compromise of tissue and protein antigen integrity. With newly developed tissue mounting and selected region-tracking methods, we established a cyclic workflow involving IF staining, tissue clearing, 3D confocal microscopy, and LED photobleaching. By registering microscope channel images generated through 3 work cycles, we produced 8-plex image data from individual 400 μm-thick tumor macrosections that visualize various vascular, immune, and cancer cells in the same TME at tissue-wide and cellular levels in 3D. Our method was also validated for quantitative 3D spatial analysis of cellular remodeling in the TME after immunotherapy. These results demonstrate that our LED photobleaching system and its workflow offer a novel approach to increase the multiplexing power of 3D IF microscopy for studying tumor heterogeneity and response to therapy.
Significance: Cellular metabolic dynamics can occur within milliseconds, yet there are no optimal tools to spatially and temporally capture these events. Autofluorescence imaging can provide metabolic information on the cellular level due to the intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD).
Aim: Our goal is to build and evaluate a widefield microscope optimized for rapid autofluorescence imaging of metabolic changes in cells.
, renowned for its large inflorescences and a diverse range of colors, highlights a significant limitation in current gene function research, which is the lack of effective molecular genetic tools. This study utilized a (TRV)-based virus-induced gene silencing (VIGS) system to investigate gene function through posttranscriptional gene silencing in for the first time. The ortholog of () in , termed , was identified.
Embryo quality assessment by optical imaging is increasing in popularity. Among available optical techniques, light sheet microscopy has emerged as a superior alternative to confocal microscopy due to its geometry, enabling faster image acquisition with reduced photodamage to the sample. However, previous assessments of photodamage induced by imaging may have failed to measure more subtle impacts.
Over the past years, fluorescence microscopy (FM) has steadily progressed in increasing the localization precision of fluorescent emitters in biological samples and led to new claims, whose rigorous validation remains an outstanding problem. We present a novel, to the best of our knowledge, multi-parameter estimation framework that captures the full complexity of a single-emitter FM localization experiment. We showcase our method with Minimum Flux (MINFLUX) microscopy, among the highest-resolution approaches, demonstrating that (i) the localization precision can be increased only by turning the illumination intensity up, thus increasing the risk of photo-bleaching, and it is independent from the beams' separation, and (ii) in presence of background noise, the localization precision decreases with the beams' separation.
Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Science, South China Normal University, Guangzhou, 510631, China.
In eukaryotes, RNA N-methyladenosine (mA) modification and microRNA (miRNA)-mediated RNA silencing represent two critical epigenetic regulatory mechanisms. The mA methyltransferase complex (MTC) and the microprocessor complex both undergo liquid-liquid phase separation to form nuclear membraneless organelles. Although mA methyltransferase has been shown to positively regulate miRNA biogenesis, a mechanism of reciprocal regulation between the MTC and the microprocessor complex has remained elusive.
