Wide-field Fluorescence Imaging Research Articles

Improved intraoperative identification of close margins in oral squamous cell carcinoma resections using a dual aperture fluorescence ratio approach: first in-human results.

Surgical excision is the main treatment for solid tumors in oral squamous cell carcinomas, where wide local excision (achieving a healthy tissue margin of around the excised tumor) is the goal as it results in reduced local recurrence rates and improved overall survival. No clinical methods are available to assess the complete surgical margin intraoperatively while the patient is still on the operating table; and while recent intraoperative back-bench fluorescence-guided surgery approaches have shown promise for detecting "positive" inadequate margins (), they have had limited success in the detection of "close" inadequate margins (1 to 5mm). Here, a dual aperture fluorescence ratio (dAFR) approach was evaluated as a means of improving detection of close margins. The approach was evaluated on surgical specimens from patients who were administered a tumor-specific fluorescent imaging agent (cetuximab-800CW) prior to surgery. The dAFR approach was compared directly against standard wide-field fluorescence imaging and pathology measurements of margin thickness in specimens from three patients and a total of 12 margin locations (1 positive, 5 close, and 6 clear margins). The area under the receiver operating characteristic curve, representing the ability to detect close compared to clear margins () was found to be 1.0 and 0.57 for dAFR and sAF, respectively. Improvements in dAFR were found to be statistically significant (). These results provide evidence that the dAFR approach potentially improves detection of close surgical margins.

Low-frequency background estimation and noise separation from high-frequency for background and noise subtraction.

In fluorescence microscopy, background blur and noise are two main factors preventing the achievement of high-signal-to-noise ratio (SNR) imaging. Background blur primarily emanates from inherent factors including the spontaneous fluorescence of biological samples and out-of-focus backgrounds, while noise encompasses Gaussian and Poisson noise components. To achieve background blur subtraction and denoising simultaneously, a pioneering algorithm based on low-frequency background estimation and noise separation from high-frequency (LBNH-BNS) is presented, which effectively disentangles noise from the desired signal. Furthermore, it seamlessly integrates low-frequency features derived from background blur estimation, leading to the effective elimination of noise and background blur in wide-field fluorescence images. In comparisons with other state-of-the-art background removal algorithms, LBNH-BNS demonstrates significant advantages in key quantitative metrics such as peak signal-to-noise ratio (PSNR) and manifests substantial visual enhancements. LBNH-BNS holds immense potential for advancing the overall performance and quality of wide-field fluorescence imaging techniques.

Multifocal fluorescence video-rate imaging of centimetre-wide arbitrarily shaped brain surfaces at micrometric resolution.

Fluorescence microscopy allows for the high-throughput imaging of cellular activity across brain areas in mammals. However, capturing rapid cellular dynamics across the curved cortical surface is challenging, owing to trade-offs in image resolution, speed, field of view and depth of field. Here we report a technique for wide-field fluorescence imaging that leverages selective illumination and the integration of focal areas at different depths via a spinning disc with varying thickness to enable video-rate imaging of previously reconstructed centimetre-scale arbitrarily shaped surfaces at micrometre-scale resolution and at a depth of field of millimetres. By implementing the technique in a microscope capable of acquiring images at 1.68 billion pixels per second and resolving 16.8 billion voxels per second, we recorded neural activities and the trajectories of neutrophils in real time on curved cortical surfaces in live mice. The technique can be integrated into many microscopes and macroscopes, in both reflective and fluorescence modes, for the study of multiscale cellular interactions on arbitrarily shaped surfaces.

Deep learning based depth map estimation of protoporphyrin IX in turbid media using dual wavelength excitation fluorescence.

This study presents a depth map estimation of fluorescent objects in turbid media, such as biological tissue based on fluorescence observation by two-wavelength excitation and deep learning-based processing. A U-Net-based convolutional neural network is adapted for fluorophore depth maps from the ratiometric information of the two-wavelength excitation fluorescence. The proposed method offers depth map estimation from wide-field fluorescence images with rapid processing. The feasibility of the proposed method was demonstrated experimentally by estimating the depth map of protoporphyrin IX, a recognized cancer biomarker, at different depths within an optical phantom.

Wide field-of-view fluorescence imaging for organ-level lineage tracing of rare intestinal stem cell populations.

Lineage tracing using fluorescent reporters is a common tool for monitoring the expression of genes and transcription factors in stem cell populations and their progeny. The zinc-binding protein 89 (ZBP-89/Zfp148 mouse gene) is a transcription factor that plays a role in gastrointestinal (GI) stem cell maintenance and cellular differentiation and has been linked to the progression of colon cancer. While lineage tracing is a useful tool, it is commonly performed with high-magnification microscopy on a small field of view within tissue sections, thereby limiting the ability to resolve reporter expression at the organ level. Furthermore, this technique requires extensive tissue processing, which is time consuming and requires euthanizing the animal. Further knowledge could be elucidated by measuring the expression of fluorescent reporters across entire organs with minimal tissue processing. We present the application of wide-field fluorescence imaging for whole-organ lineage tracing of an inducible Zfp148-tdTomato-expressing transgenic mouse line to assess the expression of ZBP-89/Zfp148 in the GI tract. We measured tdTomato fluorescence in ex vivo organs at time points between 24h and 6 months post-induction. Fluctuations in tdTomato expression were validated by fluorescence microscopy of tissue sections. Quantification of the wide field-of-view images showed a statistically significant increase in fluorescent signal across the GI tract between transgenic mice and littermate controls. The results also showed a gradient of decreasing reporter expression from proximal to distal intestine, suggesting a higher abundance of ZBP-89 expressing stem cells, or higher expression of ZBP-89 within the stem cells, in the proximal intestine. We demonstrate that wide-field fluorescence imaging is a valuable tool for monitoring whole-organ expression of fluorescent reporters. This technique could potentially be applied in vivo for longitudinal assessment of a single animal, further enhancing our ability to resolve rare stem cell lineages spatially and temporally.

Photon-noise: is a single-pixel camera better than point scanning? A signal-to-noise ratio analysis for Hadamard and Cosine positive modulation

In a single-pixel camera, an unknown object is sequentially illuminated by intensity patterns. The total reflected or transmitted intensity is summed in a single-pixel detector from which the object is computationally reconstructed. In the situation where the measurements are limited by photon-noise, it is questionable whether a single-pixel camera performs better or worse than simply scanning the object with a focused intensity spot—a modality known as point raster scanning and employed in many laser scanning systems. Here, we solve this general question and report that positive intensity modulation based on Hadamard or Cosine patterns does not necessarily improve the single-to-noise ratio (SNR) of single-pixel cameras, as compared to point raster scanning (RS). Instead, we show that the SNR is only improved on object pixels at least k times brighter than the object mean signal , where k is a constant that depends on the modulation scheme (modulation matrix, number of detectors, etc). The constant k is derived for several widespread cases and has important consequences on the choice of the optical deign. This fundamental property is demonstrated theoretically, numerically, and is experimentally confirmed in the spatial domain (widefield fluorescence imaging) and in the spectral domain (spontaneous Raman spectral measurements). Finally, we provide user-oriented guidelines that help decide when and how multiplexing under photon-noise should be used instead of point RS.

Mecp2 deletion results in profound alterations of developmental and adult functional connectivity.

As a regressive neurodevelopmental disorder with a well-established genetic cause, Rett syndrome and its Mecp2 loss-of-function mouse model provide an excellent opportunity to define potentially translatable functional signatures of disease progression, as well as offer insight into the role of Mecp2 in functional circuit development. Thus, we applied widefield optical fluorescence imaging to assess mesoscale calcium functional connectivity (FC) in the Mecp2 cortex both at postnatal day (P)35 in development and during the disease-related decline. We found that FC between numerous cortical regions was disrupted in Mecp2 mutant males both in juvenile development and early adulthood. Female Mecp2 mice displayed an increase in homotopic contralateral FC in the motor cortex at P35 but not in adulthood, where instead more posterior parietal regions were implicated. An increase in the amplitude of connection strength, both with more positive correlations and more negative anticorrelations, was observed across the male cortex in numerous functional regions. Widespread rescue of MeCP2 protein in GABAergic neurons rescued none of these functional deficits, nor, surprisingly, the expected male lifespan. Altogether, the female results identify early signs of disease progression, while the results in males indicate MeCP2 protein is required for typical FC in the brain.

Abstract P6-01-01: Validation of an Optical Imaging Platform to Identify Metabolic Vulnerabilities in Chemo-Resistant and Sensitive Tumors

Abstract Less than 20% of Triple Negative Breast Cancer (TNBC) patients experience long-term responses to mainstay chemotherapy, as tumors develop chemo-resistance. While combinations of chemotherapies and targeted therapies show potential improvements in TNBC clinical outcomes, patient stratification and prediction of treatment response is critical. Spatio-temporal metabolic reprogramming holds promise as a biomarker of therapy response as resistant tumor subpopulations utilize alternate metabolic pathways to escape therapy, enter minimum residual disease (MRD) and recur. Currently, there are limited tools to temporally evaluate heterogeneous changes along distinct metabolic axes in vivo at a spatial resolution capable of resolving vulnerabilities of residual tumor subpopulations. Here, we utilized an optical imaging-based platform to identify in vivo, longitudinal differences in metabolic reprogramming between a resistant and sensitive tumor model at high resolutions along three metabolic axes of TNBC chemoresistance (oxidative phosphorylation, glycolysis, and fatty acid oxidation). Xenografts were established by orthotopic cell injection and mice were treated with Paclitaxel (PTX), a commonly used chemotherapeutic drug in TNBC treatment, under a conventional maximum dose density regimen once the tumor reached a volume ~150mm3. MDA-MB-231 xenografts were resistant to PTX, defined as an initial response to PTX, a period of minimal residual disease, and a resurgence in tumor volume at ~60 days post drug withdrawal (n=3). HCC-1806 xenografts were sensitive to PTX, defined as an initial response to PTX in all mice and a complete cure in 7/10 mice. A separate cohort of mice for each tumor line was implanted with window chambers and imaged longitudinally at distinct stages of the tumor’s lifecycle with previously validated fluorophores 2-NBDG, TMRE, and Bodipy to directly report on glucose uptake, mitochondrial membrane potential, or fatty acid uptake, respectively. Wide field fluorescence imaging of MDA-MB-231 mice showed a significant increase in TMRE as early as two days after the 3rd PTX dose (n=5, p< 0.05), a significant decrease in 2-NBDG as early as two days after the 5th PTX dose (n=5, p< 0.05) and no significant changes in bodipy uptake. This increase in non-glucose-driven mitochondrial respiration was sustained during MRD. An increase in heterogeneity of TMRE uptake was seen during disease regression, MRD, and recurrence (n=5, p< 0.05). HCC-1806 tumors showed increased glucose uptake, decreased fatty acid uptake, and no significant changes in mitochondrial membrane potential during acute treatment. Metabolic changes were transient, with no significant changes in probe uptakes after drug withdrawal and during MRD. Unlike the MDA-MB-231 tumors, no significant changes in the heterogeneity of TMRE uptake were seen following paclitaxel withdrawal in HCC-1806 tumors (n=5, p>0.05). Our results point towards a metabolic switch from glycolysis to non-glucose, non-fat-driven mitochondrial respiration in MDA-MB-231 mice, possibly suggesting amino acid catabolism as a fuel during MRD. In the sensitive HCC-1806 line, we observed changes in glucose and fatty acid uptake during acute treatment, but no significant changes in metabolism during MRD. Consistent with the literature, our results point toward an increase in metabolic plasticity and heterogeneity in a chemo-resistant model compared to a chemo-sensitive one. Together, our results show the potential of using metabolic changes following therapy as a biomarker of therapy response, while highlighting the importance of tracking the change (or lack of change) in metabolism longitudinally. We aim to use this system to visualize and exploit early, in vivo metabolic vulnerabilities of disease regression that accompany local and distal recurrences. Citation Format: Enakshi D. Sunassee, Elizabeth Maydew, Brian Crouch, Megan Madonna, Gregory M Palmer, Nimmi Ramanujam. Validation of an Optical Imaging Platform to Identify Metabolic Vulnerabilities in Chemo-Resistant and Sensitive Tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-01.

Precise, wide field, and low-cost imaging and analysis of core-shell beads for digital polymerase chain reaction.

Digital droplet reactors have become a valuable tool for the analysis of single cells, organisms, or molecules by discretising reagents into picolitre or nanolitre volumes. However, DNA-based assays typically require processing of samples on the scale of tens of microlitres, with the detection of as few as one or as many as a hundred thousand fragments. Through the present work, we introduce a flow-focusing microfluidic device that produces 120 picolitre core-shell beads, which are assembled into a monolayer in a Petri dish for visualization and analysis. The bead assembly is subjected to polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the DNA concentration of the sample. We use a low-cost 21-megapixel digital camera and macro lens to capture wide-field fluorescence images with a 10-30 mm2 field-of-view at magnifications ranging from 5× to 2.5×. A customised Python script analysed the acquired images. Our study demonstrates the ability to perform digital PCR analysis of the entire bead assembly through end-point imaging and compare the results with those obtained through RT-qPCR.

Three-dimensional wide-field fluorescence microscopy for transcranial mapping of cortical microcirculation

Wide-field fluorescence imaging is an indispensable tool for studying large-scale biodynamics. Limited space-bandwidth product and strong light diffusion make conventional implementations incapable of high-resolution mapping of fluorescence biodistribution in three dimensions. We introduce a volumetric wide-field fluorescence microscopy based on optical astigmatism combined with fluorescence source localization, covering 5.6×5.6×0.6 mm3 imaging volume. Two alternative configurations are proposed exploiting multifocal illumination or sparse localization of point emitters, which are herein seamlessly integrated in one system. We demonstrate real-time volumetric mapping of the murine cortical microcirculation at capillary resolution without employing cranial windows, thus simultaneously delivering quantitative perfusion information across both brain hemispheres. Morphological and functional changes of cerebral vascular networks are further investigated after an acute ischemic stroke, enabling cortex-wide observation of concurrent collateral recruitment events occurring on a sub-second scale. The reported technique thus offers a wealth of unmatched possibilities for non- or minimally invasive imaging of biodynamics across scales.

Aligned silver nanowires for plasmonically-enhanced fluorescence detection of photoactive proteins in wet and dry environment

We developed a method of aligning silver nanowires in a microchannel and fixing them to glass substrates via appropriate functionalization. The attachment of nanowires to the substrate is robust with no variation of their angles over minutes. Specific conjugation with photoactive proteins is observed using wide-field fluorescence imaging in real-time for highly concentrated protein solution, both in a microchannel and in a chip geometry. In the latter case we can detect the presence of the proteins in the dropcasted solution down to single proteins. The results point towards possible implementation of aligned silver nanowires as geometrically defined plasmonic fluorescence sensing platforms.

Functional network properties derived from wide-field calcium imaging differ with wakefulness and across cell type

To improve ‘bench-to-bedside’ translation, it is integral that knowledge flows bidirectionally—from animal models to humans, and vice versa. This requires common analytical frameworks, as well as open software and data sharing practices. We share a new pipeline (and test dataset) for the preprocessing of wide-field optical fluorescence imaging data—an emerging mode applicable in animal models—as well as results from a functional connectivity and graph theory analysis inspired by recent work in the human neuroimaging field. The approach is demonstrated using a dataset comprised of two test-cases: (1) data from animals imaged during awake and anesthetized conditions with excitatory neurons labeled, and (2) data from awake animals with different genetically encoded fluorescent labels that target either excitatory neurons or inhibitory interneuron subtypes. Both seed-based connectivity and graph theory measures (global efficiency, transitivity, modularity, and characteristic path-length) are shown to be useful in quantifying differences between wakefulness states and cell populations. Wakefulness state and cell type show widespread effects on canonical network connectivity with variable frequency band dependence. Differences between excitatory neurons and inhibitory interneurons are observed, with somatostatin expressing inhibitory interneurons emerging as notably dissimilar from parvalbumin and vasoactive polypeptide expressing cells. In sum, we demonstrate that our pipeline can be used to examine brain state and cell-type differences in mesoscale imaging data, aiding translational neuroscience efforts. In line with open science practices, we freely release the pipeline and data to encourage other efforts in the community.

Translational rapid ultraviolet-excited sectioning tomography for whole-organ multicolor imaging with real-time molecular staining.

Rapid multicolor three-dimensional (3D) imaging for centimeter-scale specimens with subcellular resolution remains a challenging but captivating scientific pursuit. Here, we present a fast, cost-effective, and robust multicolor whole-organ 3D imaging method assisted with ultraviolet (UV) surface excitation and vibratomy-assisted sectioning, termed translational rapid ultraviolet-excited sectioning tomography (TRUST). With an inexpensive UV light-emitting diode (UV-LED) and a color camera, TRUST achieves widefield exogenous molecular-specific fluorescence and endogenous content-rich autofluorescence imaging simultaneously while preserving low system complexity and system cost. Formalin-fixed specimens are stained layer by layer along with serial mechanical sectioning to achieve automated 3D imaging with high staining uniformity and time efficiency. 3D models of all vital organs in wild-type C57BL/6 mice with the 3D structure of their internal components (e.g., vessel network, glomeruli, and nerve tracts) can be reconstructed after imaging with TRUST to demonstrate its fast, robust, and high-content multicolor 3D imaging capability. Moreover, its potential for developmental biology has also been validated by imaging entire mouse embryos (~2 days for the embryo at the embryonic day of 15). TRUST offers a fast and cost-effective approach for high-resolution whole-organ multicolor 3D imaging while relieving researchers from the heavy sample preparation workload.

Three-dimensional fluorescence microscopy through virtual refocusing using a recursive light propagation network.

Three-dimensional fluorescence microscopy has an intrinsic performance limit set by the number of photons that can be collected from the sample in a given time interval. Here, we extend our earlier work - a recursive light propagation network (RLP-Net) - which is a computational microscopy technique that overcomes such limitations through virtual refocusing that enables volume reconstruction from two adjacent 2-D wide-field fluorescence images. RLP-Net employs a recursive inference scheme in which the network progressively predicts the subsequent planes along the axial direction. This recursive inference scheme reflects that the law of physics for the light propagation remains spatially invariant and therefore a fixed function (i.e., a neural network) for a short distance light propagation can be recursively applied for a longer distance light propagation. In addition, we employ a self-supervised denoising method to enable accurate virtual light propagation over a long distance. We demonstrate the capability of our method through high-speed volumetric imaging of neuronal activity of a live zebrafish brain. The source code used in the paper is available at https://github.com/NICALab/rlpnet.

The miEye: Bench-top super-resolution microscope with cost-effective equipment

Commercial super-resolution (SR) imaging systems require a high budget, while current more affordable open source microscopy systems lack modularity and sometimes are too complex or lack reliability. We present miEye – a cost-effective microscope designed for high-resolution wide-field fluorescence imaging. The build is constructed using a CNC milled aluminum microscope body and commercially available optomechanics, with open-source Python-based microscope control, data visualization, and analysis software integration. The data acquisition software works robustly with commonly used industrial-grade complementary metal oxide semiconductor (iCMOS) cameras, performs IR beam back-reflection-based automatic focus stabilization, and allows for laser control via an Arduino-based laser relay. The open-source nature of the design is aimed to facilitate adaptation by the community. The build can be constructed for a cost of roughly 50 k €. It contains SM-fiber and MM-fiber excitation paths that are easy to interchange and an adaptable emission path. Also, it ensures <5 nm/min stability of the sample on all axes, and allows achieving <30 nm lateral resolution for dSTORM and DNA-PAINT single-molecule localization microscopy (SMLM) experiments. Thus it serves as a cost-effective and adaptable addition to the open source microscopy community and potentially will allow high-quality SR imaging even for limited-budget research groups.

A method for single particle tracking through a multimode fiber

Multimode optical fiber (MMF) endoscopes have recently gained widespread attention as a novel tool for imaging deep within tissue using light microscopy. We here present a method for particle tracking through the MMF, which overcomes the lack of a fast enough wide-field fluorescence imaging modality for this type of endoscope, namely a discrete implementation of orbital particle tracking. We achieve biologically relevant tracking speeds (up to 1.2 μm/s) despite using a slow SLM for the wavefront shaping. We demonstrate a tracking accuracy of λ/50 for a 0.3 NA fiber and show tracking of a pinhole moving to mimic Brownian motion with diffusion rates of up to 0.3 μm2/s.

Deciphering modes of long-range energy transfer in perovskite crystals using confocal excitation and wide-field fluorescence spectral imaging

Excitation energy migration beyond mesoscale is of contemporary interest for both solar photovoltaic and light-emissive devices, especially in context of organometal halide perovskites (OMHPs) which have been shown to have very long (charge carrier) diffusion lengths. While understanding the energy propagation pathways in OMHPs is crucial for further advancement of material design and improvement of opto-electronic features, the simultaneous existence of multiple processes like carrier diffusion, photon recycling, and photon transport makes it often complex to differentiate them. In this study, we unravel the diverse yet dominant excitation energy transfer mode(s) in crystalline MAPbBr3 micron-sized 1D rods and plates by localized (confocal) laser excitation coupled with spectrally-resolved wide-field fluorescence imaging. While rarely used, this technique can efficiently probe excitation migration beyond the diffraction limit and can be realized by simple modification of existing epifluorescence microscopy setups. We find that in rods of length below ∼2 microns, carrier diffusion dominates amongst various energy transfer processes. However, the transient non-radiative defects severely inhibit the extent of carrier migration and also temporarily affect the radiative recombination dynamics of the photo-carriers. For MAPbBr3 plates of several tens of micrometers, we find that the photoluminescence (PL) spectral characteristics remain unaltered at short distances (< ∼3 μm) while at a larger distance, the spectral profile is gradually red-shifted. This implies that carrier diffusion dominates over small distances, while photon recycling, i.e., repeated re-absorption and re-emission of photons, propagates excitation energy transfer over extended length scales with assistance from wave-guided photon transport. Our findings can potentially be used for future studies on the characterization of energy transport mechanisms in semiconductor solids as well as for organic (molecular) self-assembled microstructures.

Computational microscopy for fast widefield deep-tissue fluorescence imaging using a commercial dual-cannula probe.

A solid-glass cannula serves as a micro-endoscope that can deliver excitation light deep inside tissue while also collecting emitted fluorescence. Then, we utilize deep neural networks to reconstruct images from the collected intensity distributions. By using a commercially available dual-cannula probe, and training a separate deep neural network for each cannula, we effectively double the field of view compared to prior work. We demonstrated ex vivo imaging of fluorescent beads and brain slices and in vivo imaging from whole brains. We clearly resolved 4 μm beads, with FOV from each cannula of 0.2 mm (diameter), and produced images from a depth of ~1.2 mm in the whole brain, currently limited primarily by the labeling. Since no scanning is required, fast widefield fluorescence imaging limited primarily by the brightness of the fluorophores, collection efficiency of our system, and the frame rate of the camera becomes possible.

Application of a wavelength-swept laser for spectrally resolved wide-field near-infrared fluorescence imaging

We propose the proof-of-concept of a novel method for wide-field spectrally resolved near-infrared fluorescence (NIRF) imaging using a wavelength-swept laser. The performance of our method is evaluated on a biotissue-like phantom bearing two inclusions, one filled with indocyanine green (ICG) dissolved in distilled water and the second one in dimethyl sulfoxide (DMSO). A near-infrared wavelength-swept laser covering wavelengths around the peak absorption of ICG was used. The difference in the absorption spectra of these two ICG solutions gives rise to an additional spectral contrast. The distinction between the emitted fluorescence light from the two different solutions is performed using a principal component analysis (PCA)-based method. Results show that the two different ICG solutions were successfully resolved using this approach. This technique can be a powerful method to simultaneously spatio-spectrally image multiple near-infrared fluorescence agents.

Bright single-photon sources in the telecom band by deterministically coupling single quantum dots to a hybrid circular Bragg resonator

High-performance solid-state quantum sources in the telecom band are of paramount importance for long-distance quantum communications and the quantum Internet by taking advantage of a low-loss optical fiber network. Here, we demonstrate bright telecom-wavelength single-photon sources based on In(Ga)As/GaAs quantum dots (QDs) deterministically coupled to hybrid circular Bragg resonators (h-CBRs) by using a wide-field fluorescence imaging technique. The QD emissions are redshifted toward the telecom O-band by using an ultra-low InAs growth rate and an InGaAs strain reducing layer. Single-photon emissions under both continuous wave (CW) and pulsed operations are demonstrated, showing high brightness with count rates of 1.14 MHz and 0.34 MHz under saturation powers and single-photon purities of g ( 2 ) ( 0 ) = 0.11 ± 0.02 (CW) and g ( 2 ) ( 0 ) = 0.087 ± 0.003 (pulsed) at low excitation powers. A Purcell factor of 4.2 with a collection efficiency of 11.2 % ± 1 % at the first lens is extracted, suggesting efficient coupling between the QD and h-CBR. Our work contributes to the development of highly efficient single-photon sources in the telecom band for fiber-based quantum communication and future distributed quantum networks.