A new widefield fluorescence lifetime imaging technique, achieved with a time-gated, single-photon avalanche diode (SPAD) camera, enables thousands of molecules to be characterized rapidly, accurately, and at the same time.
Developed by a team at the Swiss Federal Institute of Technology in Lausanne (EPFL), single-molecule fluorescence lifetime imaging microscopy (smFLIM) could be a significant advancement for multitarget, single-molecule localization microscopy.
Traditional FLIM typically relies on time-correlated single-photon counting (TCSPC), a precise but low-throughput method, to discriminate molecules or probe their nanoscale environment.
Unlike conventional imaging methods, smFLIM detects molecules at a specific point in time immediately after they are subjected to an excitation pulse. It captures an alternating series of images — one image immediately after excitation and another a few nanoseconds later — with picosecond-scale resolution.
The images are analyzed to determine the molecule’s fluorescence lifetime — that is, the short delay between the excitation laser pulse and the fluorescence emitted by the molecule — and the individual molecules in the sample are characterized.
Using smFLIM, the researchers obtained measurements with a precision only about 3x less than TCSPC — but with a system capable of imaging with multiple pixels (512 × 512) to enable the spatial multiplexing of more than 3000 molecules. The new technique can take precise measurements of a molecule’s unique light-emission signature at the scale of a billionth of a second.
SmFLIM can provide precise information on thousands of molecules in less than one minute, compared to the one hour required for existing techniques. “Our method is slightly less accurate than conventional ones, but it is faster and can detect an unprecedented number of molecules at once,” professor Aleksandra Radenovic said.
For the design of smFLIM, the researchers revisited a gated imaging scheme introduced 35 years ago. Although time-gated cameras have shown potential for high-throughput FLIM, until now their use in single-molecule microscopy has not been explored extensively. The time-gated SPAD camera used for smFLIM has almost one million sensors, each of which detect a photon.
The team used smFLIM to demonstrate parallelized lifetime measurements of many labeled, pore-forming proteins on supported lipid bilayers, and temporal, single-molecule Förster resonance energy transfer (FRET) measurements to detect the distance between molecules.
“Measuring the fluorescence lifetime of a pair of molecules provides information on the distance between them at a scale of just a few nanometers,” researcher Nathan Ronceray said. “The current approach can only be applied to small samples, but our system can expand it to allow for the rapid study of dynamic phenomena on thousands of molecules.”
The team said that, although it chose single-molecule FRET for its demonstration, smFLIM can be used with other lifetime-changing phenomena, such as non-radiative energy transfer to bulk metal or 2D materials.
Based on the results of the study, smFLIM is a promising tool for diverse areas of science and technology. “One promising direction is its potential to improve multiplexed analyses, that is, to measure several parameters simultaneously in a single sample,” Radenovic said. “It is likely to be useful in fields such as spatial transcriptomics, which aims to measure gene expression in a tissue while preserving spatial information about the exact location of cells or structures in the tissue.”
This approach could benefit lifetime-based assays of biomolecules for structural biology, diagnostic assays, biopolymer sequencing, and single-molecule superresolution microscopy.
By enabling the simultaneous reading of many molecular species throughout life, the method could serve as a powerful complement to high-resolution omics tools used to study the different biological layers of an organism in a comprehensive and systematic way, often on a cellular or molecular scale.
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Developed by a team at the Swiss Federal Institute of Technology in Lausanne (EPFL), single-molecule fluorescence lifetime imaging microscopy (smFLIM) could be a significant advancement for multitarget, single-molecule localization microscopy.
Traditional FLIM typically relies on time-correlated single-photon counting (TCSPC), a precise but low-throughput method, to discriminate molecules or probe their nanoscale environment.
Unlike conventional imaging methods, smFLIM detects molecules at a specific point in time immediately after they are subjected to an excitation pulse. It captures an alternating series of images — one image immediately after excitation and another a few nanoseconds later — with picosecond-scale resolution.
The images are analyzed to determine the molecule’s fluorescence lifetime — that is, the short delay between the excitation laser pulse and the fluorescence emitted by the molecule — and the individual molecules in the sample are characterized.
Using smFLIM, the researchers obtained measurements with a precision only about 3x less than TCSPC — but with a system capable of imaging with multiple pixels (512 × 512) to enable the spatial multiplexing of more than 3000 molecules. The new technique can take precise measurements of a molecule’s unique light-emission signature at the scale of a billionth of a second.
SmFLIM can provide precise information on thousands of molecules in less than one minute, compared to the one hour required for existing techniques. “Our method is slightly less accurate than conventional ones, but it is faster and can detect an unprecedented number of molecules at once,” professor Aleksandra Radenovic said.
For the design of smFLIM, the researchers revisited a gated imaging scheme introduced 35 years ago. Although time-gated cameras have shown potential for high-throughput FLIM, until now their use in single-molecule microscopy has not been explored extensively. The time-gated SPAD camera used for smFLIM has almost one million sensors, each of which detect a photon.
The team used smFLIM to demonstrate parallelized lifetime measurements of many labeled, pore-forming proteins on supported lipid bilayers, and temporal, single-molecule Förster resonance energy transfer (FRET) measurements to detect the distance between molecules.
“Measuring the fluorescence lifetime of a pair of molecules provides information on the distance between them at a scale of just a few nanometers,” researcher Nathan Ronceray said. “The current approach can only be applied to small samples, but our system can expand it to allow for the rapid study of dynamic phenomena on thousands of molecules.”
The team said that, although it chose single-molecule FRET for its demonstration, smFLIM can be used with other lifetime-changing phenomena, such as non-radiative energy transfer to bulk metal or 2D materials.
Based on the results of the study, smFLIM is a promising tool for diverse areas of science and technology. “One promising direction is its potential to improve multiplexed analyses, that is, to measure several parameters simultaneously in a single sample,” Radenovic said. “It is likely to be useful in fields such as spatial transcriptomics, which aims to measure gene expression in a tissue while preserving spatial information about the exact location of cells or structures in the tissue.”
This approach could benefit lifetime-based assays of biomolecules for structural biology, diagnostic assays, biopolymer sequencing, and single-molecule superresolution microscopy.
By enabling the simultaneous reading of many molecular species throughout life, the method could serve as a powerful complement to high-resolution omics tools used to study the different biological layers of an organism in a comprehensive and systematic way, often on a cellular or molecular scale.
Bio Photonics Research Award
Visit: biophotonicsresearch.com
Nominate Now: https://biophotonicsresearch.com/award-nomination/?ecategory=Awards&rcategory=Awardee
#MeatAnalysis #FluorescenceTech #FoodQuality #FoodSafety #SpectroscopyInFood #MeatAuthentication #RapidDetection #FoodScience #MeatFreshness #MolecularDetection #FoodIndustryInnovation #NonDestructiveTesting #FoodMonitoring #SpectroscopyApplications #QualityControl #AdvancedSpectroscopy #MeatSpoilageDetection #FoodIntegrity #SmartFoodTesting #RealTimeAnalysis #FoodAuthenticity #FoodSafetyInnovation #SpectroscopyResearch #NextGenFoodSafety #InnovativeFoodScience,
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