Xiangnan Zhou

SignificanceAccurate identification of residual tumor during head and neck cancer surgery is essential for preventing local recurrences and improving patient outcomes, yet current methods for intraoperative margin assessment remain limited. Fluorescence lifetime imaging (FLIm), a label-free optical technique, has shown strong potential for distinguishing cancerous from noncancerous tissue in vivo. However, the effect of tissue resection on autofluorescence properties is not well defined, raising the question of whether diagnostic signatures establishedin vivo translate reliably to ex vivo specimens.AimThe aim is to characterize how fluorescence lifetime properties change between in vivo and ex vivo conditions across multiple head and neck anatomical sites and to assess how these changes affect diagnostic separability between cancerous and noncancerous tissue.ApproachA custom fiber-based, point-scanning FLIm system with phasor-based analysis was used to characterize fluorescence lifetime features in vivo and ex vivo across oral tongue, base of the tongue, and palatine tonsil tissue from 75 patients undergoing oropharyngeal surgery. A separability index based on phasor distributions was used to quantify cancer versus noncancer contrast across multiple spectral channels.ResultsResection-induced changes in decay dynamics and fluorescence lifetime values varied by anatomy: oral tongue and tonsil showed reduced contrast ex vivo, whereas the base of tongue demonstrated improved separation after excision. The base of the tongue showed a higher separability index ex vivo (0.43 versus 0.15 in vivo), whereas oral tongue and tonsil maintained superior contrast in vivo (0.27 versus 0.18 and 0.46 versus 0.28, respectively).ConclusionsSurgical resection substantially alters autofluorescence signatures in an anatomy-dependent manner, emphasizing the need to train and validate diagnostic algorithms independently for in vivo surgical guidance and ex vivo pathology assessment.

SignificanceCollagen autofluorescence provides valuable intrinsic contrast for assessing tissue structure, composition, and pathology. However, a comprehensive understanding of the fluorescence properties across different collagen types remains limited. This knowledge gap may limit the development of advanced label-free fluorescence spectroscopy and imaging techniques for specific tissue characterization and diagnostic applications.AimThis study aims to comprehensively characterize the fluorescence intensity excitation-emission matrices (I-EEMs) and time-resolved excitation-emission matrices (TR-EEMs) of collagen standards from types I, II, III, IV, and V obtained from various organ sources under both dry and hydrated conditions, to identify optimal excitation-emission parameters for each collagen type discrimination, and to establish a reference dataset that supports future research in label-free tissue characterization.ApproachWe employed a time-resolved fluorescence spectroscopy system equipped with an optical parametric oscillator laser (excitation: 200 to 2000 nm, pulse width: 30 ps) as an excitation source to generate I-EEMs and TR-EEMs of human and bovine collagen types I–V. The fluorescence light was obtained by a multichannel plate photomultiplier tube through a monochromator (spectral range: 200 to 1000 nm). Measurements were conducted using collagen standards, under both dry and hydrated states. In addition, photobleaching effects were assessed to ensure the reliability and reproducibility of fluorescence data.ResultsEach collagen type exhibited distinct I-EEM and TR-EEM signatures, with fluorescence lifetimes ranging from 2.5 ns (type III, bovine skin) to 5.3 ns (types II and V). Fibrillar collagens (types I and V) displayed broader I-EEMs, whereas basement membrane collagen (type IV) showed the narrowest spectral distribution. Organ-source-dependent variations were evident within the same collagen type. Type I collagen from human placenta exhibited an inverse lifetime–emission wavelength relationship compared with bovine sources. Hydration consistently red-shifted emission peaks into the 395 to 420 nm range and reduced fluorescence lifetimes across all collagen types (e.g., type I bovine Achilles tendon: 3.2 to 5.0 ns dry versus 3.0 to 4.5 ns hydrated). Despite excitation wavelength- and fluence-dependent photobleaching of fluorescence intensity, fluorescence lifetimes remained relatively stable, confirming the robustness of lifetime-based measurements.ConclusionsThis study establishes a comprehensive reference dataset for the fluorescence properties of collagen types I–V and demonstrates the potential of combined I-EEMs and TR-EEMs analysis for tissue characterization. The results highlight species-, organ-, type-, and environment-specific optical fingerprints of similar collagens, which must be considered before implementing more in-depth studies on how the optical properties of collagen change in different medical applications.

SignificanceProstate biopsy remains the gold standard for prostate cancer (PCa) diagnosis and treatment planning. However, current techniques suffer from low cancer detection rates, with most biopsy cores sampling benign tissue, leading to undergrading and repeat procedures. Label-free fluorescence lifetime imaging (FLIm) offers a potential solution by enabling real-time discrimination between malignant and benign tissue during biopsy collection, potentially reducing both the number of cores required and the repeat biopsy rates.AimThis pilot study evaluates the feasibility of label-free FLIm for rapid discrimination of malignant from benign prostate tissue in freshly obtained core needle biopsies.ApproachTwenty patients undergoing prostate biopsy were enrolled. FLIm measurements were performed immediately after sample collection (∼10 s) using a custom fiber-optic probe. For each point measurement, FLIm parameters from four spectral bands associated with the emission of distinct endogenous fluorophores including structural proteins and metabolic cofactors (e.g., NADH and FAD) were entered in the analysis. Each FLIm point measurement was labeled based on histological annotation. These data were analyzed to characterize tissue-type differences and to train and evaluate support vector machine (SVM) classifiers for malignancy detection.ResultsSeparation between benign tissue and Gleason pattern ≥4 PCa can already be observed using just 2 out of 56 FLIm-derived parameters. The SVM classifier, using all parameters, achieved a receiver operating characteristic of 0.88 for identifying Gleason pattern 4 PCa. A shorter lifetime value observed in the NADH-associated band was observed for Gleason pattern 4 PCa relative to benign tissue, consistent with increased free NADH from upregulated glycolysis, supporting the biochemical basis for optical differentiation.ConclusionsFLIm demonstrates strong potential for identifying high-grade PCa. Because measurements were performed using a single fiber optic, this approach can be readily integrated into standard prostate biopsy devices to enable FLIm-guided and real-time tissue characterization during the biopsy procedure and to inform targeted tissue collection.

We report a multispectral laser-scanning pulse-sampling fluorescence lifetime imaging (LSPS-FLIm) system designed for rapid, high-resolution imaging of large tissue specimens. This system provides a substantial imaging field of view (FoV) of 6 × 15 cm² with a high spatial resolution of ∼17.5 µm. The LSPS-FLIm system has been tested on a range of fluorescent dyes, endogenous tissue fluorophores, and tissue specimens with varied sizes and properties. These tests demonstrate the system’s versatility in resolving morphological and molecular features, enabling centimeter-scale FoV imaging, and distinguishing complex microstructures in tissue specimens. With the capability to maintain high imaging quality and acquisition speed while minimizing tissue damage, LSPS-FLIm represents a promising advancement in the field of fluorescence lifetime imaging for biological and clinical applications.

The clinical management of coronary artery disease and the prevention of acute coronary syndromes require knowledge of the underlying atherosclerotic plaque pathobiology. Hybrid imaging modalities capable of comprehensive assessment of biochemical and morphological plaques features can address this need. Here we report the first implementation of an intravascular catheter system combining fluorescence lifetime imaging (FLIm) with polarization-sensitive optical coherence tomography (PSOCT). This system provides multi-scale assessment of plaque structure and composition via high spatial resolution morphology from OCT, polarimetry-derived tissue microstructure, and biochemical composition from FLIm, without requiring any molecular contrast agent. This result was achieved with a low profile (2.7 Fr) double-clad fiber (DCF) catheter and high speed (100 fps B-scan rate, 40 mm/s pullback speed) console. Use of a DCF and broadband rotary junction required extensive optimization to mitigate the reduction in OCT performance originating from additional reflections and multipath artifacts. This challenge was addressed by the development of a broad-band (UV-visible-IR), high return loss (47 dB) rotary junction. We demonstrate in phantoms, ex vivo swine coronary specimens and in vivo swine heart (percutaneous coronary access) that the FLIm-PSOCT catheter system can simultaneously acquire co-registered FLIm data over four distinct spectral bands (380/20 nm, 400/20 nm, 452/45 nm, 540/45 nm) and PSOCT backscattered intensity, birefringence, and depolarization. The unique ability to collect complementary information from tissue (e.g., morphology, extracellular matrix composition, inflammation) with a device suitable for percutaneous coronary intervention offers new opportunities for cardiovascular research and clinical diagnosis.

This Letter presents an experimental study comparing the photon rate and photon economy of pulse sampling fluorescence lifetime imaging (PS-FLIm) with the conventional time-correlated single photon counting (TCSPC) technique. We found that PS-FLIm has a significantly higher photon detection rate (200 MHz) compared with TCSPC (2–8 MHz) but lower photon economy (4–5 versus 1–1.3). The main factor contributing to the lower photon economy in PS-FLIm is laser pulse variability. These results demonstrate that PS-FLIm offers 25× faster imaging speed than TCSPC while maintaining room light rejection in clinical settings. This makes PS-FLIm a robust technique for clinical applications.

SignificanceCartilage tissue engineering is a promising strategy for effective curative therapies for treatment of osteoarthritis. However, tissue engineers depend predominantly on time-consuming, expensive, and destructive techniques as quality control to monitor the maturation of engineered cartilage. This practice can be impractical for large-scale biomanufacturing and prevents spatial and temporal monitoring of tissue growth, which is critical for the fabrication of clinically relevant-sized cartilage constructs. Nondestructive multimodal imaging techniques combining fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) hold great potential to address this challenge.AimThe feasibility of using multimodal FLIm–OCT for nondestructive, spatial, and temporal monitoring of self-assembled cartilage tissue maturation in a preclinical mouse model is investigated.ApproachSelf-assembled cartilage constructs were developed for 4 weeks in vitro followed by 4 weeks of in vivo maturation in nude mice. Sterile and nondestructive in situ multispectral FLIm and OCT imaging were carried out at multiple time points (t = 2, 4, and 8 weeks) during tissue development. FLIm and 3D volumetric OCT images were reconstructed and used for the analysis of tissue biochemical homogeneity, morphology, and structural integrity. A biochemical homogeneity index was computed to characterize nonhomogeneous tissue growth at different time points. OCT images were validated against histology.ResultsFLIm detects heterogenous extracellular matrix (ECM) growth of tissue-engineered cartilage. The outer edge of the tissue construct exhibited longer fluorescence lifetime in 375 to 410 and 450 to 485 nm spectral channels, indicating increase in collagen content. Significant (p < 0.05) decrease of construct homogeneity index was observed between t = 2 weeks and t = 4 weeks. Both FLIm and OCT images revealed defects (voids) at the center of the tissue construct during in vitro culture (t = 2 and 4 weeks). Cyst formation during in vivo culture was detected by OCT and confirmed with histology.ConclusionsThe ability of multimodal FLIm–OCT to nondestructively monitor the heterogenous growth of engineered tissue constructs in situ is demonstrated. Spatial and temporal variation of construct ECM component was detected by FLIm. OCT reveals structural defects (voids and cysts). This multimodal approach has great potential to replace costly destructive tests in the manufacturing of tissue-engineered medical products, facilitating their clinical translation.

Cardiovascular disease (CVD) is currently the leading cause of death globally.¹ Early detection and improved characterization of atherosclerosis is necessary for the preventive care of CVD to protect patients from the clinical manifestation such as myocardial infarction, stroke, and peripheral artery disease. Therefore, how to effectively detect and characterize the nature of the atherosclerotic plaques has become a major topic in CVD studies and has driven the development of a variety of cardiovascular imaging technologies.²

Fluorescence-guided surgery (FGS) for primary brain tumor (glioma) resection using 5-aminolevulinic acid (5-ALA) improves the extent of tumor resection in high-grade glioma patients, directly impacting patient survival. Its use is a widespread practice in Europe and is rapidly gaining popularity in the United States.¹ Oral administration of 5-ALA results in selective accumulation of protoporphyrin IX (PpIX) in tumor cells.² In glioblastoma (GBM) patients, PpIX fluorescence (emission peak at 635 nm upon 405 nm excitation³) is strong enough for visual assessment using a surgical microscope, and it enables guided tumor resection. However, for the low-cellularity infiltrating edge of high-grade gliomas and most low-grade gliomas, the PpIX fluorescence intensity decreases below the visible threshold.⁴ Current implementations of PpIX fluorescence visualization systems require working in a dark surgical field, hindering simultaneous tumor identification and resection. Additionally, visible PpIX fluorescence suffers from non-specific intensity variations (e.g., non-uniform illumination) that complicate the quantitative assessment of tumor with high accuracy.⁵ Recent studies conducted on excised brain tissue have suggested that fluorescence spectroscopy and lifetime imaging enable further quantification of PpIX fluorescence below the visible detection threshold.^6,7 Time-resolved fluorescence is expected to increase the detection sensitivity of areas with weak fluorescence intensity because fluorescence lifetime is largely independent of tissue absorption and scattering properties, and unlike intensity alone, it is robust to variations in tissue-detector distance.

Controlling light penetration depth in Avalanche Photodiodes (APDs) and Single Photon Avalanche Diodes (SPADs) play a major role in achieving high multiplication gain by delivering light near the multiplication region where the electric field is the strongest. Such control in the penetration depth for a particular wavelength of light has been previously demonstrated using integrated photon-trapping nanostructures. In this paper, we show that an optimized periodic nanostructure design can control the penetration depth for a wide range of visible and near-infrared wavelengths simultaneously. A conventional silicon APD structure suffers from high photocarrier loss due to recombination for shorter wavelengths as they are absorbed near the surface region, while silicon has low absorption efficiency for longer wavelengths. This optimized nanostructure design allows shorter wavelengths of light to penetrate deeper into the device, circumventing recombination sites while trapping the longer wavelengths in the thin silicon device by bending the vertically propagating light into horizontal modes. This manipulation of penetration depth improves the absorption in the device, increasing light sensitivity while nanostructures reduce the reflectance from the top surface. While delivery of light near the multiplication region reduces the photogenerated carrier loss and shortens transit time, leading to high multiplication gain in APDs and SPADs over a wide spectral range. These high gain APDs and SPADs will find their potential applications in Time-Of-Flight Positron Emission Tomography (TOF-PET), Fluorescence Lifetime Imaging Microscopy (FLIM), and pulse oximetry where high detection efficiency and high gain-bandwidth is required over a multitude of wavelengths.