Analog mean-delay method: a new time-domain super-resolution technique for accurate fluorescence lifetime measurement

Kim, Dug Young; Hwang, Wonsang; Kim, Dong-Eun; Won, Young Ho; Moon, Sucbei; Lee, Sang Yoon; Kang, Min Gu; Han, Won Sub

doi:10.1117/12.2510937

Cited by 3 publications

(4 citation statements)

References 6 publications

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“…Previous studies have reported the detrimental effect of long exposure times on estimated fluorescence lifetime values using traditional FLIM techniques 17 , 70 . Therefore, we needed a faster technique to observe the effects of OAs in dynamic samples.…”

Section: Discussionmentioning

confidence: 99%

“…At these low signal levels, every photon matters; any loss to the fluorescence intensity due to loss during detection or caused by the sample can slow down image acquisition or reduce the accuracy of the estimated metabolic profiles. In an attempt to maximize speed, fast label-free MPM setups are operated at or below the limit for quantification and may require averaging several frames or pixels for extracting quantitative fluorescence parameters 9 , 11 , 15 – 17 .…”

Section: Introductionmentioning

confidence: 99%

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Label-free metabolic and structural profiling of dynamic biological samples using multimodal optical microscopy with sensorless adaptive optics

Iyer

Sorrells

Yang

et al. 2022

Sci Rep

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Label-free optical microscopy has matured as a noninvasive tool for biological imaging; yet, it is criticized for its lack of specificity, slow acquisition and processing times, and weak and noisy optical signals that lead to inaccuracies in quantification. We introduce FOCALS (Fast Optical Coherence, Autofluorescence Lifetime imaging, and Second harmonic generation) microscopy capable of generating NAD(P)H fluorescence lifetime, second harmonic generation (SHG), and polarization-sensitive optical coherence microscopy (OCM) images simultaneously. Multimodal imaging generates quantitative metabolic and morphological profiles of biological samples in vitro, ex vivo, and in vivo. Fast analog detection of fluorescence lifetime and real-time processing on a graphical processing unit enables longitudinal imaging of biological dynamics. We detail the effect of optical aberrations on the accuracy of FLIM beyond the context of undistorting image features. To compensate for the sample-induced aberrations, we implemented a closed-loop single-shot sensorless adaptive optics solution, which uses computational adaptive optics of OCM for wavefront estimation within 2 s and improves the quality of quantitative fluorescence imaging in thick tissues. Multimodal imaging with complementary contrasts improves the specificity and enables multidimensional quantification of the optical signatures in vitro, ex vivo, and in vivo, fast acquisition and real-time processing improve imaging speed by 4–40 × while maintaining enough signal for quantitative nonlinear microscopy, and adaptive optics improves the overall versatility, which enable FOCALS microscopy to overcome the limits of traditional label-free imaging techniques.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Label-free metabolic and structural profiling of dynamic biological samples using multimodal optical microscopy with sensorless adaptive optics

Iyer

Sorrells

Yang

et al. 2022

Sci Rep

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show abstract

“…Traditionally, the time delay between excitation and emission is measured using time-correlated single-photon counting (TCSPC) 13 , which is slow due to the electronic dead time after a single record of a photon count on a photomultiplier tube (PMT) or a hybrid photodetector (HPD). FLIM techniques such as analog mean delay FLIM 14,15 , frequency-domain FLIM 16,17 , and direct pulse-sampling FLIM 18,19 have avoided this problem by analog detection electronics or by sampling continuously at gigahertz speeds. However, these techniques either do not provide quantitative intensity metrics, perform poorly for low photon rates with limited dynamic range, or have inaccurate lifetime values due to the impulse response function of the single photon detector.…”

Section: Acceleration Of Each Modality In Vampire Microscopymentioning

confidence: 99%

VAMPIRE microscopy enables fast and simultaneous structural, metabolic, and chemical characterization of living tissues label-free

Iyer,

Sorrells,

Yang

et al. 2024

Multiphoton Microscopy in the Biomedical Sciences XXIV

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We present a completely label-free technology that captures eight complementary contrasts describing the metabolic, chemical, structural, and dynamic profiles of the tissue microenvironment with a single laser source, called VAMPIRE (Versatile Autofluorescence lifetime imaging, Multiharmonic generation, Polarization-sensitive Interferometry, and Raman scattering in Epi-detection) microscopy. VAMPIRE microscopy maximizes the spectral utility of light-matter interactions by creating four nonlinear (second harmonic generation, two-channel two-photon fluorescence, coherent Raman scattering) and two linear interactions (backscattering, birefringence) simultaneously with a single laser.Innovations in each modality improved the overall speed and sensitivity. Fast fluorescence lifetime imaging microscopy (FLIM) was accelerated with our computational photon counting algorithm called single-and-multi photon peak event detection (SPEED), capable of counting up to 250% photon rates. Dual-channel fast two-photon FLIM was achieved by compressed sensing of analog photocurrents on a field-programmable gate array on board the digitizer. We developed a new and faster method for hyperspectral coherent Raman microscopy using supercontinuum generation and custom pulse shapers, which facilitated rapid tuning to desired vibrational states. Polarization multiplexing in optical coherence imaging enabled compressed sensing of birefringence. Finally, computational approaches to maximize the information from these complementary contrasts yielded new insights into the processes within the tissue microenvironment. VAMPIRE microscopy is the nexus of label-free microscopy research, advances in optoelectronic technologies, and our innovations in computational and multimodal imaging for diverse applications.

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“…Traditionally, the time delay between excitation and emission is measured using time-correlated single-photon counting (TCSPC) [ 16 ], which is slow due to the electronic dead time after a single record of a photon count on a photomultiplier tube (PMT) or a hybrid photodetector (HPD). FLIM techniques such as analog mean delay FLIM [ 17 , 18 ], frequency-domain FLIM [ 19 , 20 ], and direct pulse-sampling FLIM [ 13 , 21 ] have avoided this problem by analog detection electronics or by sampling continuously at gigahertz speeds. The direct-pulse sampling and analog mean delay techniques do not have a linear readout of the fluorescence intensity.…”

Section: Introductionmentioning

confidence: 99%

Analog multiplexing of a laser clock and computational photon counting for fast fluorescence lifetime imaging microscopy

Iyer,

Sorrells,

Tan

et al. 2024

Biomed. Opt. Express

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The dynamic range and fluctuations of fluorescence intensities and lifetimes in biological samples are large, demanding fast, precise, and versatile techniques. Among the high-speed fluorescence lifetime imaging microscopy (FLIM) techniques, directly sampling the output of analog single-photon detectors at GHz rates combined with computational photon counting can handle a larger range of photon rates. Traditionally, the laser clock is not sampled explicitly in fast FLIM; rather the detection is synchronized to the laser clock so that the excitation pulse train can be inferred from the cumulative photon statistics of several pixels. This has two disadvantages for sparse or weakly fluorescent samples: inconsistencies in inferring the laser clock within a frame and inaccuracies in aligning the decay curves from different frames for averaging. The data throughput is also very inefficient in systems with repetition rates much larger than the fluorescence lifetime due to significant silent regions where no photons are expected. We present a method for registering the photon arrival times to the excitation using time-domain multiplexing for fast FLIM. The laser clock is multiplexed with photocurrents into the silent region. Our technique does not add to the existing data bottleneck, has the sub-nanosecond dead time of computational photon counting based fast FLIM, works with various detectors, lasers, and electronics, and eliminates the errors in lifetime estimation in photon-starved conditions. We demonstrate this concept on two multiphoton setups of different laser repetition rates for single and multichannel FLIM multiplexed into a single digitizer channel for real-time imaging of biological samples.

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Analog mean-delay method: a new time-domain super-resolution technique for accurate fluorescence lifetime measurement

Cited by 3 publications

References 6 publications

Label-free metabolic and structural profiling of dynamic biological samples using multimodal optical microscopy with sensorless adaptive optics

Label-free metabolic and structural profiling of dynamic biological samples using multimodal optical microscopy with sensorless adaptive optics

VAMPIRE microscopy enables fast and simultaneous structural, metabolic, and chemical characterization of living tissues label-free

Analog multiplexing of a laser clock and computational photon counting for fast fluorescence lifetime imaging microscopy

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