Fluorescence Lifetime Microscopy Research Articles

Nanosecond compressive fluorescence lifetime microscopy imaging via the RATS method with a direct reconstruction of lifetime maps.

The RAndom Temporal Signals (RATS) method has proven to be a useful and versatile method for measuring photoluminescence (PL) dynamics and fluorescence lifetime imaging (FLIM). Here, we present two fundamental development steps in the method. First, we demonstrate that by using random digital laser modulation in RATS, it is possible to implement the measurement of PL dynamics with temporal resolution in units of nanoseconds. Secondly, we propose an alternative approach to evaluating FLIM measurements based on a single-pixel camera experiment. In contrast to the standard evaluation, which requires a lengthy iterative reconstruction of PL maps for each timepoint, here we use a limited set of predetermined PL lifetimes and calculate the amplitude maps corresponding to each lifetime. The alternative approach significantly saves post-processing time and, in addition, in a system with noise present, it shows better stability in terms of the accuracy of the FLIM spectrogram. Besides simulations that confirmed the functionality of the extension, we implemented the new advancements into a microscope optical setup for mapping PL dynamics on the micrometer scale. The presented principles were also verified experimentally by mapping a LuAG:Ce crystal surface.

Lipid Membrane Alterations in Tumor Spheroids Revealed by Fluorescence Lifetime Microscopy Imaging.

Three-dimensional (3D) cultured tumor spheroid models, as one type of in vitro model, have been proven to have more physiological similarities to in vivo animal models than cells in 2D cultures. Tumor spheroids have been widely used in preclinical experiments of anticancer drug treatments, providing reliable data in pathogenetic research. Currently, different 3D cell culture conditions, even in the same cell line, generate heterogeneous spheroids in morphology and size, resulting in different growth rates or drug-killing responses. Therefore, the measurement and evaluation of the properties of tumor spheroids have become highly demanding tasks with huge challenges. For functional characterization of tumor spheroids, the microenvironment sensitivity and quantitative properties of the fluorescence lifetime microscopy imaging (FLIM) technique have great advantages for improving the reliability of cell physiological testing. In this paper, we have proposed a FLIM-based approach to observe the lipid components labeled with Nile red of cells in both 3D and 2D cultures. The imaging data and analysis provided basic information on the sizes, morphologies, and cell membrane fluorescence lifetime values of the tumor spheroids. FLIM data showed that the microenvironment of the cell membrane in the 3D model was largely altered compared to that in the 2D culture. Next, a series of parameters that may influence the lipid components of tumor cells and tumor spheroids were tested by FLIM, including pH, viscosity, and polarity. The results showed that pH and viscosity contributed little to the change in fluorescence lifetime values, while the change in cell membrane polarity was the main cause of the alterations in fluorescence lifetime data, suggesting that cell membrane polarity should be considered a marker in distinguishing tumor spheroids from cellular physiological status. In conclusion, this FLIM-based testing process has been proven to be a quantitative method for measuring the differences between the cells of the 3D model from the 2D cultured cells with satisfactory sensitivity and accuracy, providing a high potential standard assay in the quality evaluation and control of tumor spheroids for future anticancer drug development.

Improving longitudinal resolution of Airy beams two-photon volume imaging with fluorescence lifetime imaging

The non-diffracting Airy beams have been reported to increase acquisition speed by projecting the axially significantly elongated information to a two-dimensional (2D) image. Due to the elongated focal length, an Airy volumetric image can cover information as much as stack images with multiple Gaussian images at different imaging depths, which significantly increases the volumetric imaging rate. However, the projection of three-dimensional (3D) structures on a 2D plane is usually indistinguishable, especially those that overlap along the axis. Therefore, we proposed an axially resolved volume imaging technique based on a two-photon fluorescence lifetime microscopy imaging (FLIM) by using Airy beams.

Advanced Fluorescence Microscopy Methods to Study Dynamics of Fluorescent Proteins In Vivo.

Fluorescent proteins are standard tools for addressing biological questions in a cell biology laboratory. The genetic tagging of protein of interest with fluorescent proteins opens the opportunity to follow them in vivo and to understand their interactions and dynamics. In addition, the latest advances in optical microscopy image acquisition and processing allow us to study many cellular processes in vivo. Techniques such as fluorescence lifetime microscopy and hyperspectral imaging provide valuable tools for understanding fluorescent protein interactions and their photophysics. Finally, fluorescence fluctuation analysis opens the possibility to address questions of molecular diffusion, protein-protein interactions, and oligomerization, among others, yielding quantitative information on the subject of study. This chapter will cover some of the more important advances in cutting-edge technologies and methods that, combined with fluorescent proteins, open new frontiers for biological studies.

Molecular to Supramolecular Self-Assembled Luminogens for Tracking the Intracellular Organelle Dynamics.

Deciphering the dynamics of intracellular organelles has gained immense attention due to their subtle control over diverse, complex biological processes such as cellular metabolism, energy homeostasis, and autophagy. In this context, molecular materials, including small-organic fluorescent probes and their supramolecular self-assembled nano-/microarchitectures, have been employed to explore the diverse intracellular biological events. However, only a handful of fluorescent probes and self-assembled emissive structures have been successfully used to track different organelle's movements, circumventing the issues related to water solubility and long-term photostability. Thus, the water-soluble molecular fluorescent probes and the water-dispersible supramolecular self-assemblies have emerged as promising candidates to explore the trafficking of the organelles under diverse physiological conditions. In this review, we have delineated the recent progress of fluorescent probes and their supramolecular self-assemblies for the elucidation of the dynamics of diverse cellular organelles with a special emphasis on lysosomes, lipid droplets, and mitochondria. Recent advancement in fluorescence lifetime and super-resolution microscopy imaging has also been discussed to investigate the dynamics of organelles. In addition, the fabrication of the next-generation molecular to supramolecular self-assembled luminogens for probing the variation of microenvironments during the trafficking process has been outlined.

Abstract 2472: Tumor/normal and live/dead classification in live tumor fragments using label-free multiphoton microscopy

Abstract We have developed a live tumor fragment (LTF) platform for predicting clinical response to cancer drugs. The success of this approach depends on screening tissue fragments derived from biopsies and excisions before drug treatment to select those that have acceptable levels of viability and tumor content. To enable this screening, we are developing label-free methods for integrated assessment of LTF histology, viability, and metabolic status using intrinsic multiphoton-excited fluorescence lifetime microscopy (MP-FLIM). We cut live EMT6 tumors and mammary fat pad into rectangular fragments that were then sorted and cultured in glass-bottomed multi-well plates. To induce cancer cell death, we treated one group with a therapeutic agent, e.g., the multi-kinase inhibitor, staurosporine, or the alkylating agent, cisplatin. Before and after treatment, we imaged LTF structure and metabolic status based on the intrinsically fluorescent metabolic co-factors nicotinamide dinucleotides (NAD(P)H) and flavin adenine dinucleotides (FAD) fluorescence intensity and lifetime using dual-excitation multiphoton microscopy. As a ground truth viability reference, we then stained and re-imaged the fragments using nuclear and cell viability probes such as Hoechst, SYTOX, TMRE and/or propidium iodide (PI). We analyzed the data by fitting fluorescence decay curves with dual or triple exponents to generate images of lifetime parameters, including the mean and individual component lifetimes and amplitudes. Finally, we co-registered the extrinsically labeled and autofluorescent lifetime images for 3D spatial analysis using commercial and custom software. We verified the methods using monolayer cell cultures and ATP luminescence and flow cytometry viability assays. Image spatial heterogeneity was quantified utilizing entropy metrics. Intrinsic contrast from multiphoton imaging revealed cellular and tissue structures. The median entropy was higher and its distribution negatively skewed for EMT6 (p<0.01), allowing us to distinguish tumor from its corresponding normal tissue of origin. In viable cells, nuclei were distinguishable from the cytoplasm via higher cytoplasmic NADH signal. Upon loss of cell viability, nuclear NADH signals increased, with characteristically short lifetimes, and the overall NADH intensity decreased, reducing the contrast between nuclei and their surrounding cytoplasm. We detected an increase in the whole fragment mean lifetime (τm p<0.002) and concomitant decrease in the short lifetime component (α1 p< 0.002) within 24 hours of staurosporine exposure. We have demonstrated that MP-FLIM can distinguish tumor from normal and live from dead in LTFs without labels. This approach will facilitate selection of fragments with acceptable viability and tumor content for subsequent drug treatment as part of a platform built for personalizing cancer therapy. Citation Format: Jonathan Ouellette, John Rafter, Kelsey Tweed, Leung Kau Tang, Christina Scribano, Pichet Adstamongkonkul, Mikaela Schultz, Justine Coburn, Maged Aldeeb, Neil Anthony, Christin Johnson, Kevin Eliceiri, Jonathan Oliner, Tomasz Zal. Tumor/normal and live/dead classification in live tumor fragments using label-free multiphoton microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2472.

Engineering the gain and bandwidth in avalanche photodetectors.

Avalanche and Single-Photon Avalanche photodetectors (APDs and SPADs) rely on the probability of photogenerated carriers to trigger a multiplication process. Photon penetration depth plays a vital role in this process. In silicon APDs, a significant fraction of the short visible wavelengths is absorbed close to the device surface that is typically highly doped to serve as a contact. Most of the photogenerated carriers in this region can be lost by recombination, get slowly transported by diffusion, or multiplied with high excess noise. On the other hand, the extended penetration depth of near-infrared wavelengths requires thick semiconductors for efficient absorption. This diminishes the speed of the devices due to the long transit time in the thick absorption layer that is required for detecting most of these photons. Here, we demonstrate that it is possible to drive photons to a critical depth in a semiconductor film to maximize their gain-bandwidth performance and increase the absorption efficiency. This approach to engineering the penetration depth for different wavelengths in silicon is enabled by integrating photon-trapping nanoholes on the device surface. The penetration depth of short wavelengths such as 450 nm is increased from 0.25 µm to more than 0.62 µm. On the other hand, for a long-wavelength like 850 nm, the penetration depth is reduced from 18.3 µm to only 2.3 µm, decreasing the device transit time considerably. Such capabilities allow increasing the gain in APDs by almost 400× at 450 nm and by almost 9× at 850 nm. This engineering of the penetration depth in APDs would enable device designs requiring higher gain-bandwidth in emerging technologies such as Fluorescence Lifetime Microscopy (FLIM), Time-of-Flight Positron Emission Tomography (TOF-PET), quantum communications systems, and 3D imaging systems.

Bladder cancer cells shift rapidly and spontaneously to cisplatin-resistant oxidative phosphorylation that is trackable in real time

Genetic mutations have long been recognized as drivers of cancer drug resistance, but recent work has defined additional non-genetic mechanisms of plasticity, wherein cancer cells assume a drug resistant phenotype marked by altered epigenetic and transcriptional states. Currently, little is known about the real-time, dynamic nature of this phenotypic shift. Using a bladder cancer model of nongenetic plasticity, we discovered that rapid transition to drug resistance entails upregulation of mitochondrial gene expression and a corresponding metabolic shift towards the tricarboxylic acid cycle and oxidative phosphorylation. Based on this distinction, we were able to track cancer cell metabolic profiles in real time using fluorescence lifetime microscopy (FLIM). We observed single cells transitioning spontaneously to an oxidative phosphorylation state over hours to days, a trend that intensified with exposure to cisplatin chemotherapy. Conversely, pharmacological inhibition of oxidative phosphorylation significantly reversed the FLIM metabolic signature and reduced cisplatin resistance. These rapid, spontaneous metabolic shifts offer a new means of tracking nongenetic cancer plasticity and forestalling the emergence of drug resistance.

The oxygen initial dip in the brain of anesthetized and awake mice

An ongoing controversy in brain metabolism is whether increases in neural activity cause a local and rapid decrease in oxygen concentration (i.e., the “initial dip”) preceding functional hyperemia. This initial dip has been suggested to cause a transient increase in vascular deoxyhemoglobin with several imaging techniques and stimulation paradigms, but not consistently. Here, we investigate contributors to this initial dip in a distinct neuronal network, an olfactory bulb (OB) glomerulus most sensitive to a specific odorant (ethyl tiglate [ET]) and a site of strong activation and energy consumption upon ET stimulation. Combining two-photon fluorescence and phosphorescence lifetime microscopy, and calcium, blood flow, and pO2 measurements, we characterized this initial dip in pO2 in mice chronically implanted with a glass cranial window, during both awake and anesthetized conditions. In anesthetized mice, a transient dip in vascular pO2 was detected in this glomerulus when functional hyperemia was slightly delayed, but its amplitude was minute (0.3 SD of resting baseline). This vascular pO2 dip was not observed in other glomeruli responding nonspecifically to ET, and it was poorly influenced by resting pO2. In awake mice, the dip in pO2 was absent in capillaries as well as, surprisingly, in the neuropil. These high-resolution pO2 measurements demonstrate that in awake mice recovered from brain surgery, neurovascular coupling was too fast and efficient to reveal an initial dip in pO2.

Endofluorescence Imaging of Murine Hepatocellular Carcinoma Cell Culture by Fluorescence Lifetime Microscopy with Modulated CMOS Camera

In this work, we propose the results of pilot studies on using the modulated CMOS camera for the imaging of autofluorescence in murine hepatocellular carcinoma cells with excitation in UV light. Here, we assess the capabilities of the imaging system to detect changes in the NAD(P)H fractions produced and utilised in the glycolysis, pentose phosphate pathway and oxidative phosphorylation. Our results suggest that the camera and the system based on one possess a sufficient margin of SNR and sensitivity to detect the cellular metabolic changes associated with the metabolic pathways mentioned.

Single-Photon Infrared Imaging With a Silicon Camera Based on Long-Wavelength-Pumping Two-Photon Absorption

We experimentally demonstrated an ultra-sensitive imaging system for telecom photons based on the non-degenerate two-photon absorption in a silicon-based electron multiplying charge-coupled device (EMCCD). The proposed long-wavelength-pumping scheme with mid-infrared pulsed excitation could not only effectively increase the two-photon absorption coefficient, but also significantly suppress the background noise caused by the harmonic absorption of the strong pumping field. In comparison to the photoelectric response via the degenerate two-photon absorption, the implemented configuration could offer over 30-folded enhancement of the photon-counting rate in the infrared imaging. The resulting detection sensitivity up to 1 photon/pixel/pulse was unprecedentedly approached, thus facilitating the single-photon operation. The elimination of the stringent phase matching as typically required in the optical parametric conversion has led to a high spatial resolution of 13 μm. Moreover, the on-chip nonlinearity of the optical imager would enable a broadband spectral window and an enlarged field of view. In combination with the 5-ps temporal resolution due to the coincident optical gating, the presented imaging system would find various promising applications, such as low-light fluorescence lifetime microscopy and photon counting time-of-flight 3D imaging.

Coexisting Ordered and Disordered Membrane Phases Have Distinct Modes of Interaction with Disease-Associated Oligomers.

Ordered membrane domains are thought to influence the attachment and insertion of toxic amyloid oligomers, and consequently, their toxicity. However, if and how the molecular aspects of this interaction depend on the membrane order is poorly understood. Here we measure the affinity, location, and degree of insertion of the small oligomers of hIAPP (human Islet Amyloid Polypeptide, associated with Type II diabetes) at near-physiological concentrations to adjacent domains of a biphasic lipid bilayer. Using simultaneous atomic force, confocal and fluorescence lifetime microscopy (AFM-FLIM), we find that hIAPP oligomers have a nearly 8-fold higher affinity to the disordered domains over the ordered domains. To probe whether this difference indicates different modes of interaction, we measure the change of lifetime of peptide-attached fluorescent labels induced by soluble fluorescence quenchers and also measure the kinetics of localized photobleaching. We find that in the raft-like ordered domains, the oligomers primarily lie on the aqueous interface with limited membrane penetration. However, in the neighboring disordered domains, their C-termini penetrate deeper into the lipid bilayer. We conclude that local membrane order determines not only the affinity but also the mode of interaction of amyloid oligomers, which may have significant implications for disease mechanisms.

Investigating the effects of alcohols and cholesterol manipulations on bovine aortic endothelial cell membrane properties using phasor analysis of laurdan fluorescence lifetime microscopy and spectral imaging

Endothelial cell membranes are made up of a heterogenous mixture of lipids and proteins and include liquid ordered (Lo) domains called lipid rafts and caveolae. These compartments have higher concentrations of cholesterol and sphingolipids and are concentrated in signaling molecules. Perturbations in membrane fluidity, including changes in cholesterol content can lead to changes in the structure of the plasma membrane and impair signaling mechanisms. In order to study how changing membrane fluidity can lead to changes in cellular signaling, we must be able to characterize changes in fluidity.

NAD(P)H autofluorescence lifetime imaging enables single cell analyses of cellular metabolism of osteoblasts in vitro and in vivo via two-photon microscopy.

Two-photon fluorescence lifetime microscopy (2P-FLIM) is a non-invasive optical technique that can obtain cellular metabolism information based on the intrinsic autofluorescence lifetimes of free and enzyme-bound NAD(P)H, which reflect the metabolic state of single cells within the native microenvironment of the living tissue. NAD(P)H 2P-FLIM was initially performed in bone marrow stromal cell (BMSC) cultures established from Col (I) 2.3GFP or OSX-mCherry mouse models, in which osteoblastic lineage cells were labelled with green or red fluorescence protein, respectively. Measurement of the mean NAD(P)H lifetime, τM, demonstrated that osteoblasts in osteogenic media had a progressively increased τM compared to cells in regular media, suggesting that osteoblasts undergoing mineralization had higher NAD+/NAD(P)H ratio and may utilize more oxidative phosphorylation (OxPhos). In vivo NAD(P)H 2P-FLIM was conducted in conjunction with two-photon phosphorescence lifetime microscopy (2P-PLIM) to evaluate cellular metabolism of GFP+ osteoblasts as well as bone tissue oxygen at different locations of the native cranial bone in Col (I) 2.3GFP mice. Our data showed that osteocytes dwelling within lacunae had higher τM than osteoblasts at the bone edge of suture and marrow space. Measurement of pO2 showed poor correlation of pO2 and τM in native bone. However, when NAD(P)H 2P-FLIM was used to examine osteoblast cellular metabolism at the leading edge of the cranial defects during repair in Col (I) 2.3GFP mouse model, a significantly lower τM was recorded, which was associated with lower pO2 at an early stage of healing, indicating an impact of hypoxia on energy metabolism during bone tissue repair. Taken together, our current study demonstrates the feasibility of using non-invasive optical NAD(P)H 2P-FLIM technique to examine cellular energy metabolism at single cell resolution in living animals. Our data further support that both glycolysis and OxPhos are being used in the osteoblasts, with more mature osteoblasts exhibiting higher ratio of NAD+/NAD(P)H, indicating a potential change of energy mode during differentiation. Further experiments utilizing animals with genetic modification of cellular metabolism could enhance our understanding of energy metabolism in various cell types in living bone microenvironment.

Revealing the dynamics of a mitochondrial microenvironment during apoptosis under two-photon fluorescence lifetime microscopy using a cyclic iridium(iii) complex

A mitochondrial viscosity-sensitive two-photon fluorescent probe (Mito-Ap) to reflect apoptosis was rationally developed. The apoptotic process was accurately detected by two-photon fluorescence lifetime imaging in real-time.

Super-Multiplex Nonlinear Optical Imaging Unscrambles the Statistical Complexity of Cancer Subtypes and Tumor Microenvironment.

Label‐free nonlinear optical imaging (NLOI) has made tremendous inroads toward unscrambling the microcosmic complexity of cancers. However, harmonic and Raman microscopy offers throughput without redox information to reveal metabolic differentiation, and fluorescence lifetime microscopy lacks the vibrational response of molecules to visualize specific molecular constituents such as lipid. Here, a flexible, robust simultaneous multi‐nonlinear imaging and cross‐modality system that combines complementary imaging contrast mechanisms is demonstrated. This system, utilizing multiplexed ultrashort pulses, ingeniously integrates typical nonlinear processes, and high‐dimension lifetime extension in a single setup to enhance the imaging dimensions and quality. Using this system, the authors perform label‐free comprehensive evaluation of clinicopathological tissues of ovarian carcinoma due to its statistical complexity. The results show that the technology provides statistically rich, insightful information with high accuracy, sensitivity, and specificity, in contrast to standard histopathology, and can potentially be a powerful tool for fundamental cancer research and clinical applications.

Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA\u2013induced metabolic stress in reconstructed human skin

Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.

Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies.

Fluorescence microscopy is essential for a detailed understanding of cellular processes; however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction.

Resonant Electro-Optic Imaging for Microscopy at Nanosecond Resolution.

We demonstrate an electro-optic wide-field method to enable fluorescence lifetime microscopy (FLIM) with high throughput and single-molecule sensitivity. Resonantly driven Pockels cells are used to efficiently gate images at 39 MHz, allowing fluorescence lifetime to be captured on standard camera sensors. Lifetime imaging of single molecules is enabled in wide field with exposure times of less than 100 ms. This capability allows combination of wide-field FLIM with single-molecule super-resolution localization microscopy. Fast single-molecule dynamics such as FRET and molecular binding events are captured from wide-field images without prior spatial knowledge. A lifetime sensitivity of 1.9 times the photon shot-noise limit is achieved, and high throughput is shown by acquiring wide-field FLIM images with millisecond exposure and >108 photons per frame. Resonant electro-optic FLIM allows lifetime contrast in any wide-field microscopy method.

Time-magnified photon counting with 550-fs resolution

Time-correlated single-photon counting (TCSPC) is an enabling technology for applications such as low-light fluorescence lifetime microscopy and photon counting time-of-flight (ToF) 3D imaging. However, state-of-the-art TCSPC single-photon timing resolution (SPTR) is limited to 3–100 ps by single-photon detectors. Here, we experimentally demonstrate a time-magnified TCSPC (TM-TCSPC) that achieves an ultrashort SPTR of 550 fs with an off-the-shelf single-photon detector. The TM-TCSPC can resolve ultrashort pulses with a 130-fs pulse width difference at a 22-fs accuracy. When applied to photon counting ToF 3D imaging, the TM-TCSPC greatly suppresses the range walk error that limits all photon counting ToF 3D imaging systems by 99.2% and thus provides high depth accuracy and precision of 26 µm and 3 µm, respectively.