Quantitative collagen analysis using second harmonic generation images for the detection of basal cell carcinoma with ex vivo multiphoton microscopy.

Basal cell carcinoma (BCC) is the most common skin cancer, and its incidence is rising. Millions of benign biopsies are performed annually for BCC diagnosis, increasing morbidity, and healthcare costs. Non-invasive in vivo technologies such as multiphoton microscopy (MPM) can aid in diagnosing BCC, reducing the need for biopsies. Furthermore, the second harmonic generation (SHG) signal generated from MPM can classify and prognosticate cancers based on extracellular matrix changes, especially collagen type I. We explored the potential of MPM to differentiate collagen changes associated with different BCC subtypes compared to normal skin structures and benign lesions. Quantitative analysis such as frequency band energy analysis in Fourier domain, CurveAlign and CT-FIRE fibre analysis was performed on SHG images from 52 BCC and 12 benign lesions samples. Our results showed that collagen distribution is more aligned surrounding BCCs nests compared to the skin's normal structures (p < 0.001) and benign lesions (p < 0.001). Also, collagen was orientated more parallelly surrounding indolent BCC subtypes (superficial and nodular) versus those with more aggressive behaviour (infiltrative BCC) (p = 0.021). In conclusion, SHG signal from type I collagen can aid not only in the diagnosis of BCC but could be useful for prognosticating these tumors. Our initial results are limited to a small number of samples, requiring large-scale studies to validate them. These findings represent the groundwork for future in vivo MPM for diagnosis and prognosis of BCC.

COVID-19 management landscape: A need for an affordable platform to manufacture safe and efficacious biotherapeutics and prophylactics for the developing countries.

To gain world-wide control over COVID-19 pandemic, it is necessary to have affordable and accessible vaccine and monoclonal antibody technologies across the globe. In comparison to the western countries, Asian and African countries have less percentage of vaccination done which warrants urgent attention. Global manufacturer production capacities, dependency on advanced nations for the supply of vaccines or the raw material, national economy, limited research facilities, and logistics could be the factors. This review article elaborates the existing therapeutic and prophylactic strategies available for COVID-19, currently adopted vaccine and monoclonal antibody platforms for SARS-CoV-2 along with the approaches to bridge the gap prevailing in the challenges faced by low- and middle-income countries. We believe adoption of yeast-derived P. pastoris technology can help in developing safe, proven, easy to scale-up, and affordable recombinant vaccine or monoclonal antibodies against SARS-CoV-2. This platform has the advantage of not requiring a dedicated or specialized facility making it an affordable option using existing manufacturing facilities, without significant additional capital investments. Besides, the technology platform of multiantigen vaccine approach and monoclonal antibody cocktail will serve as effective weapons to combat the threat posed by the SARS-CoV-2 variants. Successful development of vaccines and monoclonal antibodies using such a technology will lead to self-sufficiency of these nations in terms of availability of vaccines and monoclonal antibodies.

Capacity Building for Vaccine Manufacturing Across Developing Countries: The Way Forward.

Approved vaccines prevent 2 to 3 million deaths per year. There is a lack of equitable access to vaccines in the low- and middle-income developing nations. Challenges in the life cycle of vaccine production include process development, lead time, intellectual property, and local vaccine production. A robust and stable manufacturing process and constant raw material supplies over decades is critical. In a continuously evolving vaccine landscape, the need of the hour for developing nations is to manufacture their own vaccines besides having supply security, control over production scheduling and sustainability, control of costs, socio-economic development, and rapid response to local epidemics. There is a need for capacity building of workforce development, technology transfer, and financial support. Technology transfer has improved vaccine access and reduced prices of vaccines. Capacity building for the manufacturing of vaccines in developing countries has always been an area of paramount importance and more so in a pandemic situation.

Facilitated self-assembly of a prevascularized dermal/epidermal collagen scaffold.

Introduction: Resurfacing complex full thickness wounds requires free tissue transfer which creates donor site morbidity. We describe a method to fabricate a skin flap equivalent with a hierarchical microvascular network. Materials & methods: We fabricated a flap of skin-like tissue containing a hierarchical vascular network by sacrificing Pluronic® F127 macrofibers and interwoven microfibers within collagen encapsulating human pericytes and fibroblasts. Channels were seeded with smooth muscle and endothelial cells. Constructs were topically seeded with keratinocytes. Results: After 28 days in culture, multiphoton microscopy revealed a hierarchical interconnected network of macro- and micro-vessels; larger vessels (>100 µm) were lined with a monolayer endothelial neointima and a subendothelial smooth muscle neomedia. Neoangiogenic sprouts formed in the collagen protodermis and pericytes self-assembled around both fabricated vessels and neoangiogenic sprouts. Conclusion: We fabricated a prevascularized scaffold containing a hierarchical 3D network of interconnected macro- and microchannels within a collagen protodermis subjacent to an overlying protoepidermis with the potential for recipient microvascular anastomosis.

B7-H3 as a Prognostic Biomarker and Therapeutic Target in Pediatric central nervous system Tumors.

B7-H3 (CD276), a member of the B7 superfamily, is an important factor in downregulating immune responses against tumors. It is also aberrantly expressed in many human malignancies. Beyond immune regulatory roles, its overexpression has been linked to invasive metastatic potential and poor prognosis in patients with cancer. Antibody-dependent cell-mediated cytotoxicity strategies targeting B7-H3 are currently in development, and early-phase clinical trials have shown encouraging preliminary results. To understand the role of B7-H3 in pediatric central nervous system (CNS) malignancies, a comprehensive panel of primary CNS tumors of childhood was examined by immunohistochemistry for levels and extent of B7-H3 expression. In addition, B7-H3 m-RNA expression status and association with overall survival in various pediatric CNS tumor types was accessed by curating publicly available patient gene expression data sets derived from bioinformatics analysis and visualization platforms (GlioVis). We demonstrate that B7-H3 is broadly expressed in pediatric glial and nonglial CNS tumors, and its aberrant expression, as determined by immunohistochemical staining intensity, correlates with tumor grade. Moreover, high B7-H3 m-RNA expression is significantly associated with worse survival and could potentially improve prognostication in various brain tumor types of childhood. B7-H3 can be used as a therapeutic target, given its tumor selectivity and the availability of targeted therapeutic agents to this antigen.

Adipsin preserves beta cells in diabetic mice and associates with protection from type 2 diabetes in humans.

Type 2 diabetes is characterized by insulin resistance and a gradual loss of pancreatic beta cell mass and function1,2. Currently, there are no therapies proven to prevent beta cell loss and some, namely insulin secretagogues, have been linked to accelerated beta cell failure, thereby limiting their use in type 2 diabetes3,4. The adipokine adipsin/complement factor D controls the alternative complement pathway and generation of complement component C3a, which acts to augment beta cell insulin secretion5. In contrast to other insulin secretagogues, we show that chronic replenishment of adipsin in diabetic db/db mice ameliorates hyperglycemia and increases insulin levels while preserving beta cells by blocking dedifferentiation and death. Mechanistically, we find that adipsin/C3a decreases the phosphatase Dusp26; forced expression of Dusp26 in beta cells decreases expression of core beta cell identity genes and sensitizes to cell death. In contrast, pharmacological inhibition of DUSP26 improves hyperglycemia in diabetic mice and protects human islet cells from cell death. Pertaining to human health, we show that higher concentrations of circulating adipsin are associated with a significantly lower risk of developing future diabetes among middle-aged adults after adjusting for body mass index (BMI). Collectively, these data suggest that adipsin/C3a and DUSP26-directed therapies may represent a novel approach to achieve beta cell health to treat and prevent type 2 diabetes.

Non-reversible tissue fixation retains extracellular vesicles for in situ imaging.

Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.

Selenium Drives a Transcriptional Adaptive Program to Block Ferroptosis and Treat Stroke.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.

Supplemental estrogen and caloric restriction reduce obesity-induced periprostatic white adipose inflammation in mice.

Obesity is associated with an increased incidence of high-grade prostate cancer (PC) and worse prognosis for PC patients. Recently, we showed in men that obesity-related periprostatic white adipose tissue (WAT) inflammation, characterized by macrophages surrounding dead or dying adipocytes forming crown-like structures, was associated with high-grade PC. Possibly, interventions that suppress periprostatic WAT inflammation will improve outcomes for men with PC. Here, we tested the hypothesis that supplemental 17ß-estradiol (E2) could decrease periprostatic WAT inflammation in obese male mice. Mice were fed a high-fat diet to induce periprostatic WAT inflammation before being treated with supplemental E2. E2 supplementation suppressed caloric intake, induced weight loss, decreased periprostatic WAT inflammation and downregulated the expression of genes linked to inflammation including Cd68, Mcp1 and Tnf. Similar to the effects of E2 supplementation, treatment with diethylstilbestrol, a synthetic estrogen, also suppressed caloric intake and reduced periprostatic WAT inflammation. To determine whether the observed effects of supplemental estrogen could be reproduced by caloric restriction (CR) alone, obese mice were put on a 30% CR diet. Like estrogen treatment, CR was effective in reducing body weight, periprostatic WAT inflammation and the expression of pro-inflammatory genes. Transcriptomic analyses of periprostatic fat showed that obesity was associated with enrichment in inflammatory response pathways, which were normalized by both supplemental E2 and CR. Taken together, these findings strengthen the rationale for future efforts to determine whether either CR or supplemental estrogen will decrease periprostatic WAT inflammation and thereby improve outcomes for men with PC.

Exploring Multiphoton Microscopy as a Novel Tool to Differentiate Chromophobe Renal Cell Carcinoma From Oncocytoma in Fixed Tissue Sections.

CONTEXT: - Distinguishing chromophobe renal cell carcinoma (chRCC), especially in the presence of eosinophilic cytoplasm, from oncocytoma on hematoxylin-eosin can be difficult and often requires time-consuming ancillary procedures that ultimately may not be informative. OBJECTIVE: - To explore the potential of multiphoton microscopy (MPM) as an alternative and rapid diagnostic tool in differentiating oncocytoma from chRCC at subcellular resolution without tissue processing. DESIGN: - Unstained, deparaffinized tissue sections from 27 tumors (oncocytoma [n = 12], chRCC [n = 12], eosinophilic variant of chRCC [n = 1], and atypical oncocytic renal neoplasm [n = 2]) were imaged with MPM. Morphologic evaluation and automated quantitative morphometric analysis were conducted to distinguish between chRCC and oncocytoma. RESULTS: - The typical cases of oncocytomas (12 of 12) and chRCC (12 of 12) could be readily differentiated on MPM based on the morphologic features similar to hematoxylin-eosin. The most striking MPM signature of both of the tumors was the presence of autofluorescent intracytoplasmic granules, which are not seen on hematoxylin-eosin-stained slides. Although we saw these granules in both types of tumors, they appeared distinct, based on their size, shape, cytoplasmic distribution, and autofluorescence wavelengths, and were valuable in arriving at a definitive diagnosis. For oncocytomas and chRCC, high diagnostic accuracies of 100% and 83.3% were achieved on blinded MPM and morphometric analysis, respectively. CONCLUSIONS: - To the best of our knowledge, this is the first demonstration of MPM to distinguish chRCC from oncocytoma in fixed tissues. Our study was limited by small sample size and only a few variants of oncocytic tumors. Prospective studies are warranted to assess the utility of MPM as a diagnostic aid in oncocytic renal tumors.

Fabrication of capillary-like structures with Pluronic F127® and Kerria lacca resin (shellac) in biocompatible tissue-engineered constructs.

The fabrication of large cellular tissue-engineered constructs is currently limited by an inability to manufacture internal vasculature that can be anastomosed to the host circulatory system. Creation of synthetic tissues with microvascular networks that adequately mimic the size and density of in vivo capillaries remains one of the foremost challenges within tissue engineering, as cells must reside within 200-300 µm of vasculature for long-term survival. In our previous work, we used a sacrificial microfibre technique whereby Pluronic® F127 fibres were embedded and then sacrificed within a collagen matrix, leaving behind a patent channel, which was subsequently seeded with endothelial and smooth muscle cells, forming a neointima and neomedia. We now have extended our technique and describe two approaches to synthesize a biocompatible tissue-engineered construct with macro-inlet and -outlet vessels, bridged by a dense network of cellularized microvessels, recapitulating the hierarchical organization of an arteriole, venule and capillary bed, respectively. Copyright © 2016 John Wiley & Sons, Ltd.

Multifunctional in vivo imaging of pancreatic islets during diabetes development.

Pancreatic islet dysfunction leading to insufficient glucose-stimulated insulin secretion triggers the clinical onset of diabetes. How islet dysfunction develops is not well understood at the cellular level, partly owing to the lack of approaches to study single islets longitudinally in vivo Here, we present a noninvasive, high-resolution system to quantitatively image real-time glucose metabolism from single islets in vivo, currently not available with any other method. In addition, this multifunctional system simultaneously reports islet function, proliferation, vasculature and macrophage infiltration in vivo from the same set of images. Applying our method to a longitudinal high-fat diet study revealed changes in islet function as well as alternations in islet microenvironment. More importantly, this label-free system enabled us to image real-time glucose metabolism directly from single human islets in vivo for the first time, opening the door to noninvasive longitudinal in vivo studies of healthy and diabetic human islets.

Multiphoton microscopy for rapid histopathological evaluation of kidney tumours.

OBJECTIVE: To explore the potential of multiphoton microscopy (MPM) for rapid evaluation and triaging of ex vivo kidney tissue. MATERIALS AND METHODS: Fresh neoplastic and non-neoplastic tissues from nephrectomy specimens (n = 40) were imaged with MPM and later submitted for routine histopathology. RESULTS: On MPM, normal kidney architecture was evident and clearly distinguishable from tumour. Forty malignant tumours (20 clear-cell renal cell carcinomas [RCCs], 10 papillary RCCs, five chromophobe RCCs and five papillary urothelial carcinomas [UCs], as diagnosed by haematoxylin and eosin staining) were imaged and subtyped as non-papillary and papillary, based on their architecture. Non-papillary tumours were further classified based on their unique cytoplasmic signatures. Clear-cell RCCs had a predominant population of cells with fat droplets in cytoplasm. Chromophobe RCCs had cells with non-fatty/homogeneous cytoplasm and distinct intra-cytoplasmic granules. Papillary RCCs had single-cell-lined papillae with often abundant histiocytes in their core, whereas PUC had multi-layered urothelium-lined papillae. The diagnostic accuracy of tumour subtyping by two independent uropathologists was 95%. CONCLUSIONS: We showed that MPM can reliably differentiate neoplastic from non-neoplastic kidney tissue and subtype kidney tumours in fresh, unprocessed tissue, MPM might therefore be useful as a rapid real-time diagnostic tool for the evaluation of kidney biopsies, and surgical margins in partial nephrectomies, to improve overall patient management.

Rapid evaluation of fresh ex vivo kidney tissue with full-field optical coherence tomography.

BACKGROUND: Full-field optical coherence tomography (FFOCT) is a real-time imaging technique that rapidly generates images reminiscent of histology without any tissue processing, warranting its exploration for evaluation of ex vivo kidney tissue. METHODS: Fresh tissue sections from tumor and adjacent nonneoplastic kidney (n = 25 nephrectomy specimens; clear cell renal cell carcinoma (CCRCC) = 12, papillary RCC (PRCC) = 4, chromophobe RCC (ChRCC) = 4, papillary urothelial carcinoma (PUC) = 1, angiomyolipoma (AML) = 2 and cystic nephroma = 2) were imaged with a commercial FFOCT device. Sections were submitted for routine histopathological diagnosis. RESULTS: Glomeruli, tubules, interstitium, and blood vessels were identified in nonneoplastic tissue. In tumor sections, the normal architecture was completely replaced by either sheets of cells/trabeculae or papillary structures. The former pattern was seen predominantly in CCRCC/ChRCC and the latter in PRCC/PUC (as confirmed on H&E). Although the cellular details were not very prominent at this resolution, we could identify unique cytoplasmic signatures in some kidney tumors. For example, the hyper-intense punctate signal in the cytoplasm of CRCC represents glycogen/lipid, large cells with abundant hyper-intense cytoplasm representing histiocytes in PRCC, and signal-void large polygonal cell representing adipocytes in AML. According to a blinded analysis was performed by an uropathologist, all nonneoplastic tissues were differentiated from neoplastic tissues. Further, all benign tumors were called benign and malignant were called malignant. A diagnostic accuracy of 80% was obtained in subtyping the tumors. CONCLUSION: The ability of FFOCT to reliably differentiate nonneoplastic from neoplastic tissue and identify some tumor types makes it a valuable tool for rapid evaluation of ex vivo kidney tissue e.g. for intraoperative margin assessment and kidney biopsy adequacy. Recently, higher resolution images were achieved using an experimental FFOCT setup. This setup has the potential to further increase the diagnostic accuracy of FFOCT.

Multiphoton microscopy: a potential intraoperative tool for the detection of carcinoma in situ in human bladder.

CONTEXT: Urothelial carcinoma in situ (CIS) is a precursor of invasive bladder cancer, which if left untreated, will likely progress to more aggressive disease. Approximately 50% of CIS lesions are missed on routine cystoscopy owing to their flat architecture. Furthermore, many benign but abnormal-appearing areas may be biopsied owing to lack of cellular resolution of cystoscopes. Multiphoton microscopy (MPM) is an optical imaging technique that generates subcellular-resolution three-dimensional images from unfixed tissue without using exogenous dyes. OBJECTIVE: To assess the diagnostic potential of MPM in identifying and differentiating benign from malignant flat bladder lesions, especially CIS. DESIGN: Seventy-eight specimens (benign = 46, CIS = 23, invasive = 9, as diagnosed on histopathology) were obtained from flat bladder mucosa via transurethral resection of bladder, cold cup biopsy, or cystectomy, imaged fresh with a commercial benchtop MPM, and submitted for routine histopathology. Multiphoton microscopy and hematoxylin-eosin diagnoses were compared. RESULTS: In 77 of 78 specimens (99%), accurate MPM diagnoses (benign/malignant) were given on the basis of their architectural and cytologic features (nuclear to cytoplasmic ratio, pleomorphism, polarity/organization of urothelial layers, etc). The sensitivity and specificity were 97% and 100%, respectively, with positive (malignant) and negative (benign) predictive values of 100% and 98%, respectively. The interobserver agreement, κ, was 0.93. CONCLUSIONS: Our study demonstrates the capability of MPM to identify and differentiate benign from malignant flat bladder lesions, especially CIS. With the advent of MPM endoscopes, we foresee their potential as a biopsy guidance tool for early detection and treatment of CIS, thus reducing the rate of biopsies with benign diagnoses and their associated complications.

Real-time in vivo periprostatic nerve tracking using multiphoton microscopy in a rat survival surgery model: a promising pre-clinical study for enhanced nerve-sparing surgery.

OBJECTIVES: To assess the ability of multiphoton microscopy (MPM) to visualise, differentiate and track periprostatic nerves in an in vivo rat model, mimicking real-time imaging in humans during RP and to investigate the tissue toxicity and reproducibility of in vivo MPM on prostatic glands in the rat after imaging and final histological correlation study. MATERIALS AND METHODS: In vivo prostatic rat imaging was carried out using a custom-built bench-top MPM system generating real-time three-dimensional histological images, after performing survival surgery consisting of mini-laparotomies under xylazine/ketamine anaesthesia exteriorising the right prostatic lobe. The acquisition time and the depth of anaesthesia were adjusted for collecting multiple images in order to track the periprostatic nerves in real-time. The rats were then monitored for 15 days before undergoing a new set of imaging under similar settings. After humanely killing the rats, their prostates were submitted for routine histology and correlation studies. RESULTS: In vivo MPM images distinguished periprostatic nerves within the capsule and the prostatic glands from fresh unprocessed prostatic tissue without the use of exogenous contrast agents or biopsy sample. Real-time nerve tracking outlining the prostate was feasible and acquisition was not disturbed by motion artefacts. No serious adverse event was reported during rat monitoring; no tissue damage due to laser was seen on the imaged lobe compared with the contralateral lobe (control) allowing comparison of their corresponding histology. CONCLUSIONS: For the first time, we have shown that in vivo tracking of periprostatic nerves using MPM is feasible in a rat model. Development of a multiphoton endoscope for intraoperative use in humans is currently in progress and must be assessed.

Genetic variations in colorectal cancer risk and clinical outcome.

Colorectal cancer (CRC) has an apparent hereditary component, as evidenced by the well-characterized genetic syndromes and family history associated with the increased risk of this disease. However, in a large fraction of CRC cases, no known genetic syndrome or family history can be identified, suggesting the presence of "missing heritability" in CRC etiology. The genome-wide association study (GWAS) platform has led to the identification of multiple replicable common genetic variants associated with CRC risk. These newly discovered genetic variations might account for a portion of the missing heritability. Here, we summarize the recent GWASs related to newly identified genetic variants associated with CRC risk and clinical outcome. The findings from these studies suggest that there is a lack of understanding of the mechanism of many single nucleotide polymorphisms (SNPs) that are associated with CRC. In addition, the utility of SNPs as prognostic markers of CRC in clinical settings remains to be further assessed. Finally, the currently validated SNPs explain only a small fraction of total heritability in complex-trait diseases like CRC. Thus, the "missing heritability" still needs to be explored further. Future epidemiological and functional investigations of these variants will add to our understanding of CRC pathogenesis, and may ultimately lead to individualized strategies for prevention and treatment of CRC.

Photophysics of glycosylated derivatives of a chlorin, isobacteriochlorin and bacteriochlorin for photodynamic theragnostics: discovery of a two-photon-absorbing photosensitizer.

The photophysical properties of a chlorin, isobacteriochlorin and bacteriochlorin built on a core tetrapentafluorophenylporphyrin (TPPF20 ) and the nonhydrolyzable para thioglycosylated conjugates of these chromophores are presented. The photophysical characterization of these compounds was done in three different solvents to correlate with different environments in cells and tissues. Compared with TPPF20 other dyes have greater absorption in the red region of the visible spectrum and greater fluorescence quantum yields. The excited state lifetimes are from 3 to 11 ns. The radiative and nonradiative rate constants for deactivation of the excited state were estimated from the fluorescence quantum yield and excited state lifetime. The data indicate that the bacteriochlorin has strong absorption bands near 730 nm and efficiently enters the triplet manifold. The isobacteriochlorin has a 40-70% fluorescence quantum yield depending on solvent, so it may be a good fluorescent tag. The isobacteriochlorins also display enhanced two-photon absorption, thereby allowing the use of 860 nm light to excite the compound. While the two-photon cross section of 25 GM units is not large, excitation of low chromophore concentrations can induce apoptosis. The glycosylated compounds accumulate in cancer cells and a head and neck squamous carcinoma xenograft tumor model in mice. These compounds are robust to photobleaching.

Microsurgical denervation of rat spermatic cord: safety and efficacy data.

OBJECTIVE: To describe a microsurgical technique for denervation of the spermatic cord and use of multiphoton microscopy (MPM) laser to identify and ablate residual nerves after microsurgical denervation. To evaluate structural and functional changes in the rat testis and vas deferens after denervation. MATERIALS AND METHODS: Nine Sprague-Dawley rats were divided into three experimental groups: sham, microsurgical denervation of the spermatic cord (MDSC), and MDSC immediately followed by laser ablation with MPM. At 2 months after surgery, we assessed testicular volume, functional circulation of the testicular artery with Doppler, patency of the vas deferens, and histology of the testis and vas deferens. RESULTS: There was a significant decrease in the median number of nerves remaining around the vas deferens with MDSC alone (3.5 nerves) or MDSC with MPM (1.5 nerves) compared with sham rats (15.5 nerves) (P = 0.003). Although, MDSC with MPM resulted in the fewest remaining nerves, this result was similar to MDSC alone (P = 0.29). No deleterious effects on spermatogenesis or vas patency were seen in the experimental groups when compared with the sham rats. CONCLUSION: A microsurgical approach can be used to effectively and safely denervate the rat spermatic cord with minimal changes to structure and function of the testis and vas deferens. MPM can be used as an adjunct to identify and ablate residual nerves after MDSC.

Multiphoton microscopy: a potential "optical biopsy" tool for real-time evaluation of lung tumors without the need for exogenous contrast agents.

CONTEXT: Multiphoton microscopy (MPM) is an emerging, nonlinear, optical-biopsy technique, which can generate subcellular-resolution images from unprocessed and unstained tissue in real time. OBJECTIVE: To assess the potential of MPM for lung tumor diagnosis. DESIGN: Fresh sections from tumor and adjacent nonneoplastic lung were imaged with MPM and then compared with corresponding hematoxylin-eosin slides. RESULTS: Alveoli, bronchi, blood vessels, pleura, smokers' macrophages, and lymphocytes were readily identified with MPM in nonneoplastic tissue. Atypical adenomatous hyperplasia (a preinvasive lesion) was identified in tissue adjacent to the tumor in one case. Of the 25 tumor specimens used for blinded pathologic diagnosis, 23 were diagnosable with MPM. Of these 23 cases, all but one adenocarcinoma (15 of 16; 94%) was correctly diagnosed on MPM, along with their histologic patterns. For squamous cell carcinoma, 4 of 7 specimens (57%) were correctly diagnosed. For the remaining 3 squamous cell carcinoma specimens, the solid pattern was correctly diagnosed in 2 additional cases (29%), but it was not possible to distinguish the squamous cell carcinoma from adenocarcinoma. The other squamous cell carcinoma specimen (1 of 7; 14%) was misdiagnosed as adenocarcinoma because of pseudogland formation. Invasive adenocarcinomas with acinar and solid pattern showed statistically significant increases in collagen. Interobserver agreement for collagen quantification (among 3 observers) was 80%. CONCLUSIONS: Our pilot study provides a proof of principle that MPM can differentiate neoplastic from nonneoplastic lung tissue and identify tumor subtypes. If confirmed in a future, larger study, we foresee real-time intraoperative applications of MPM, using miniaturized instruments for directing lung biopsies, assessing their adequacy for subsequent histopathologic analysis or banking, and evaluating surgical margins in limited lung resections.