A wide field fluorescence lifetime imaging system using a light sheet microscope

Birch, Philip; Moore, Lamar; Li, Xiaofei; Phillips, Roger; Young, Rupert; Chatwin, Chris

doi:10.1117/12.2230908

Cited by 5 publications

(3 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, super‐resolution microscopy (STED‐FLIM) increases the number of simultaneously measured probes , while live cell FLIM allows quantitative measurement of O 2 , pH, Ca 2+ , NADH, cell cycle, and other parameters . Current FLIM hardware enables fast measurements and is compatible with major microscopy platforms, one‐ and multiphoton excitation modes and with emerging light‐sheet FLIM microscopy . In contrast to the “traditional” intensity‐based measurements, in vivo FLIM and PLIM allow visualization of oxygenation and metabolism in live animals , while ex vivo models benefit from the antibody‐free labeling of proliferating cells, autofluorescence FLIM and related methods .…”

mentioning

confidence: 99%

Estimation of the Mitochondrial Membrane Potential Using Fluorescence Lifetime Imaging Microscopy

2019

View full text Add to dashboard Cite

Monitoring of cell metabolism represents an important application area for fluorescence lifetime imaging microscopy (FLIM). In particular, assessment of mitochondrial membrane potential (MMP) in complex three-dimensional multicellular in vitro, ex vivo, and in vivo models would enable improved segmentation and functional discrimination of cell types, directly report on the mitochondrial function and complement the quenched-phosphorescence detection of cellular O 2 and two-photon excited FLIM of endogenous NAD(P)H. Here, we report the green and orange-emitting fluorescent dyes SYTO and tetramethylrhodamine methyl ester (TMRM) as potential FLIM probes for MMP. In addition to nuclear, SYTO 16 and 24 dyes also display mitochondrial accumulation. FLIM with the culture of human colon cancer HCT116 cells allowed observation of the heterogeneity of mitochondrial polarization during the cell cycle progression. The dyes also demonstrated good performance with 3D cultures of Lgr5-GFP mouse intestinal organoids, providing efficient and quick cell staining and compatibility with two-photon excitation. Multiplexed imaging of Lgr5-GFP, proliferating cells (Hoechst 33342-aided FLIM), and TMRM-FLIM allowed us to identify the population of metabolically active cells in stem cell niche. TMRM-FLIM enabled to visualize the differences in membrane potential between Lgr5-positive and other proliferating and differentiated cell types. Altogether, SYTO 24 and TMRM dyes represent promising markers for advanced FLIM-based studies of cell bioenergetics with complex 3D and in vivo models.Key terms FLIM; intestinal organoids; Lgr5-GFP; mitochondrial membrane potential; SYTO; tetramethylrhodamine methyl ester Energy production and regulation of cell metabolism are essential for the living cell. Corresponding biomarkers are important for cancer, stem and basic cell biology, drug screening and discovery, cell death and toxicity, and tissue engineering (1-4). Quantitative assessment of cell metabolism and mitochondrial function is possible via a number of techniques, which include analyses of oxygenation and oxygen consumption rate (OCR), mitochondrial membrane potential (MMP), glycolytic flux, ATP and redox status, analysis of metabolome and other approaches (5-8). Photoluminescence (i.e., fluorescence and phosphorescence)-based methods enable quantification of these parameters in formats of a plate reader, fluorescence microscope, and other readouts compatible with various cells and tissues. For in vivo and advanced microscopy applications, quantitative analysis of mitochondrial function is possible only by limited number of probes and approaches, such as measurement of cell oxygenation by phosphorescence quenching method (9,10), analysis of redox status by imaging of NAD(P)H, FAD + , and tryptophan autofluorescence by FLIM or by using various genetically-encoded fluorescent biosensors (11-15). The quantitative assessment of the direct indicator of mitochondrial function, MMP, remains a challenging task (16). This situation creates a gap in the...

show abstract

mentioning

confidence: 99%

Estimation of the Mitochondrial Membrane Potential Using Fluorescence Lifetime Imaging Microscopy

2019

View full text Add to dashboard Cite

show abstract

“…FLIM as a contrast enhancement methodology has become more popular with the advent of super-resolution microscopy (Auksorius et al, 2008;Bückers et al, 2011;Hell and Wichmann, 1994;Niehörster et al, 2016;Masullo et al, 2020;Thiele et al, 2020), providing a new way to multiplex fluorescence imaging. 3D FLIM for contrast enhancement has been demonstrated in proof-ofconcept light-sheet microscopy with optically cleared and fixed samples Greger et al, 2011;Favreau et al, 2020;Funane et al, 2018;Mitchell et al, 2017;Weber et al, 2015;Li et al, 2020;Birch et al, 2016).…”

Section: Contrast Enhancement For High-performance 3d Imaging and Super-resolution Microscopymentioning

confidence: 99%

Luminescence lifetime imaging of three-dimensional biological objects

Dmitriev

Intes

Barroso

2021

Journal of Cell Science

View full text Add to dashboard Cite

A major focus of current biological studies is to fill the knowledge gaps between cell, tissue and organism scales. To this end, a wide array of contemporary optical analytical tools enable multiparameter quantitative imaging of live and fixed cells, three-dimensional (3D) systems, tissues, organs and organisms in the context of their complex spatiotemporal biological and molecular features. In particular, the modalities of luminescence lifetime imaging, comprising fluorescence lifetime imaging (FLI) and phosphorescence lifetime imaging microscopy (PLIM), in synergy with Förster resonance energy transfer (FRET) assays, provide a wealth of information. On the application side, the luminescence lifetime of endogenous molecules inside cells and tissues, overexpressed fluorescent protein fusion biosensor constructs or probes delivered externally provide molecular insights at multiple scales into protein–protein interaction networks, cellular metabolism, dynamics of molecular oxygen and hypoxia, physiologically important ions, and other physical and physiological parameters. Luminescence lifetime imaging offers a unique window into the physiological and structural environment of cells and tissues, enabling a new level of functional and molecular analysis in addition to providing 3D spatially resolved and longitudinal measurements that can range from microscopic to macroscopic scale. We provide an overview of luminescence lifetime imaging and summarize key biological applications from cells and tissues to organisms.

show abstract

“…Nipkow-disk, [107][108][109] light sheet systems, [110][111][112][113][114] and total internal re°ection (TIR) illumination 115 can inherently provide depth resolution. For the Nipkow systems described by Grant et al 107 and G€ orlitz et al 109 the acquisition times for 256 Â 256 pixels range from 1 s for low-quality images recorded in two time gates and 30 s for high-quality images sequentially recorded in eight time gates.…”

Section: Nipkow-disk Systemsmentioning

confidence: 99%

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Liu

Lin

Becker

et al. 2019

J. Innov. Opt. Health Sci.

View full text Add to dashboard Cite

Fluorescence lifetime imaging microscopy (FLIM) is increasingly used in biomedicine, material science, chemistry, and other related research fields, because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments, studying interaction between proteins, metabolic state, screening drugs and analyzing their efficacy, characterizing novel materials, and diagnosing early cancers. Understandably, there is a large interest in obtaining FLIM data within an acquisition time as short as possible. Consequently, there is currently a technology that advances towards faster and faster FLIM recording. However, the maximum speed of a recording technique is only part of the problem. The acquisition time of a FLIM image is a complex function of many factors. These include the photon rate that can be obtained from the sample, the amount of information a technique extracts from the decay functions, the efficiency at which it determines fluorescence decay parameters from the recorded photons, the demands for the accuracy of these parameters, the number of pixels, and the lateral and axial resolutions that are obtained in biological materials. Starting from a discussion of the parameters which determine the acquisition time, this review will describe existing and emerging FLIM techniques and data analysis algorithms, and analyze their performance and recording speed in biological and biomedical applications.

show abstract

A wide field fluorescence lifetime imaging system using a light sheet microscope

Cited by 5 publications

References 7 publications

Estimation of the Mitochondrial Membrane Potential Using Fluorescence Lifetime Imaging Microscopy

Estimation of the Mitochondrial Membrane Potential Using Fluorescence Lifetime Imaging Microscopy

Luminescence lifetime imaging of three-dimensional biological objects

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Contact Info

Product

Resources

About