Your LSM, Now in 4D

발달 중인 zebrafish 심장의 형태와 심장벽 움직임 관찰

Analyzing embryonic heart morphology and movement in 3D is challenging as the heart is continually beating. The data was recorded from a 3 days post fertilization zebrafish larvae embedded in agarose. ZEISS Lightfield 4D allowed to image the heartbeat with 80 volumes per second. The movie shows 3 full heartbeats in 1.2 seconds, during which cardiomyocytes are temporally and spatially resolved. This allows for cell segmentation and tracking using ZEISS arivis Pro. It is clearly visible that the cardiomyocytes follow exactly the same trajectory in every heartbeat.

Courtesy of Stone Elworthy and Emily Noël, School of Biosciences, University of Sheffield, UK. Data acquired at Wolfson Light Microscopy Facility in the School of Biosciences at the University of Sheffield.

발달 중인 zebrafish 배아의 꼬리 혈관을 따라 흐르는 적혈구 실시간 관찰

Imaging the behavior of the circulatory system in real time in three dimensions is far not possible, as the heart pumps blood through the circulatory system at a speed of 3 beats per second, giving cells too fast of speeds to capture their movement in more than 2 dimensions at a time. Lightfield 4D with speeds up to 80 volumes per second offer the first opportunity to track blood cells throughout this network in 3D and visualize just exactly how this network is organized with respect to relative speed and directionality of blood in different vessels throughout the volume.

Courtesy of Toby Andrews and Rashmi Priya, The Francis Crick Institute, London, UK

Drosophila 혈 림프 내 곤충 혈구세포 hemocyte의 흐름 관찰

Investigating flow of hemocytes, the insect blood cells, through the hemolymph in vivo was almost impossible for researchers due to the fast three-dimensional movement. Lightfield 4D offers the unique opportunity to image a large volume fast enough to follow this process under physiological in vivo conditions. Thanks to the unparalleled imaging speed of 80 volumes per second, cells are reliably resolved both spatially and temporally. The acquired data allow subsequent segmentation and automated tracking using ZEISS arivis Pro.

Courtesy of Iwan Robert Evans, University of Sheffield, UK. Data acquired at Wolfson Light Microscopy Facility in the School of Biosciences at the University of Sheffield.

민감한 Drosophila 근육 활성 3D 이미징

Drosophila melanogaster 1st instar larvae, semi-constrained, expressing Calcium sensitive fluorescent proteins in all body wall muscles. Analysis with ZEISS arivis Pro to visualize muscle activity.

Courtesy of Sean Sweeney, Department of Biology, University of York, UK

Arabidopsis thaliana에서 빛에 의해 유도되는 단백질 재분포 포착

Investigating how plants respond to light appropriately on the molecular level is important for understanding how they carry out many of their light-dependent functions. In this situation, a re-localization of a mobile Arabidopsis thaliana protein upon blue-light stimulation is observed in the hypocotyl (stem) of a 3-day old seedling. The molecular dynamics are too fast for traditional confocal imaging. The high speed of Lightfield 4D enables an entire 3D volume to be imaged in a single snap, perfectly preserving the spatial localizations of protein aggregates. This provides reliable positioning information, which enables accurate reconstructions.

Courtesy of Hannah Walters, Cellular Analysis Facility, MVLS-Shared Research Facilities, University of Glasgow. Data acquired at the Cellular Analysis Facility, University of Glasgow, UK

발달 중인 Drosophila 번데기에서 지방체 조직 형성 관찰

Visualizing development of tissues and organs in intact animals enables better understanding of factors involved in their regulation and dysfunction. One example is the developing fat body forming during the pupal stage of Drosophila. Lightfield 4D allows to keep pace with the cell movement, to provide robust data for 4D tracking. Illumination is gentle enough to image overnight without sacrificing organism viability or fluorophore strength.

15-hour overnight imaging including 12 positions and 10 animals, 500 ms exposure times per volume with 2-minute intervals.

Courtesy of Ignacio Manuel Fernández Guerrero, Cellular Analysis Facility, MVLS-Shared Research Facilities, University of Glasgow. Data acquired at the Cellular Analysis Facility, University of Glasgow

발생 형태형성이 진행 중인 zebrafish 귀 관찰

Morphogenesis of developing organs requires a complex coordination of varying regulators and genomic elements. Lightfield 4D enables acquisition of light-sensitive processes with sufficient resolution to track epithelial cell morphological patterning. Its one-snap-one-volume imaging not only ensures that no developmental processes are missed or lost amidst the z-stacks but even allows multiple animals to be imaged in batch mode to capture all events and increase experimental throughput.

Zebrafish embryo, timelapse movie of developing otic vesicle, 2 – 3 days post fertilization. Every 2 minutes, volumes of 4 different zebrafish embryo ears were imaged over the course of 16 hours.

Courtesy of Tanya Whitfield, Sarah Baxendale, School of Biosciences, University of Sheffield, UK. Data acquired at the Wolfson Light Microscopy Facility, University of Sheffield.

Drosophila 배아의 장 발달 과정 추적

The Drosophila embryonic midgut is particularly challenging to live image due to its depth combined with the different refractive indices of the eggshell and surrounding tissues. The current practice is to either use multiphoton microscopy and risk phototoxicity, or to image mutants in which the refractive index of the embryo is shifted and risk biological side effects during characterization.

Lightfield 4D allows to image the formation of the midgut throughout much of embryogenesis without any risk of phototoxicity, and with unparalleled clarity. The dataset was recorded for close to 7 hours with 1 volume every 10 seconds.

Courtesy of Andrew T Plygawko, School of Biosciences, University of Sheffield, UK. Data acquired at the Wolfson Light Microscopy Facility, University of Sheffield.

Cleared spheroid의 효율적인 볼륨 이미징 및 후속 세포 카운팅

Traditional acquisition methods like confocal point scanning or the use of spinning disk systems take considerable time for the acquisition of z-stacks. The speed of Lightfield 4D imaging enables advanced screening applications where higher throughput is required and facilitates faster screening of many spheroids under similar and different conditions, like in compound screens and drug treatments.

Cleared spheroid of a co-culture of HCT-116-GFP (colon cancer) / NIH-3T3-RFP (fibroblasts) cells stained with Hoechst for nuclei. Imaged in an InSphero Akura plate. Dataset was segmented using arivis Pro.

Sample courtesy of InSphero AG. Schlieren, Switzerland

Perturbation 평가를 위한 cancer organoid 고속 이미징

Organoids are popular biological models for analyzing properties within cancer systems, such as responses to drug treatments, extracellular environments, and immune cell interactions. Image acquisition of such large 3D structures and screening through large sample sets is particularly time consuming. Lightfield 4D enables 3D image acquisition of organoids at a rate of several per second, dramatically increasing the throughput for screening through large numbers compared to traditional microscopy methods.

Colorectal cancer organoids, actin cytoskeleton labelled with phalloidin (magenta), nuclei labelled with DAPI (blue). Captured with a 40× objective using 100 ms exposure time for each fluorophore.

Courtesy of Nikki R. Paul, Cancer Research UK Scotland Institute, Glasgow. Data acquired at the Cellular Analysis Facility, University of Glasgow.