Calcium Indicators with Fluorescence Lifetime-Based Signal Readout: A Structure–Function Study DOI Open Access
Tatiana R. Simonyan, Larisa A. Varfolomeeva, Anastasia V. Mamontova

и другие.

International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(23), С. 12493 - 12493

Опубликована: Ноя. 21, 2024

The calcium cation is a crucial signaling molecule involved in numerous cellular pathways. Beyond its role as messenger or modulator intracellular cascades, calcium's function excitable cells, including nerve impulse transmission, remarkable. central of nervous activity has driven the rapid development fluorescent techniques for monitoring this living cells. Specifically, genetically encoded indicators (GECIs) are most in-demand molecular tools their class. In work, we address two issues imaging by designing based on successful GCaMP6 backbone and protein BrUSLEE. first indicator variant (GCaMP6s-BrUS), with reduced, calcium-insensitive fluorescence lifetime, potential dynamics high temporal resolution combination advanced microscopy techniques, such light beads microscopy, where lifetime limits acquisition speed. Conversely, second (GCaMP6s-BrUS-145), flexible, calcium-sensitive relevant static measurements, particularly determining absolute concentration values using (FLIM). To identify structural determinants sensitivity these variants, determine spatial structures. A comparative analysis allowed optimization GCaMP6s-BrUS construct, resulting an combining behavior time domain enhanced brightness. Our data may serve starting point further engineering efforts towards improved GECI variants fine-tuned lifetimes.

Язык: Английский

High-throughput wide-field multispectral FLIM system based on a 16-channel silicon photomultiplier array DOI Creative Commons
Elisabetta Avanzi,

Valerio Gandolfi,

A. Costa

и другие.

APL Photonics, Год журнала: 2025, Номер 10(6)

Опубликована: Июнь 1, 2025

Fluorescence microscopy is nowadays one of the most diffused techniques to study photophysical processes and molecular interactions in both biomedical material science fields. Since each fluorophore characterized by a specific emission spectrum lifetime, it essential not only spatially localize its position but also measure spectral temporal properties with multispectral fluorescence lifetime imaging microscope (λFLIM). Moreover, rapidly evolving samples, measurement system capable fast acquisitions needed. λFLIM systems are currently limited on acquisition speed cannot reach high throughput. In this work, we propose novel wide-field based 16-channel silicon photomultiplier (SiPM) array. Our system, thanks SiPM technology along single pixel camera compress sensing approaches able acquire multidimensional measurements (space, spectrum, time) at frame rate. We validate moving fluorescent samples capturing snapshots rate up 13 fps. The developed could enable enhanced specificity real-time biological biosensing applications.

Язык: Английский

Процитировано

0

Recent advances in label-free imaging techniques based on nonlinear optical microscopy to reveal the heterogeneity of the tumor microenvironment DOI
Ishita Chakraborty, Nirmal Mazumder, Ankur Gogoi

и другие.

Biophysical Reviews, Год журнала: 2024, Номер 16(5), С. 581 - 590

Опубликована: Сен. 28, 2024

Язык: Английский

Процитировано

2

Light-field tomographic fluorescence lifetime imaging microscopy DOI Creative Commons
Yayao Ma, Jongchan Park, Luzhe Huang

и другие.

Proceedings of the National Academy of Sciences, Год журнала: 2024, Номер 121(40)

Опубликована: Сен. 25, 2024

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique that enables the visualization of biological samples at molecular level by measuring fluorescence decay rate fluorescent probes. This provides critical information about interactions, environmental changes, and localization within systems. However, creating high-resolution maps using conventional FLIM systems can be challenging, as it often requires extensive scanning significantly lengthen acquisition times. issue further compounded in three-dimensional (3D) because demands additional along depth axis. To tackle this challenge, we developed computational called light-field tomographic (LIFT-FLIM). Our approach allows for volumetric images highly data-efficient manner, reducing number steps required compared to point-scanning or line-scanning imagers. Moreover, LIFT-FLIM measurement high-dimensional data low-dimensional detectors, which are typically low cost feature higher temporal bandwidth. We demonstrated linear single-photon avalanche diode array on various systems, showcasing unparalleled detection sensitivity. Additionally, expanded functionality our method spectral its application high-content multiplexed lung organoids. has potential open up broad avenues both basic translational biomedical research.

Язык: Английский

Процитировано

1

Fluorescence modulation of pyridinium betaines: a mechanofluorochromic investigation DOI
Peter W. McDonald, Jingjing Xu, Dale R. Lonsdale

и другие.

Journal of Materials Chemistry C, Год журнала: 2024, Номер 12(48), С. 19371 - 19385

Опубликована: Янв. 1, 2024

The mechanism of new mechanofluorochromic is resolved using established and unexplored techniques, including electron diffraction fluorescence lifetime mapping. Driving factors posited include conformational flexibility partial amorphisation.

Язык: Английский

Процитировано

1

Benefits of Combined Fluorescence Lifetime Imaging Microscopy and Fluorescence Correlation Spectroscopy for Biomedical Studies Demonstrated by Using a Liposome Model System DOI Creative Commons

Kristina Bruun,

Hans‐Gerd Löhmannsröben, Carsten Hille

и другие.

Biophysica, Год журнала: 2024, Номер 4(2), С. 207 - 226

Опубликована: Апрель 25, 2024

Drug delivery systems play a pivotal role in targeted pharmaceutical transport and controlled release at specific sites. Liposomes, commonly used as drug carriers, constitute fundamental part of these systems. Moreover, the drug–liposome model serves robust platform for investigating interaction processes both cellular molecular levels. To advance our understanding carrier uptake mechanisms, we employed fluorescence lifetime imaging microscopy (FLIM) correlation spectroscopy (FCS), leveraging unique benefits two-photon (2P) excitation. Our approach utilized giant unilamellar vesicles (GUVs) simplified system cell membranes, labelled with amphiphilic fluorescent dye 3,3′-dioctadecyloxa-carbocyanine (DiOC18(3)). Additionally, large (LUVs) functioned system, incorporating spectrally distinct sulforhodamine 101 (SRh101) surrogate drug. The investigation emphasized diverse interactions between GUVs LUVs based on charged lipids employed. We examined exchange kinetics structural alterations liposome carriers during process. study underscores significance employing 2P excitation conjunction FLIM FCS. This powerful combination offers valuable methodological studying interactions, positioning them an exceptionally versatile technical advantage.

Язык: Английский

Процитировано

0

Calcium Indicators with Fluorescence Lifetime-Based Signal Readout: A Structure–Function Study DOI Open Access
Tatiana R. Simonyan, Larisa A. Varfolomeeva, Anastasia V. Mamontova

и другие.

International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(23), С. 12493 - 12493

Опубликована: Ноя. 21, 2024

The calcium cation is a crucial signaling molecule involved in numerous cellular pathways. Beyond its role as messenger or modulator intracellular cascades, calcium's function excitable cells, including nerve impulse transmission, remarkable. central of nervous activity has driven the rapid development fluorescent techniques for monitoring this living cells. Specifically, genetically encoded indicators (GECIs) are most in-demand molecular tools their class. In work, we address two issues imaging by designing based on successful GCaMP6 backbone and protein BrUSLEE. first indicator variant (GCaMP6s-BrUS), with reduced, calcium-insensitive fluorescence lifetime, potential dynamics high temporal resolution combination advanced microscopy techniques, such light beads microscopy, where lifetime limits acquisition speed. Conversely, second (GCaMP6s-BrUS-145), flexible, calcium-sensitive relevant static measurements, particularly determining absolute concentration values using (FLIM). To identify structural determinants sensitivity these variants, determine spatial structures. A comparative analysis allowed optimization GCaMP6s-BrUS construct, resulting an combining behavior time domain enhanced brightness. Our data may serve starting point further engineering efforts towards improved GECI variants fine-tuned lifetimes.

Язык: Английский

Процитировано

0