The crosstalk between two fluorescent species causes problems in fluorescence microscopy imaging, especially for quantitative measurements such as co-localization, Förster resonance energy transfer (FRET), fluorescence cross correlation spectroscopy (FCCS). In laser scanning confocal microscopy, the lasers can be switched on and off by acousto-optic tunable filters (AOTF) in the microsecond scale for alternative line scanning in order to avoid the crosstalk while minimizing the time delay between two lasers on the same pixel location. In contrast, the pulsed interleaved excitation (PIE) technique synchronizes two pulsed lasers of different wavelengths in the nanosecond scale to enable measuring superfast dynamics of two fluorescent species simultaneously and yet quantitatively without the crosstalk contamination. This feature is critical for many cell biology applications, e.g. accurate determination of stoichiometry in FRET measurements for studying protein-protein interactions or cell signal events, detection of weaker bindings in FCCS by eliminating the false cross correlation due to the crosstalk. The PIE has been used with the time correlated single photon counting (TCSPC) electronics. Here, we describe a novel PIE development using the digital frequency domain (DFD) technique — FastFLIM, which provides tunable PIE setups and synchronized gating detections, tailored and optimized to specific applications. A few PIE setups by FastFLIM and measurement examples are described. Combined with the sensitivity of Alba and Q2 systems, the PIE allowed us to quantitatively measure the fast dynamics of single molecules.

Dynamics

10.1117/12.2217434

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Quantitative tools in the Multi-image Phasor Analysis (MiPA)

Yuansheng Sun Ulas Coskun Shih‐Chu Liao Beniamino Barbieri

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique that provides a totally new dimension to quantitate the fluorescent probe, in addition to its steady-state intensity and spectral profiles. As the fluorescence lifetime is a parameter specific of each fluorophore, its measurement is instrumental in the identification of the fluorophore in a mixture: this opens a new opportunity for multiplex imaging and improves imaging sensitivity (signal-to-noise ratio) and resolution in many applications. More importantly, with peculiar selectivity of probes, FLIM can provide quantitative information of the probe microenvironment (ions, pH, oxygen content, local electrical fields, index of refractions, etc.); FLIM is one of the most robust ways of quantifying FRET for studying protein-protein interactions in live specimens. Today, turn-key FLIM instruments are a mature technology available from several companies, making the technique easily accessible. However, a major challenge for the user is still the analysis and interpretation of FLIM data. In combination with the digital frequency domain FLIM (known as FastFLIM), the phasor plots approach has been demonstrated to be an effective solution to tackle many challenges in FLIM data analysis. In this report, we describe several tools using the phasor plots method available in the ISS VistaVision software multiimage phasor analysis module, and demonstrate their utilities with examples for various applications including time-resolved multiplexing, NADH imaging, STED and FRET.

Fluorescence-lifetime imaging microscopy

10.1117/12.2626879

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Measuring upconversion nanoparticles photoluminescence lifetime with FastFLIM and phasor plots

Yuansheng Sun Ulas Coskun Shih‐Chu Liao Beniamino Barbieri Hsien‐Ming Lee

Photon upconversion is a nonlinear process in which the sequential of absorption of two or more photons leads to the anti-stoke emission. Different than the conventional multiphoton excitation process, upconversion can be efficiently performed at low excitation densities. Recent developments in lanthanide-doped upconversion nanoparticles (UCNPs) have led to a diversity of applications, including detecting and sensing of biomolecules, imaging of live cells, tissues and animals, cancer diagnostic and therapy, etc. Measuring the upconversion lifetime provides a new dimension of its imaging and opens a new window for its applications. Due to the long metastable intermediate excited state, UCNP typically has a long excited state lifetime ranging from sub-microseconds to milliseconds. Here, we present a novel development using the FastFLIM technique to measure UCNP lifetime by laser scanning confocal microscopy. FastFLIM is capable of measuring lifetime from 100 ps to 100 ms and features the high data collection efficiency (up to 140-million counts per second). Other than the traditional nonlinear least-square fitting analysis, the raw data acquired by FastFLIM can be directly processed by the model-free phasor plots approach for instant and unbiased lifetime results, providing the ideal routine for the UCNP photoluminescence lifetime microscopy imaging.

Photon Upconversion

10.1117/12.2290451

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Morphological evolution of hnRNPA1 pathological condensates

Biophysical Journal (2022)

Allan Chris M. Ferreon My Diem Quan Shih‐Chu Liao Phoebe S. Tsoi Josephine C. Ferreon

10.1016/j.bpj.2021.11.973

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Frequency domain phosphorescence lifetime Imaging measurements and applications by ISS FastFLIM and multi pulse excitation

Proceedings of SPIE, the International Society for Optical Engineering/Proceedings of SPIE (2017)

Ulas Coskun Sandra Lam Yuansheng Sun Shih‐Chu Liao Steven C. George

Phosphorescence probes can have significantly long lifetimes, on the order of micro- to milli-seconds or longer. In addition, environmental changes can affect the lifetimes of these phosphorescence probes. Thus, Phosphorescence Lifetime Imaging Microscopy (PLIM) is a very useful tool to localize the phosphorescence probes based on their lifetimes to study the variance in the lifetimes due to the micro environmental changes. Since the probes respond to the biologically relevant parameters like oxygen concentration, they can be used to study various biologically relevant processes like cellular metabolism, protein interaction etc. In this case, we study the effects of oxygen on Oxyphor G4 with PLIM. Since The Oxyphor G4 can be quenched by O₂, it is a good example of such a probe and has a lifetime around 250us. Here we present the digital frequency domain PLIM technique and study the lifetime of the Oxyphor G4 as a function of the O2 concentration. The lifetime data are successfully presented in a phasor plot for various O₂ concentrations and are consistent with the time domain data. Overall, we can analyze the oxygen consumption of varying cells using this technique.

10.1117/12.2255481

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Enhancing the resolution of STED microscopy by SPLIT and flimGANE

Yuansheng Sun Giorgio Tortarolo Yuan‐I Chen Yin-Jui Chang Ulas Coskun

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of sub-cellular structures with spatial resolution well below the diffraction limit. The higher the STED beam intensity, the higher the resolution, but at the cost of increased photo-damage, which significantly limits the application of STED microscopy in live specimens. The separation by lifetime tuning (SPLIT) technique uses a time-resolved acquisition and a phasor-based analysis to efficiently distinguish photons emitted from the center and from the periphery of the effective fluorescent region, thus improves the resolution of STED microscopy without increasing the STED beam intensity. Furthermore, the SPLIT method is combined with a deep learning-based phasor analysis algorithm termed flimGANE (fluorescence lifetime imaging based on a generative adversarial network), to improve the robustness of SPLIT-STED allowing improving the resolution up to 1.45 folds at the half of the depletion laser beam intensity.

STED microscopy

Temporal resolution

10.1117/12.3002745

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Front Matter: Volume 9712

Proceedings of SPIE, the International Society for Optical Engineering/Proceedings of SPIE (2016)

Peter Andersen Enrico Baria Ana Batista Hope T. Beier Jasmin Breymayer

identifier (CID) number is assigned to each article at the time of

10.1117/12.2239754

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Time-resolved mesoscopic imaging of a whole animal by FastFLIM

Yuansheng Sun Chien‐Sing Poon Hailin Qiu Ulas Coskun Shih‐Chu Liao

The mesoscopic scale is between microscopic and macroscopic scales. In life sciences, mesoscopic imaging allows scientists to record, track and study details of biological systems in the context of an organ, body part, or organism. Mesoscopic imaging techniques have been developed for medical and clinical research, such as drug delivery, cancer diagnosis, etc. Especially when combined with novel nanoparticles and organic dyes in the near-infrared spectral regime, the mesoscopic imaging can probe deeper parts of the animal body. Here, we describe a timeresolved mesoscopic imaging approach, which can image deep inside of the whole mouse noninvasively. In addition, it uses the FastFLIM technique to measure the lifetime of the fluorescent probe. Since the lifetime carries information about the probe's local microenvironment such as temperature, pH, ion concentration, etc., the lifetime imaging map obtained by the FastFLIM-mesoscope allows tracking quantitative dynamics of the probes in the whole animal body. The technique can also be used for quantitative intrinsic NADH metabolism mapping for real time monitoring of mitochondrial function. Here, we will show mesoscopic-scale NADH imaging in an oral cancer model.

Mesoscopic physics

10.1117/12.2546110

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Single actomyosin motor interactions in skeletal muscle

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research (2011)

Zeno Földes‐Papp Shih‐Chu Liao Ben Barbieri Karol Gryczynski Rafał Luchowski

We present a study of intramuscular motion during contraction of skeletal muscle myofibrils. Myofibrillar actin was labeled with fluorescent dye so that the ratio of fluorescently labeled to unlabeled protein was 1:105. Such sparse labeling assured that there was on average only one actin-marker present in the focus at a given time. From the intensity signal in the two orthogonal detection channels, significant fluctuations, similar to fluorescent burst in diffusion-based single-molecule detection schemes, were identified via a threshold algorithm and analyzed with respect to their intensity and polarization. When only rigor complexes were formed, the fluctuations of polarized intensity were characterized by unimodal Gaussian photon distributions. During contraction, in contrast, bimodal Gaussian photon distributions were observed above the rigor background threshold. This suggests that the bimodal Gaussian photon distributions represent pre- and post-power stroke conformations. Clusters of polarized photons indicated an anisotropy decay of single actomyosin motors of ~ 9 s during muscle contraction.

Myofibril

10.1016/j.bbamcr.2011.02.001

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