Autofluorescence Detection Research Articles

Autofluorescence of blood and its application in biomedical and clinical research.

Autofluorescence of blood has been explored as a label free approach for detection of cell types, as well as for diagnosis and detection of infection, cancer, and other diseases. Although blood autofluorescence is used to indicate the presence of several physiological abnormalities with high sensitivity, it often lacks disease specificity due to use of a limited number of fluorophores in the detection of several abnormal conditions. In addition, the measurement of autofluorescence is sensitive to the type of sample, sample preparation, and spectroscopy method used for the measurement. Therefore, while current blood autofluorescence detection approaches may not be suitable for primary clinical diagnosis, it certainly has tremendous potential in developing methods for large scale screening that can identify high risk groups for further diagnosis using highly specific diagnostic tests. This review discusses the source of blood autofluorescence, the role of spectroscopy methods, and various applications that have used autofluorescence of blood, to explore the potential of blood autofluorescence in biomedical research and clinical applications.

Near-infrared fluorescent imaging techniques for the detection and preservation of parathyroid glands during endocrine surgery.

In over 30% of all thyroid surgeries, complications arise from transient and definitive hypoparathyroidism, underscoring the need for real-time identification and preservation of parathyroid glands (PGs). Here, we evaluate the promising intraoperative optical technologies available for the identification, preservation, and functional assessment of PGs to enhance endocrine surgery. We performed a review of the literature to identify published studies on fluorescence imaging in thyroid and parathyroid surgery. Fluorescence imaging is a well-demonstrated approach for both in vivo and in vitro localization of specific cells or tissues, and is gaining popularity as a technique to detect PGs during endocrine surgery. Autofluorescence (AF) imaging and indocyanine green (ICG) angiography are two emerging optical techniques to improve outcomes in thyroid and parathyroid surgeries. Near-infrared-guided technology has significantly contributed to the localization of PGs, through the detection of glandular AF. Perfusion through the PGs can be visualized with ICG, which can also reveal the blood supply after dissection. Near infrared AF and ICG angiography, providing a valuable spatial and anatomical information, can decrease the incidence of complications in thyroid surgery.

Near-infrared autofluorescence-based parathyroid glands identification in the thyroidectomy or parathyroidectomy: a systematic review and meta-analysis.

To evaluate the diagnostic accuracy of near-infrared autofluorescence-based identification in the identification of parathyroid glands during thyroidectomy or parathyroidectomy. The clinical studies were retrieved from PubMed, the Cochrane Central Register of Controlled Trials, Embase, Web of Science, SCOPUS, and Google Scholar. The study protocol was registered on Open Science Framework ( https://osf.io/um8rj/ ). The search period ranged from the date of each database's inception to May 2021. Cohort studies dealing with patients of whom parathyroid glands were detected by near-infrared autofluorescence and confirmed clinically or pathologically during thyroidectomy or parathyroidectomy were included. Editorials, letters, "how-I-do-it" descriptions, other site head and neck tumors, and articles with lack of diagnostic identification data were excluded. True positive, true negative, false positive, and false negative were extracted. The QUDAS ver. 2 was used to evaluate the methodological quality. Seventeen studies with 1198 participants were evaluated in this analysis. Near-infrared autofluorescence-based identification of parathyroid glands showed a diagnostic odds ratio of 228.8759 (95% confidence interval, 134.1099; 390.6063). The area under the summary receiver operating characteristic curve was 0.967. The sensitivity, specificity, negative predictive value, and positive predictive value were 0.9693 (0.9491; 0.9816), 0.9248 (0.8885; 0.9499), 0.9517 (0.8981; 0.9778), and 0.9488 (0.9167; 0.9689), respectively. Subgroup analyses were performed to compare two autofluorescence detection methods, because there was high heterogeneity in the outcomes. The diagnostic accuracy was higher in probe-based detection than in image-based detection. Near-infrared autofluorescence-based identification is valuable for identifying the parathyroid glands of patients during thyroidectomy or parathyroidectomy.

Comparing intraoperative parathyroid identification based on surgeon experience versus near infrared autofluorescence detection – A surgeon-blinded multi-centric study

BackgroundNear infrared autofluorescence (NIRAF) detection has previously demonstrated significant potential for real-time parathyroid gland identification. However, the performance of a NIRAF detection device - PTeye® - remains to be evaluated relative to a surgeon's own ability to identify parathyroid glands. MethodsPatients eligible for thyroidectomy and/or parathyroidectomy were enrolled under 6 endocrine surgeons at 3 high-volume institutions. Participating surgeons were categorized based on years of experience. All surgeons were blinded to output of PTeye® when identifying tissues. The surgeon's performance for parathyroid discrimination was then compared with PTeye®. Histology served as gold standard for excised specimens, while expert surgeon's opinion was used to validate in-situ tissues. ResultsPTeye® achieved 92.7% accuracy across 167 patients recruited. Junior surgeons (<5 years of experience) were found to have lower confidence in parathyroid identification and higher tissue misclassification rate per specimen when compared to PTeye® and senior surgeons (>10 years of experience). ConclusionsNIRAF detection with PTeye® can be a valuable intraoperative adjunct technology to aid in parathyroid identification for surgeons.

Initial clinical experiences using the intraoperative probe-based parathyroid autofluorescence identification system-PTeye™ during thyroid and parathyroid procedures.

The Food and Drug Administration has cleared a probe-based near-infrared autofluorescence (NIRAF) detection system called PTeye™ as an adjunct tool for label-free intraoperative parathyroid gland (PG) identification. Since PTeye™ has been investigated only in a "blinded" manner to date, this study describes the preliminary impressions of PTeye™ when used by surgeons without being blinded to the device output. Patients undergoing thyroid and parathyroid procedures were prospectively recruited. Target tissues were intraoperatively assessed with PTeye™. The surgeon's confidence in PG identification was recorded concomitantly with NIRAF parameters that were output in real-time from PTeye™. A retrospective review of prospectively collected data on 83 patients was performed. PTeye™ was used for interrogating 336 target tissues in 46 parathyroid and 37 thyroid procedures. PTeye™ yielded an overall accuracy of 94.3% with a positive predictive value of 93.0% and a negative predictive value of 100%. An increase in confidence for intraoperative PG identification with PTeye™ was observed by all three participating high-volume surgeons, irrespective of their level of accrued surgical experience. Probe-based NIRAF detection with PTeye™ can be a valuable adjunct device to intraoperatively identify PGs for surgeons of varied training and experience.

Reducing lipofuscin accumulation and cardiomyocytic senescence of aging heart by enhancing autophagy

Cardiomyocytes are particularly prone to lipofuscin accumulation. In the aging heart, lipofuscin accumulation is augmented. This study examined distribution of lipofuscin and senescent cardiomyocytes and evaluated improvement of lipofuscin accumulation and cardiomyocytic senescence of the aging heart after treatment with rapamycin. The results of Schmorl staining, Sudan black staining and autofluorescence detection showed that there was more lipofuscin in the myocardium of the ventricles especially in the left ventricle. The conductive tissue contained less lipofuscin than the myocardium. In the aged hearts, lipofuscin accumulation and senescent cardiomyocytes were increased, and the level of autophagy was reduced. In double staining of Sudan black B and senescence-associated β-galactosidase, 10%–20% lipofuscin-loaded cardiomyocytes became senescent. All senescent cardiomyocytes contained lipofuscin deposits. After enhancing autophagy with feed of rapamycin for six months, lipofuscin accumulation and senescence of cardiomyocytes were improved in old rats. Colocalization of autophagic structure and lipofuscin as well as electron micrographs showed that some lipofuscin-loaded lysosomes were sequestrated by autophagic structures. This study suggests that rapamycin-enhanced autopahgy is effective for reducing lipofuscinogenesis and promoting degradation of lipofuscin. Therefore, enhancing autophagy is a novel therapy for alleviating lipofuscin accumulation and myocardial senescence.

Unbiased analysis of obesity related, fat depot specific changes of adipocyte volumes and numbers using light sheet fluorescence microscopy.

In translational obesity research, objective assessment of adipocyte sizes and numbers is essential to characterize histomorphological alterations linked to obesity, and to evaluate the efficacies of experimental medicinal or dietetic interventions. Design-based quantitative stereological techniques based on the analysis of 2D-histological sections provide unbiased estimates of relevant 3D-parameters of adipocyte morphology, but often involve complex and time-consuming tissue processing and analysis steps. Here we report the application of direct 3D light sheet fluorescence microscopy (LSFM) for effective and accurate analysis of adipocyte volumes and numbers in optically cleared adipose tissue samples from a porcine model of diet-induced obesity (DIO). Subcutaneous and visceral adipose tissue samples from DIO-minipigs and lean controls were systematically randomly sampled, optically cleared with 3DISCO (3-dimensional imaging of solvent cleared organs), stained with eosin, and subjected to LSFM for detection of adipocyte cell membrane autofluorescence. Individual adipocytes were unbiasedly sampled in digital 3D reconstructions of the adipose tissue samples, and their individual cell volumes were directly measured by automated digital image analysis. Adipocyte numbers and mean volumes obtained by LSFM analysis did not significantly differ from the corresponding values obtained by unbiased quantitative stereological analysis techniques performed on the same samples, thus proving the applicability of LSFM for efficient analysis of relevant morphological adipocyte parameters. The results of the present study demonstrate an adipose tissue depot specific plasticity of adipocyte growth responses to nutrient oversupply. This was characterized by an exclusively hypertrophic growth of visceral adipocytes, whereas adipocytes in subcutaneous fat tissue depots also displayed a marked (hyperplastic) increase in cell number. LSFM allows for accurate and efficient determination of relevant quantitative morphological adipocyte parameters. The applied stereological methods and LSFM protocols are described in detail and can serve as a guideline for unbiased quantitative morphological analyses of adipocytes in other studies and species.

Correlation between temperature rise and auto-fluorescence: ex vivo study with Er:YAG laser, Quantum Molecular Resonance scalpel and Nd:YAG laser on hard and soft tissues.

Surgical techniques of soft and hard oral tissues highly benefited from new technologies such as the Quantic Molecular Resonance (QMR) lancet, the Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser and the Erbium-doped Yttrium Aluminum Garnet (Er:YAG) laser. Increasingly, these technologies replace scalpel, conventional electrosurgery and traditional rotary surgery instruments due to their proven advantages. Features such as reduction of the surgical time, more efficient bleeding control resulting in higher intra-operative visibility and improvement of postoperative course with better Quality of Life score (QoL) are highlighted in numerous studies published in the literature. The thermal rise of tissues during surgical incision, performed with other instruments rather than traditional cold blade scalpels, is not to be ignored by the operator and it must take into consideration first when choosing the surgical instrument and then throughout all the surgical act. Auto-fluorescence (AF) is a property possessed by every cell that exposed to a specific wavelength can absorbance or reflect with peculiar characteristics and its direct examination has been proposed as a non-invasive visual tool for investigation of suspicious changes in oral mucosa. At the limit of our knowledge, few studies have been published in the literature regarding tissue's temperature variations and the interest in Infra-Red temperature detection has been shown in various medicine fields and none of published studies investigated the possible correlation between temperature raise and AF variations. This ex vivo study aims to analyse and compare through the use of a thermal imaging camera and simultaneous detection of AF, the possible correlation between temperature increase and auto-fluorescence.

Development of an imaging device for label-free parathyroid gland identification and vascularity assessment.

During thyroid surgeries, it is important for surgeons to accurately identify healthy parathyroid glands and assess their vascularity to preserve their function postoperatively, thus preventing hypoparathyroidism and hypocalcemia. Near infrared autofluorescence detection enables parathyroid identification, while laser speckle contrast imaging allows assessment of parathyroid vascularity. Here, we present an imaging system combining the two techniques to perform both functions, simultaneously and label-free. An algorithm to automate the segmentation of a parathyroid gland in the fluorescence image to determine its average speckle contrast is also presented, reducing a barrier to clinical translation. Results from imaging ex vivo tissue samples show that the algorithm is equivalent to manual segmentation. Intraoperative images from representative procedures are presented showing successful implementation of the device to identify and assess vascularity of healthy and diseased parathyroid glands.

Spatially-Resolved Multiply-Excited Autofluorescence and Diffuse Reflectance Spectroscopy: SpectroLive Medical Device for Skin In Vivo Optical Biopsy

This contribution presents the development of an optical spectroscopy device, called SpectroLive, that allows spatially-resolved multiply-excited autofluorescence and diffuse reflectance measurements. Besides describing the device, this study aims at presenting the metrological and safety regulation validations performed towards its aimed application to skin carcinoma in vivo diagnosis. This device is made of six light sources and four spectrometers for detection of the back-scattered intensity spectra collected through an optical probe (made of several optical fibers) featuring four source-to-detector separations (from 400 to 1000 µm). In order to be allowed by the French authorities to be evaluated in clinics, the SpectroLive device was successfully checked for electromagnetic compatibility and electrical and photobiological safety. In order to process spectra, spectral correction and metrological calibration were implemented in the post-processing software. Finally, we characterized the device’s sensitivity to autofluorescence detection: excitation light irradiance at the optical probe tip in contact with skin surface ranges from 2 to 11 W/m², depending on the light source. Such irradiances combined to sensitive detectors allow the device to acquire a full spectroscopic sequence within 6 s which is a short enough duration to be compatible with optical-guided surgery. All these results about sensitivity and safety make the SpectroLive device mature enough to be evaluated through a clinical trial that aims at evaluating its diagnostic accuracy for skin carcinoma diagnosis.

Boosting the properties of a fluorescent dye by encapsulation into halloysite nanotubes

The synthesis of a new biocompatible bichromophoric system (CURBO) was developed, by connecting the skeleton of the naturally occurring curcumin to a BODIPY derivative. The system exhibited an intense fluorescence band with maximum at about 510 nm in organic solvent, while its emission spectra in aqueous solution were more complicated and slightly red-shifted, due to the effect of aggregation for the poor solubility of the dyad. To overcome these problems, the bichomophoric system has been loaded into the halloysite nanotubes (HNT). The HNT/CURBO nanocomposite, suspended in aqueous solution, showed an intensity of emission in the red region of the spectrum higher than the one exhibited by the pristine CURBO. The biocompatibility and photophysical behavior of HNT/CURBO nanocomposite were studied using ASC52Telo, h-TERT immortalized adipose-derived mesenchymal stem cells. To investigate the localization of the CURBO alone and in HNT/CURBO, fluorescence epimicroscopy was performed. The HNT/CURBO nanocomposite showed a high propensity to cross cell membranes, resulting in a massive cell uptake around the cell nucleus. Moreover, it lighted up ASC52Telo cells both with intense green and red fluorescence while excited in the blue portion of the electromagnetic spectrum, a phenomenon that could not be observed for the free CURBO. Dual-channel emissions dyes and, in particular, materials, such as the HNT/CURBO nanocomposite, emitting in the red portion of the spectrum have become, in recent decades, particularly fascinating since they can minimize the autofluorescence detection of the cell medium and allow interesting microscopy applications.

Clinical Application of Near-infrared Autofluorescence Detection of Parathyroid Glands in Thyroid Surgery

The purpose of this paper is to compare and analyze the advantages and disadvantages of near-infrared autofluorescence (NIRAF) imaging, nano-carbon negative imaging and conventional naked eye recognition technology in recognizing parathyroid glands during surgery, and to preliminary evaluate the effectiveness, safety and feasibility of NIRAF imaging in the recognition of parathyroid glands. The clinical trials retrospectively analyzed 36 patients, including 12 patients in Group A (NIRAF imaging group), 12 patients in Group B (nano-carbon negative imaging group), and 12 patients in Group C (conventional naked eye recognition group). The number of parathyroid glands recognized during surgery in Group A was more than that in Group B and C. The time taken to recognize parathyroid glands in Group A was shorter than that in Group B and C. There was no significant difference in the average calcium ion levels of the three groups A, B and C before surgery. After surgery, the average calcium ion level of Group A was not significantly different from that of Group B, but it was higher than that of Group C, and the difference was significant. The incidence of postoperative hypocalcemia in Group A was not significantly different from that in Group B, but it was lower than that in Group C. The incidence of postoperative symptomatic hypocalcemia in Group A was lower than that in Group B and C. Compared with traditional technology, NIRAF can recognize more parathyroid glands during surgery, take less time, and make the probability of postoperative hypocalcemia and symptomatic hypocalcemia lower during thyroid surgery. It needs noninvasive and simple operation. It is a kind of safe, effective and feasible parathyroid gland recognition technology, which is worth further exploration.

Usefulness of Autofluorescence Video-Monitoring to Enhanced Localization of Parathyroid Glands

Background and Objectives Near-infrared (NIR) fluorescence photo imaging provides real time parathyroid anatomy enhancement. Moreover, autofluorescence enables intraoperative virtual reality parathyroid exploration of the optical characteristics of the parathyroid gland. This study was performed to demonstrate the new technique of visualizing the parathyroid gland using video-guided autofluorescence during thyroid and parathyroid surgery and to evaluate the outcomes. This is the first study that introduces the video-monitoring technique for intraoperative parathyroid mapping.Subjects and Method A total of 26 patients underwent 18 total thyroidectomies and 8 hemithyroidectomies in 2016. Fifty-six parathyroid glands were enrolled in this study. Surgery was performed by NIR video-monitoring via thyroid lateral side dissection to find the parathyroid tissues and extract the thyroid glands. With the operation room light turned on, the parathyroid glands were identified by the video-guided autofluorescence detection technique carried out in 3 stages (P1, P2, and P3), which are imaging with surgeon’s eyes before parathyroids exposure (P1), after identification (P2), and in extracted specimen (P3).Results The parathryoid autofluorescence could be video-monitored in real time by our NIR camera system with the indoor room light turned on. Of the total 56 parathyroids, 52 were detected by fluorescence. Of these, the location of 43 glands were predicted by using the high signal in a before-exposure state and the glands were confirmed as containing parathyroid tissues [in P1, sensitivity=82.69%, positive predictive value (PPV)=100.00%]. Of the nine glands that did not show high signals in P1, seven glands visually showed fluorescence signals (in P1 and P2, sensitivity=96.15%, PPV=100.00%). One of the two glands that showed high signals in the extracted tissue was identified as parathyroid, but the other one was proved not by histologic examination by despite high intensity fluorescence signal (in P1-P3, sensitivity=100.00%, PPV=98.08%). The accuracy of video-guided parathyroid mapping in P1, P2, and P3 were 83.93%, 96.43%, and 96.43%, respectively.Conclusion This is the first study that demonstrates the parathyroid gland autofluorescence as a real-time video-monitoring technique and shows that it could be applied to real surgery. Although parathyroid autofluorescence is a phenomenon seen in the invisible wavelength, our data suggest that the operator can see the parathyroid fluorescent signal in real time on the video-monitor. This technique could help the operator to predict the gland location and preserve them safely.

Higher skin autofluorescence detection using AGE-Reader™ technology as a measure of increased tissue accumulation of advanced glycation end products in dialysis patients with diabetes: a meta-analysis.

This meta-analysis was conducted to compare skin autofluorescence (SAF) levels as a measure of advanced glycation end products (AGEs) accumulation between diabetic and non-diabetic dialysis patients. Relevant studies were identified primarily by searching PubMed/MEDLINE and other online databases and manual searching of cross-references. Random-effects model was used to obtain standardized mean differences (SMD) as a summary statistic. Heterogeneity was tested with meta-regression and sub-group analyses. Publication bias and robustness of this meta-analysis were tested using funnel plot and sensitivity analyses. A total of 11 reports comparing SAF between diabetic and non-diabetic dialysis patients were finally included in this meta-analysis. Dialysis patients with diabetes showed significantly higher circulatory SAF levels than their non-diabetic counterparts (SMD = 0.39; P < 0.0001). The difference was more evident in hemodialysis modality (SMD = 0.40; P < 0.0001). A one-study leave-out sensitivity analysis validated the robustness of this meta-analysis. The presence of diabetes in dialysis patients is associated with increased AGEs accumulation reflected through SAF measurements using a novel and non-invasive AGE-Reader™ technology. Our findings are in support of considering anti-AGE therapeutic strategies in addition to standard glycemic management in dialysis patients with diabetes.

Dual-band fluorosensor for discriminating non-eating from algae-eating zooplankton in aquatic environments

We present a fluorosensor for the detection of laser-induced autofluorescence of zooplankton in marine environments. The sensor uses an inexpensive 410 nm laser diode as excitation source and simultaneously measures two fluorescence bands, 500-550 nm and 675-725 nm, using two identical 16-bit linear array detectors. We show continuous measurements at 200 Hz of zooplankton swimming through a water volume illuminated by the 410 nm laser. The sensor can distinguish salmon lice (Lepeophtheirus salmonis) larvae from an algae-eating reference species (Acartia tonsa) with a sensitivity of up to 99%. The system successfully differentiates the two species using mixed-species cultures at different ratios. This work shows the potential of fluorescent pest monitoring in the salmon farming industry and paves the way for single-ended aquatic lidars.

Recent Advances and the Potential for Clinical Use of Autofluorescence Detection of Extra-Ophthalmic Tissues.

The autofluorescence (AF) characteristics of endogenous fluorophores allow the label-free assessment and visualization of cells and tissues of the human body. While AF imaging (AFI) is well-established in ophthalmology, its clinical applications are steadily expanding to other disciplines. This review summarizes clinical advances of AF techniques published during the past decade. A systematic search of the MEDLINE database and Cochrane Library databases was performed to identify clinical AF studies in extra-ophthalmic tissues. In total, 1097 articles were identified, of which 113 from internal medicine, surgery, oral medicine, and dermatology were reviewed. While comparable technological standards exist in diabetology and cardiology, in all other disciplines, comparability between studies is limited due to the number of differing AF techniques and non-standardized imaging and data analysis. Clear evidence was found for skin AF as a surrogate for blood glucose homeostasis or cardiovascular risk grading. In thyroid surgery, foremost, less experienced surgeons may benefit from the AF-guided intraoperative separation of parathyroid from thyroid tissue. There is a growing interest in AF techniques in clinical disciplines, and promising advances have been made during the past decade. However, further research and development are mandatory to overcome the existing limitations and to maximize the clinical benefits.

Detection of parathyroid gland auto-fluorescence using a near infrared fluorescence imaging: comparison with pde-neo® and FLUOBEAM 800®

Detection of parathyroid gland auto-fluorescence using a near infrared fluorescence imaging: comparison with pde-neo® and FLUOBEAM 800®

Modification of the technique for detection and follow-up of diseases of the anterior ocular segment using the conjunctival autofluorescence method

Every person is in constant interaction with the environmental factors, with the sun being the highest energy source. The electromagnetic spectrum includes rays of different wavelengths, with shortwave ultraviolet rays being an invisible, potential source of damage. For the purpose of objectively documenting and tracking the UV changes on the anterior ocular surface, a detection method based on conjunctival autofluorescence has been developed. The purpose of this presentation is to present a modified technique for the diagnosis of some common diseases and to demonstrate the results of a study among patients with pterygium monitored in the clinic with a conjunctival autofluorescence detection camera.

The Influence of Volume and Anatomic Location of Optic Disc Drusen on the Sensitivity of Autofluorescence.

Optic disc drusen (ODD) are acellular deposits in the optic nerve head. ODD can be diagnosed using different imaging modalities, including enhanced depth imaging optical coherence tomography (EDI-OCT) and autofluorescence (AF). It is unknown which factors determine the sensitivity of AF. The aim of this study was to investigate the effect of volume and anatomic location of ODD on the sensitivity of AF. Cross-sectional study. A total of 38 patients (75 eyes) with ODD were included. In 12 of 75 eyes (16%) and in 11 of 38 patients (29%), EDI-OCT detected ODD that were not detected by AF. In 24 distinctly solitary ODD, both increase in ODD volume (P = 0.0388) and a more superficial ODD location (P < 0.0001) increased the possibility of AF detection of ODD, when performing a multivariate analysis. EDI-OCT is superior to AF in the diagnosis of ODD. Volume and anatomic location of ODD have a significant impact on the sensitivity of AF.

Enhancing Parathyroid Gland Visualization Using a Near Infrared Fluorescence-Based Overlay Imaging System

Misidentifying parathyroid glands (PGs) during thyroidectomies or parathyroidectomies could significantly increase postoperative morbidity. Imaging systems based on near infrared autofluorescence (NIRAF) detection can localize PGs with high accuracy. These devices, however, depict NIRAF images on remote display monitors, where images lack spatial context and comparability with actual surgical field of view. In this study, we designed an overlay tissue imaging system (OTIS) that detects tissue NIRAF and back-projects the collected signal as a visible image directly onto the surgical field of view instead of a display monitor, and tested its ability for enhancing parathyroid visualization. The OTIS was first calibrated with a fluorescent ink grid and initially tested with parathyroid, thyroid, and lymph node tissues exvivo. For invivo measurements, the surgeon's opinion on tissue of interest was first ascertained. After the surgeon looked away, the OTIS back-projected visible green light directly onto the tissue of interest, only if the device detected relatively high NIRAF as observed in PGs. System accuracy was determined by correlating NIRAF projection with surgeon's visual confirmation for in situ PGs or histopathology report for excised PGs. The OTIS yielded 100% accuracy when tested exvivo with parathyroid, thyroid, and lymph node specimens. Subsequently, the device was evaluated in 30 patients who underwent thyroidectomy and/or parathyroidectomy. Ninety-seven percent of exposed tissue of interest was visualized correctly as PGs by the OTIS, without requiring display monitors or contrast agents. Although OTIS holds novel potential for enhancing label-free parathyroid visualization directly within the surgical field of view, additional device optimization is required for eventual clinical use.