Piston-based specimen holder for rapid surgical and biopsy specimen imaging.

Advanced fluorescence imaging modalities such as confocal microscopy and two photon fluorescence microscopy can provide rapid, real-time histology images, but the mounting of fresh tissue specimens in standard orientations required for diagnosis without embedding and sectioning remains an unsolved problem. Here, we introduce a piston-based specimen holder designed for consistent, even pressure distribution. We improve upon previous designs by incorporating an air piston system with a flexible membrane and wick that extracts fluid during compression. We combine this with support fixtures to aid in the distribution of pressure, enabling imaging of specimens with small surface areas relative to their thickness, such as bisected shave skin biopsies in standard orientation without embedding or sectioning. We image both fresh biopsy specimens and diagnostic Mohs first stage specimens during clinical procedures, demonstrating improved visualization of the tissue surface in real time. Finally, we show that conventional cryosectioning can exaggerate the extent of margin positivity, which can be avoided using the piston-based holder.

Rapid clearing and imaging of Mohs and melanoma surgical margins using a low-cost tissue processor.

Tissue clearing methods render biological tissues transparent while maintaining tissue structure, enabling visualization of entire tissues. Recent developments in tissue clearing have predominantly emphasized preserving intrinsic fluorescent proteins or aqueous-based tissue clearing and so typically involve complex procedures and long processing times. The utilization of tissue clearing protocols in standard of care histology settings has been less well explored, and protocols for rapid clearing of human tissue specimens are limited. This study presents a novel rapid clearing protocol and demonstrates a low-cost tissue processor for high volume rapid tissue clearing that can be intergraded into standard histology workflow. We demonstrate rapid clearing in dermatological specimens, including both nonmelanoma and melanoma excisions.

Suppression of Subpixel Jitter in Resonant Scanning Systems With Phase-locked Sampling.

Resonant scanning is critical to high speed and in vivo imaging in many applications of laser scanning microscopy. However, resonant scanning suffers from well-known image artifacts due to scanner jitter, limiting adoption of high-speed imaging technologies. Here, we introduce a real-time, inexpensive and all electrical method to suppress jitter more than an order of magnitude below the diffraction limit that can be applied to most existing microscope systems with no software changes. By phase-locking imaging to the resonant scanner period, we demonstrate an 86% reduction in pixel jitter, a 15% improvement in point spread function with resonant scanning and show that this approach enables two widely used models of resonant scanners to achieve comparable accuracy to galvanometer scanners running two orders of magnitude slower. Finally, we demonstrate the versatility of this method by retrofitting a commercial two photon microscope and show that this approach enables significant quantitative and qualitative improvements in biological imaging.

Optimal real-time resonant scanner linearization using filtered Hermite interpolation.

High-speed laser scanning microscopy frequently relies on resonant scanners due to their order of magnitude increase in imaging rate compared to conventional galvanometer scanners. However, the use of a nonlinear scan trajectory introduces distortion that must be corrected. This manuscript derives a new algorithm based on filtered Hermite polynomial interpolation that provides the optimal shot-noise-limited SNR for a fixed number of photons and provides higher spatial accuracy than previous methods. An open-source library is presented using the Intel advanced vector instruction set (AVX) to process up to 32 samples in parallel. Using this approach, I simultaneously demonstrate lower shot noise variance, moderately higher spatial accuracy and greater than 1 gigapixel per second interpolation rate on a desktop CPU.

Design for a low-cost heterodyne frequency domain-diffuse optical spectroscopy system.

A design for a low-cost, heterodyne, frequency domain-diffuse optical spectroscopy system is presented and validated. The system uses a single wavelength of 785 nm and a single detector to illustrate the capability, but is built in a modular fashion to make it easily expandable to additional wavelengths and detectors. The design incorporates methods to allow software-based control over the system operating frequency, laser diode output amplitude, and detector gain. Validation methods include characterization of electrical designs as well as determination of the system stability and accuracy using tissue-mimicking optical phantoms. The system requires only basic equipment for its construction and can be built for under $600.

Real-time Analysis of Skin Biopsy Specimens With 2-Photon Fluorescence Microscopy.

Importance: Nonmelanoma skin cancers (NMSCs) are primarily diagnosed through paraffin section histologic analysis of skin biopsy specimens that requires days to weeks before a formal diagnosis is reported. Two-photon fluorescence microscopy (TPFM) has the potential for point-of-care diagnosis of NMSC and other dermatologic conditions, which could enable same-visit diagnosis and treatment. Objective: To demonstrate that TPFM imaging of NMSC can occur within minutes of obtaining biopsies and provide similar histological features to those of conventional histology and evaluate TPFM diagnostic performance with respect to conventional histology. Design, Setting, and Participants: This comparative effectiveness pilot study examined 29 freshly excised biopsies from confirmed NMSC lesions in patients presenting for treatment. Biopsies underwent imaging immediately with TPFM on site at Rochester Dermatologic Surgery (Victor, New York) between October 2019 and August 2021. The imaged biopsies were subsequently submitted for paraffin histology to produce coregistered images. Twelve of these coregistered image pairs (41.4%) were used as a training set. Fifteen (51.7%) were used in a masked evaluation by a board-certified dermatopathologist. Two (6.9%) were excluded from the study before evaluation because they could not be coregistered. Main Outcomes and Measures: Sensitivity, specificity, and accuracy of TPFM for NMSC biopsies were evaluated compared with conventional histology. Results: Fourteen of the 15 biopsy specimens (93.3%) in the evaluation set were identically diagnosed with TPFM and paraffin histology. The TPFM had 100% sensitivity (95% CI, 48%-100%), 100% specificity (95% CI, 69%-100%), and 100% accuracy (95% CI, 78%-100%) for basal cell carcinoma diagnosis. For squamous cell carcinoma diagnosis, TPFM had 89% sensitivity (95% CI, 52%-100), 100% specificity (95% CI, 54%-100%), and 93% accuracy (95% CI, 68%-100%). For overall NMSC diagnosis, TPFM had a 93% sensitivity (95% CI, 66%-100%), 100% specificity (95% CI, 3%-100%), and 93% accuracy (95% CI, 68%-100%). Examination of the 1 discordant pair revealed mismatched imaging planes as the source of error. Conclusions and Relevance: The results of this comparative effectiveness pilot study suggest that TPFM captures histological characteristics of NMSC that are present in conventional histology, which reveals its potential as a rapid, point-of-care diagnostic alternative that does not need extensive sample preparation or retraining for image evaluation. Further validation of TPFM imaging performed for a larger cohort is needed to fully evaluate its diagnostic accuracy and potential effect within the field.

Tendon mechanical properties are enhanced via recombinant lysyl oxidase treatment.

Tendon mechanical properties are significantly compromised in adult tendon injuries, tendon-related birth defects, and connective tissue disorders. Unfortunately, there currently is no effective treatment to restore native tendon mechanical properties after postnatal tendon injury or abnormal fetal development. Approaches to promote crosslinking of extracellular matrix components in tendon have been proposed to enhance insufficient mechanical properties of fibrotic tendon after healing. However, these crosslinking agents, which are not naturally present in the body, are associated with toxicity and significant reductions in metabolic activity at concentrations that enhance tendon mechanical properties. In contrast, we propose that an effective method to restore tendon mechanical properties would be to promote lysyl oxidase (LOX)-mediated collagen crosslinking in tendon during adult tissue healing or fetal tissue development. LOX is naturally occurring in the body, and we previously demonstrated LOX-mediated collagen crosslinking to be a critical regulator of tendon mechanical properties during new tissue formation. In this study, we examined the effects of recombinant LOX treatment on tendon at different stages of development. We found that recombinant LOX treatment significantly enhanced tensile and nanoscale tendon mechanical properties without affecting cell viability or collagen content, density, and maturity. Interestingly, both tendon elastic modulus and LOX-mediated collagen crosslink density plateaued at higher recombinant LOX concentrations, which may have been due to limited availability of adjacent lysine residues that are near enough to be crosslinked together. The plateau in crosslink density at higher concentrations of recombinant LOX treatments may have implications for preventing over-stiffening of tendon, though this requires further investigation. These findings demonstrate the exciting potential for a LOX-based therapeutic to enhance tendon mechanical properties via a naturally occurring crosslinking mechanism, which could have tremendous implications for an estimated 32 million acute and chronic tendon and ligament injuries each year in the U.S.

High-speed mosaic imaging using scanner-synchronized stage position sampling.

SIGNIFICANCE: Two-photon and confocal microscopy can obtain high frame rates; however, mosaic imaging of large tissue specimens remains time-consuming and inefficient, with higher imaging rates leading to a larger fraction of time wasted translating between imaging locations. Strip scanning obtains faster mosaic imaging rates by translating a specimen at constant velocity through a line scanner at the expense of more complex stitching and geometric distortion due to the difficulty of translating at completely constant velocity. AIM: We aim to develop an approach to mosaic imaging that can obtain higher accuracy and faster imaging rates while reducing computational complexity. APPROACH: We introduce an approach based on scanner-synchronous position sampling that enables subwavelength accurate imaging of specimens moving at a nonuniform velocity, eliminating distortion. RESULTS: We demonstrate that this approach increases mosaic imaging rates while reducing computational complexity, retaining high SNR, and retaining geometric accuracy. CONCLUSIONS: Scanner synchronous strip scanning enables accurate, high-speed mosaic imaging of large specimens by reducing acquisition and processing time.

Improved microscopy with ultraviolet surface excitation (MUSE) using high-index immersion illumination.

Microscopy with ultraviolet surface excitation (MUSE) typically has an optical sectioning thickness significantly larger than standard physical sectioning thickness, resulting in increased background fluorescence and higher feature density compared to formalin-fixed, paraffin-embedded physical sections. We demonstrate that high-index immersion with angled illumination significantly reduces optical sectioning thickness through increased angle of refraction of excitation light at the tissue interface. We present a novel objective dipping cap and waveguide-based MUSE illuminator design with high-index immersion and quantify the improvement in optical sectioning thickness, demonstrating an e-1 section thickness reduction to 6.67 µm in tissue. Simultaneously, the waveguide illuminator can be combined with high or low magnification objectives, and we demonstrate a 6 mm2 field of view, wider than a conventional 10x pathology objective. Finally, we show that resolution and contrast can be further improved using deconvolution and focal stacking, enabling imaging that is robust to irregular surface profiles on surgical specimens.

Ultrahigh-speed point scanning two-photon microscopy using high dynamic range silicon photomultipliers.

Conventional two-photon microscopes use photomultiplier tubes, which enable high sensitivity but can detect relatively few photons per second, forcing longer pixel integration times and limiting maximum imaging rates. We introduce novel detection electronics using silicon photomultipliers that greatly extend dynamic range, enabling more than an order of magnitude increased photon detection rate as compared to state-of-the-art photomultiplier tubes. We demonstrate that this capability can dramatically improve both imaging rates and signal-to-noise ratio (SNR) in two-photon microscopy using human surgical specimens. Finally, to enable wider use of more advanced detection technology, we have formed the OpenSiPM project, which aims to provide open source detector designs for high-speed two-photon and confocal microscopy.

Nonlinear microscopy for detection of prostate cancer: analysis of sensitivity and specificity in radical prostatectomies.

Intraoperative evaluation of specimens during radical prostatectomy using frozen sections can be time and labor intensive. Nonlinear microscopy (NLM) is a fluorescence microscopy technique that can rapidly generate images that closely resemble H&E histology in freshly excised tissue, without requiring freezing or microtome sectioning. Specimens are stained with nuclear and cytoplasmic/stromal fluorophores, and NLM evaluation can begin within 3 min of grossing. Fluorescence signals can be displayed using an H&E color scale, facilitating pathologist interpretation. This study evaluates the accuracy of prostate cancer detection in a blinded reading of NLM images compared with the gold standard of formalin-fixed, paraffin-embedded H&E histology. A total of 122 freshly excised prostate specimens were obtained from 40 patients undergoing radical prostatectomy. The prostates were grossed, dissected into specimens of ~10 × 10 mm with 1-4 mm thickness, stained for 2 min for nuclear and cytoplasmic/stromal contrast, and then rinsed with saline for 30 s. NLM images were acquired and multiple images were stitched together to generate large field of view, centimeter-scale digital images suitable for reading. Specimens were then processed for standard paraffin H&E. The study protocol consisted of training, pretesting, and blinded reading phases. After a washout period, pathologists read corresponding paraffin H&E slides. Three pathologists achieved a 95% or greater sensitivity with 100% specificity for detecting cancer on NLM compared with paraffin H&E. Pooled sensitivity and specificity was 97.3% (93.7-99.1%; 95% confidence interval) and 100.0% (97.0-100.0%), respectively. Interobserver agreement for NLM reading had a Fleiss κ = 0.95. The high cancer detection accuracy and rapid specimen preparation suggest that NLM may be useful for intraoperative evaluation in radical prostatectomy.

Evaluation of silicon photomultipliers for multiphoton and laser scanning microscopy.

The silicon photomultiplier (SIPM) is an emerging detector technology that enables both high sensitivity and high dynamic range detection of visible and near-infrared light at a fraction of the cost of conventional vacuum tube photomultiplier tubes (PMTs). A low-cost detection circuit is presented and the performance of a commercial SIPM is evaluated for high-speed laser scanning microscopy applications. For moderate-to-high-speed fluorescent imaging applications, the measurements and imaging results indicate that the SIPM exceeds the sensitivity of GaAsP PMTs, while providing higher dynamic range and better saturation behavior. For low speed or applications requiring large detector areas, the GaAsP PMT retains a sensitivity advantage due to large area and lower dark counts. The calculations presented show that, above a critical detection bandwidth, the SIPM sensitivity exceeds that of a GaAsP PMT.

Comparison of nonlinear microscopy and frozen section histology for imaging of Mohs surgical margins.

Mohs surgery uses en face frozen section analysis (FSA) with complete margin examination for the excision of select basal cell carcinomas (BCC), obtaining excellent cosmetic outcomes and extremely low recurrence rates. However, Mohs with FSA is time-consuming because of the need to iteratively perform cryosectioning on sequential excisions. Fluorescent microscopies can image tissue specimens without requiring physical sectioning, potentially reducing the time to perform Mohs surgery. We demonstrate a protocol for nonlinear microscopy (NLM) imaging of surgical specimens that combines dual agent staining, virtual H&E rendering, and video rate imaging. We also introduce a novel protocol that enables micron-level co-registration of NLM images with FSA histology, and demonstrate that NLM can reproduce similar features similar to FSA in BCC specimens with both negative and positive surgical margins. We show that the fluorescent labels can be extracted with conventional vacuum infiltration processing, enabling subsequent immunohistochemistry on fluorescently labeled tissue. This protocol can also be applied to evaluate the performance of NLM compared with FSA in a wide range of pathologies for intraoperative consultation.

Comparing histologic evaluation of prostate tissue using nonlinear microscopy and paraffin H&E: a pilot study.

Rapid histological assessment of large areas of prostate tissue is required for many intraoperative consultation scenarios such as margin evaluation. Nonlinear microscopy (NLM) enables imaging of large (whole mount) specimens without freezing or cryotoming. This study demonstrates rapid histological imaging of unsectioned prostate cancer surgical specimens using nonlinear microscopy and compares features of prostate pathology to standard paraffin embedded H&E histology. Fresh or formalin fixed specimens were stained in 2.5 min with fluorescent nuclear and stromal dyes. Nonlinear microscopy images of unsectioned tissues were generated by nonlinear (two-photon) excitation of the fluorophores, where fluorescence is only emitted from tissue at the microscope focus, avoiding the need for physical sectioning. The images were displayed in real time using a color scale similar to H&E, then tissues were processed for standard paraffin embedded H&E histology. Seventy nonlinear microscopy and corresponding paraffin H&E images of fresh and fixed prostate specimens (15 cancer, 55 benign) from 24 patients were read by genitourinary pathologists to assess if nonlinear microscopy could achieve an equivalent evaluation to paraffin embedded H&E histology. Differences between nonlinear microscopy images and paraffin H&E slides, including cytoplasmic color and stromal density, were observed, however nonlinear microscopy images could be interpreted with minimal training. Nonlinear microscopy enabled visualization of benign, atrophic and hyperplastic glands and stroma, ejaculatory ducts, vasculature and inflammatory changes. Nonlinear microscopy enabled identification of typical and variants of adenocarcinoma, as well as Gleason patterns. Perineural invasion and extraprostatic extension could also be assessed. Nonlinear microscopy images closely resemble paraffin H&E slides and enable rapid assessment of normal prostate architecture, benign conditions, and carcinoma in freshly excised and fixed specimens. Nonlinear microscopy can image large regions of tissue, equivalent to multiple frozen section tissue blocks, within minutes because cryotoming/microtoming are not required, making it a promising technique for intraoperative consultation.

Assessment of Barrett's esophagus and dysplasia with ultrahigh-speed volumetric en face and cross-sectional optical coherence tomography.

BACKGROUND: This study aimed to evaluate the use of ultrahigh-speed volumetric en face and cross-sectional optical coherence tomography (OCT) with micromotor catheters for the in vivo assessment of Barrett's esophagus and dysplasia. METHODS: 74 OCT datasets with correlated biopsy/endoscopic mucosal resection histology (49 nondysplastic Barrett's esophagus [NDBE], 25 neoplasia) were obtained from 14 patients with Barrett's esophagus and a history of dysplasia and 30 with NDBE. The associations between irregular mucosal patterns on en face OCT, absence of mucosal layering, surface signal > subsurface, and > 5 atypical glands on cross-sectional OCT vs. histology and treatment history were assessed by three blinded readers. RESULTS: Atypical glands under irregular mucosal patterns occurred in 75 % of neoplasia (96 % of treatment-naïve neoplasia) vs. 30 % of NDBE datasets (43 % of short- and 18 % of long-segment NDBE). Mucosal layering was absent in 35 % of neoplasia and 50 % of NDBE datasets, and surface signal > subsurface occurred in 29 % of neoplasia and 30 % of NDBE datasets. CONCLUSIONS: Atypical glands under irregular mucosal patterns are strongly associated with neoplasia, suggesting potential markers for dysplasia and a role in pathogenesis.

Multiscale nonlinear microscopy and widefield white light imaging enables rapid histological imaging of surgical specimen margins.

The ability to histologically assess surgical specimens in real-time is a long-standing challenge in cancer surgery, including applications such as breast conserving therapy (BCT). Up to 40% of women treated with BCT for breast cancer require a repeat surgery due to postoperative histological findings of close or positive surgical margins using conventional formalin fixed paraffin embedded histology. Imaging technologies such as nonlinear microscopy (NLM), combined with exogenous fluorophores can rapidly provide virtual H&E imaging of surgical specimens without requiring microtome sectioning, facilitating intraoperative assessment of margin status. However, the large volume of typical surgical excisions combined with the need for rapid assessment, make comprehensive cellular resolution margin assessment during surgery challenging. To address this limitation, we developed a multiscale, real-time microscope with variable magnification NLM and real-time, co-registered position display using a widefield white light imaging system. Margin assessment can be performed rapidly under operator guidance to image specific regions of interest located using widefield imaging. Using simulated surgical margins dissected from human breast excisions, we demonstrate that multi-centimeter margins can be comprehensively imaged at cellular resolution, enabling intraoperative margin assessment. These methods are consistent with pathology assessment performed using frozen section analysis (FSA), however NLM enables faster and more comprehensive assessment of surgical specimens because imaging can be performed without freezing and cryo-sectioning. Therefore, NLM methods have the potential to be applied to a wide range of intra-operative applications.

Rapid histopathological imaging of skin and breast cancer surgical specimens using immersion microscopy with ultraviolet surface excitation.

Rapid histopathological evaluation of fresh, unfixed human tissue using optical sectioning microscopy would have applications to intraoperative surgical margin assessment. Microscopy with ultraviolet surface excitation (MUSE) is a low-cost optical sectioning technique using ultraviolet illumination which limits fluorescence excitation to the specimen surface. In this paper, we characterize MUSE using high incident angle, water immersion illumination to improve sectioning. Propidium iodide is used as a nuclear stain and eosin yellow as a counterstain. Histologic features of specimens using MUSE, nonlinear microscopy (NLM) and conventional hematoxylin and eosin (H&E) histology were evaluated by pathologists to assess potential application in Mohs surgery for skin cancer and lumpectomy for breast cancer. MUSE images of basal cell carcinoma showed high correspondence with frozen section H&E histology, suggesting that MUSE may be applicable to Mohs surgery. However, correspondence in breast tissue between MUSE and paraffin embedded H&E histology was limited due to the thicker optical sectioning in MUSE, suggesting that further development is needed for breast surgical applications. We further demonstrate that the transverse image resolution of MUSE is limited by the optical sectioning thickness and use co-registered NLM to quantify the improvement in MUSE optical sectioning from high incident angle water immersion illumination.

Introduction to the Biophotonics Congress 2018 feature issue.

The guest editors introduce a feature issue containing papers based on research presented at the OSA Biophotonics Congress (the former BIOMED) held in Hollywood, FL, 2-6 April, 2018.

Rapid virtual hematoxylin and eosin histology of breast tissue specimens using a compact fluorescence nonlinear microscope.

Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 µm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.