More than double the fun with two-photon excitation microscopy

Communications Biology, Journal Year: 2024, Volume and Issue: 7(1)

Published: March 26, 2024

Abstract For generations researchers have been observing the dynamic processes of life through lens a microscope. This has offered tremendous insights into biological phenomena that span multiple orders time- and length-scales ranging from pure magic molecular reorganization at membrane immune cells, to cell migration differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses such in vitro, however, when applied intact systems, they are challenged by reduced signal strengths signal-to-noise ratios result deeper imaging. As remedy, two-photon excitation (TPE) takes special place, because it allows us investigate vivo, their natural environment, even living animal. Here, we review fundamental principles underlying TPE aimed basic advanced users interested adopting for intravital We focus on applications neurobiology, present current trends towards faster, wider imaging, discuss combination with photon counting technologies metabolic imaging spectroscopy, as well highlight outstanding issues drawbacks application these methodologies.

Language: Английский

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Phasor plots and the future of spectral and lifetime imaging

Nature Methods, Journal Year: 2023, Volume and Issue: 20(7), P. 965 - 967

Published: July 1, 2023

Language: Английский

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Multispectral live-cell imaging with uncompromised spatiotemporal resolution

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 13, 2024

Abstract Multispectral imaging is an established method to extend the number of colours usable in fluorescence beyond typical limit three or four, but standard approaches are poorly suited live-cell imaging. We introduce approach for multispectral live cells, comprising iterative spectral unmixing algorithm and eight channel camera-based image acquisition hardware. This enables accurate low signal-to-noise ratio datasets, imaging, captured at video rates. use this on a commercial spinning disk confocal microscope home-built oblique plane light sheet 1–7 spectrally distinct fluorophore species simultaneously, using both fluorescent protein fusions small-molecule dyes. further de novo designed protein-binding proteins (minibinders), labelled with organic fluorophores, these combination our study endosomal trafficking cell-surface receptors endogenous levels.

Language: Английский

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METAPHOR: Metabolic evaluation through phasor-based hyperspectral imaging and organelle recognition for mouse blastocysts and oocytes

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(28)

Published: July 5, 2024

Only 30% of embryos from in vitro fertilized oocytes successfully implant and develop to term, leading repeated transfer cycles. To reduce time-to-pregnancy stress for patients, there is a need diagnostic tool better select based on their physiology. The current standard employs brightfield imaging, which provides limited physiological information. Here, we introduce METAPHOR: Metabolic Evaluation through Phasor-based Hyperspectral Imaging Organelle Recognition. This non-invasive, label-free imaging method combines two-photon illumination AI deliver the metabolic profile intrinsic autofluorescence signals. We used it classify i) mouse blastocysts cultured under conditions or with depletion selected metabolites (glucose, pyruvate, lactate); ii) young old females, vitro-aged oocytes. process was safe METAPHOR classification control vs. metabolites-depleted reached an area ROC curve (AUC) 93.7%, compared 51% achieved human grading using imaging. binary old/in blastulation prediction AUC 96.2% 82.2%, respectively. Finally, organelle recognition segmentation flavin adenine dinucleotide signal revealed that quantification mitochondria size distribution can be as biomarker embryos. performance safety highlight accuracy noninvasive complementary approach evaluate

Language: Английский

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Quantitative assessment of near-infrared fluorescent proteins

Nature Methods, Journal Year: 2023, Volume and Issue: 20(10), P. 1605 - 1616

Published: Sept. 4, 2023

Language: Английский

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Multiplexed imaging in live cells using pulsed interleaved excitation spectral FLIM

Optics Express, Journal Year: 2024, Volume and Issue: 32(3), P. 3290 - 3290

Published: Jan. 2, 2024

Multiplexed fluorescence detection has become increasingly important in the fields of biosensing and bioimaging. Although a variety excitation/detection optical designs unmixing schemes have been proposed to allow for multiplexed imaging, rapid reliable differentiation quantification multiple fluorescent species at each imaging pixel is still challenging. Here we present pulsed interleaved excitation spectral lifetime microscopic (PIE-sFLIM) system that can simultaneously image six tags live cells single hyperspectral snapshot. Using an alternating laser scheme two different wavelengths synchronized 16-channel time-resolved detector, our PIE-sFLIM effectively excite fluorophores collect their emission over broad spectrum analysis. Combining with advanced live-cell labeling techniques lifetime/spectral phasor analysis, approach well unmix acquired measurement, thus improving speed live-specimen investigation.

Language: Английский

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Assessment of unmixing approaches for the quantitation of SERS nanoparticles in highly multiplexed spectral images

Journal of Raman Spectroscopy, Journal Year: 2024, Volume and Issue: 55(5), P. 566 - 580

Published: Jan. 22, 2024

Surface-enhanced Raman scattering nanoparticles (SERS NPs) offer powerful optical contrast features for imaging assays. Their gold core enhances the inelastic cross section, allowing highly sensitive and rapid detection, their characteristic sets of narrow spectral bands give them unsurpassed multiplexing capabilities. Multiplexed hyperspectral images are commonly unmixed using a compensation matrix reference spectra to produce quantitative image channels illustrating distribution each material. It is these that fit interpretation from assays utilizing SERS NP agents. Some factors may impact dynamic range capabilities include endogenous background heterogeneity, ability unmixing algorithms account signal variances, linear system conditioning imposed by agent signals. We report on mixtures NPs an expanded library study increasing plexity varying degrees as inputs diverse set classical, non-negatively constrained, regularized regression investigate which methods deliver most promising quantitation performance with least error. performed controlled substrates representative biological specimens, experimental results compared against ground truth data. evaluate fitting fidelity, quantitation, specificity correlations conditioning. Spectral hybrid squares principal component analysis (HLP) algorithm approximated 3.5× better fidelity 3× robustness tissue simpler routines.

Language: Английский

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Assessment of Essential Information in the Fourier Domain to Accelerate Raman Hyperspectral Microimaging

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(42), P. 15497 - 15504

Published: Oct. 11, 2023

In the context of multivariate curve resolution (MCR) and spectral unmixing, essential information (EI) corresponds to most linearly dissimilar rows and/or columns a two-way data matrix. recent works, assessment EI has been revealed be very useful practical tool select relevant before MCR analysis, key features being speed compression ability. However, canonical approach relies on principal component analysis evaluate convex hull that encapsulates structure in normalized score space. This implies evaluation essentiality each spectrum can only achieved after all spectra have acquired by instrument. paper proposes new extract Fourier domain (EIFD). Spectral is transformed into coefficients, assessed from point cloud 2D phasor plots few selected harmonics. Because coordinate system plot does not depend themselves, carried individually independently others. As result, time-consuming operations like Raman imaging significantly accelerated exploiting chemometric-driven (i.e., based content pixel) procedure for acquisition targeted sampling. The usefulness EIFD shown analyzing hyperspectral microimaging data, demonstrating potential 50-fold acceleration acquisition.

Language: Английский

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Multidimensional quantitative characterization of basal cell carcinoma by spectral- and time-resolved two-photon microscopy

Nanophotonics, Journal Year: 2024, Volume and Issue: 13(2), P. 217 - 227

Published: Jan. 2, 2024

Basal cell carcinoma (BCC) is a common type of skin cancer. Conventional approaches to BCC diagnosis often involve invasive histological examinations that can distort or even destroy information derived from the biomolecules in sample. Therefore, non-invasive, label-free examination method for clinical represents critical advance. This study combined spectral- and time-resolved two-photon microscopy with spectral phasor extract rich biochemical describing macroscopic tumor morphology microscopic metabolism. The proposed optical imaging technique achieved rapid efficient separation structures systematic research conducted on normal human tissues. results demonstrate combination multidimensional data (e.g., fluorescence intensity, spectrum, lifetime) accurately identify boundaries achieve separation. label-free, real-time, serves as complement conventional diagnostic toolbox demonstrates significant potential early wider applications intraoperative auxiliary imaging.

Language: Английский

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On-the-fly spectral unmixing based on Kalman filtering

Chemometrics and Intelligent Laboratory Systems, Journal Year: 2024, Volume and Issue: 255, P. 105252 - 105252

Published: Oct. 31, 2024

Language: Английский

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