Autofluorescence Intensity Research Articles

Computational macroscopic lifetime imaging and concentration unmixing of autofluorescence.

Single-pixel computational imaging can leverage highly sensitive detectors that concurrently acquire data across spectral and temporal domains. For molecular imaging, such methodology enables to collect rich intensity and lifetime multiplexed fluorescence datasets. Herein we report on the application of a single-pixel structured light-based platform for macroscopic imaging of tissue autofluorescence. The super-continuum visible excitation and hyperspectral single-pixel detection allow for parallel characterization of autofluorescence intensity and lifetime. Furthermore, we exploit a deep learning based data processing pipeline, to perform autofluorescence unmixing while yielding the autofluorophores' concentrations. The full scheme (setup and processing) is validated in silico and in vitro with clinically relevant autofluorophores flavin adenine dinucleotide, riboflavin, and protoporphyrin. The presented results demonstrate the potential of the methodology for macroscopically quantifying the intensity and lifetime of autofluorophores, with higher specificity for cases of mixed emissions, which are ubiquitous in autofluorescence and multiplexed in vivo imaging.

Anterior cruciate ligament microfatigue damage detected by collagen autofluorescence in situ

PurposeCertain types of repetitive sub-maximal knee loading cause microfatigue damage in the human anterior cruciate ligament (ACL) that can accumulate to produce macroscopic tissue failure. However, monitoring the progression of that ACL microfatigue damage as a function of loading cycles has not been reported. To explore the fatigue process, a confocal laser endomicroscope (CLEM) was employed to capture sub-micron resolution fluorescence images of the tissue in situ. The goal of this study was to quantify the in situ changes in ACL autofluorescence (AF) signal intensity and collagen microstructure as a function of the number of loading cycles.MethodsThree paired and four single cadaveric knees were subjected to a repeated 4 times bodyweight landing maneuver known to strain the ACL. The paired knees were used to compare the development of ACL microfatigue damage on the loaded knee after 100 consecutive loading cycles, relative to the contralateral unloaded control knee, through second harmonic generation (SHG) and AF imaging using confocal microscopy (CM). The four single knees were used for monitoring progressive ACL microfatigue damage development by AF imaging using CLEM.ResultsThe loaded knees from each pair exhibited a statistically significant increase in AF signal intensity and decrease in SHG signal intensity as compared to the contralateral control knees. Additionally, the anisotropy of the collagen fibers in the loaded knees increased as indicated by the reduced coherency coefficient. Two out of the four single knee ACLs failed during fatigue loading, and they exhibited an order of magnitude higher increase in autofluorescence intensity per loading cycle as compared to the intact knees. Of the three regions of the ACL - proximal, midsubstance and distal - the proximal region of ACL fibers exhibited the highest AF intensity change and anisotropy of fibers.ConclusionsCLEM can capture changes in ACL AF and collagen microstructures in situ during and after microfatigue damage development. Results suggest a large increase in AF may occur in the final few cycles immediately prior to or at failure, representing a greater plastic deformation of the tissue. This reinforces the argument that existing microfatigue damage can accumulate to induce bulk mechanical failure in ACL injuries. The variation in fiber organization changes in the ACL regions with application of load is consistent with the known differences in loading distribution at the ACL femoral enthesis.

Modulation of calcium signaling by nanosecond electric pulses and cell death through apoptosis in A549 lung cancerous cells

Calcium ion (Ca2+), which serves as one of the important signaling agents, is imperative to control normal cell function. The altered Ca2+ signaling is known to play a crucial role in the resistance of cancerous cells against apoptosis. Herein, nanosecond pulsed electric field (nsPEF) was applied to non-small lung cancerous cells A549, and Ca2+ mobilization along with mitochondria-mediated apoptotic cell death that accompanies morphological changes was investigated. In the presence of nsPEF, intracellular mobilization of Ca2+ ions through the efflux from endoplasmic reticulum storage was dependent on the pulse-width of the applied field. The field-induced Ca2+ mobilization has shown to correlate with the superoxide anion (O2−) generation, which probably occurred in mitochondria utilizing the nicotinamide adenine dinucleotide (NADH), as supported by the field-induced change in autofluorescence intensity and lifetime of NADH. Mitochondrial dysfunction and O2− generation, which promoted apoptotic cell death, were also induced by nsPEF with strong correlation with intracellular Ca2+ ions.

Could trees change future behaviour in xylogenesis to improve fitness based on past and current conditions? A tropical case of study

Carbon allocation is the Achilles' heel of forest models, due to the difficulty to predict responses induced by environmental changes. Structural-functional models involve the plant's strategy to split its resources between organs and functions. An important functional characteristic is xylem architecture, which is connected to water use strategy and plant productivity besides one of the most important C-sink (that absorbs carbon). Our goal is to use the information present in the tree rings of Schizolobium parahyba, to expand the possibilities of teleonomic approaches in structural-functional models of tree species. In a comparative xylem rings analyses, we established relationships among specific gravity, vessel traits as hydraulic conductivity potential, double wall thickness, and autofluorescence intensity of lignin. The analyses showed that growth ring characteristics seem to have been modulated not only by water availability of the period of wood formation but also by the water availability of previous years. This relationship would be occurring due to the greater capacity of accumulation of reserve carbohydrates in years whose climatic conditions were favourable. This pattern of behaviour entails the formation of two distinct growth ring patterns, one with high and the other with low structural cost. This case study shows that the species can change future behaviour in xylogenesis to improve fitness based on past and current conditions. Our study may help to extend the possibilities of teleonomic approaches in structural-functional models of tree species, which will assist in a greater understanding of how trees balance their carbon allocation in wood according to changes in the environment.

Inhibition of B-cell lymphoma 2 family proteins alters optical redox ratio, mitochondrial polarization, and cell energetics independent of cell state.

.SignificanceThe optical redox ratio (ORR) [autofluorescence intensity of the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)/flavin adenine dinucleotide (FAD)] provides a label-free method to quantify cellular metabolism. However, it is unclear whether changes in the ORR with B-cell lymphoma 2 (Bcl-2) family protein inhibition are due to metabolic stress alone or compromised cell viability.AimDetermine whether ABT-263 (navitoclax, Bcl-2 family inhibitor) changes the ORR due to changes in mitochondrial function that are independent of changes in cell viability.ApproachSW48 colon cancer cells were used to investigate changes in ORR, mitochondrial membrane potential, oxygen consumption rates, and cell state (cell growth, viability, proliferation, apoptosis, autophagy, and senescence) with ABT-263, TAK-228 [sapanisertib, mammalian target of rapamycin complex 1/2 (mTORC 1/2) inhibitor], and their combination at 24 h.ResultsChanges in the ORR with Bcl-2 inhibition are driven by increases in both NAD(P)H and FAD autofluorescence, corresponding with increased basal metabolic rate and increased mitochondrial polarization. ABT-263 treatment does not change cell viability or induce autophagy but does induce a senescent phenotype. The metabolic changes seen with ABT-263 treatment are mitigated by combination with mTORC1/2 inhibition.ConclusionsThe ORR is sensitive to increases in mitochondrial polarization, energetic state, and cell senescence, which can change independently from cell viability.

Decreased green autofluorescence of lung parenchyma is a biomarker for lung cancer tissues.

It is highly valuable to discover novel biomarkers for differentiating noninvasively the cancerous tissues from the nonneoplastic tissues of lung cancer. In current study, we determined the green autofluorescence (AF) of the pulmonary parenchyma of lung cancer patients, indicating that decreased green AF of pulmonary parenchyma may be the biomarker of this type: First, the green AF intensity of the cancerous tissues was significantly lower than that of the nonneoplastic tissues of the lung cancer patients; second, the green AF intensity of the nonneoplastic tissues of the lung squamous cell carcinoma was significantly lower than that of the lung adenocarcinoma; and third, "decreased green AF intensity" could be used for differentiating the nonneoplastic tissues and the cancerous tissues. Collectively, our study has suggested that decreased green AF of lung parenchyma is a biomarker for differentiating the cancerous tissues from the nonneoplastic tissues of lung cancer.

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The skin’s green autofluorescence (AF) at the Dorsal Centremetacarpus of lung cancer patients was significantly higher than that of healthy subjects and pulmonary infection patients. Using the AF intensity as the variable, ROC analyses showed that the AUC for differentiating the lung cancer patients and the pulmonary infection patients was 0.87, suggesting that the skin’s AF may become a novel biomarker for non‐invasive and economical diagnosis of lung cancer.Further details can be found in the article by Mingchao Zhang, Yue Tao, Qing Chang, Kaixuan Wang, Tianqing Chu, and Weihai Ying (e202100389)image

Application of BiVO4–Microalgae Combined Treatment to Remove High Concentration Mixture of Sulfamethazine and Sulfadiazine

Sulfonamides (SAs) are the most common and bio-refractory antibiotics detected in surface water systems, which cause long-term toxic effects on aquatic organisms. This study used the combination of a BiVO4 photocatalyst and freshwater micro-green alga (Dictyosphaerium sp.) to remove sulfadiazine (SD) and sulfamethazine (SM2) at an initial concentration of 5 mg/L (1:1 v/v) for 7 days. We set up three gradient concentrations of BiVO4 (0.5, 1 and 2 g/L) combined with the same concentration (80 mg/L) of Dictyosphaerium sp. and then prepared corresponding concentrations of pure BiVO4 and pure microalgae as controls. We evaluated the ability of BiVO4 and Dictyosphaerium sp. combined technology to remove SAs by observing the removal efficiency of antibiotics and explained the degradation mechanism of antibiotics and the key role of microalgae by studying the changes of reactive oxygen species (ROS) and inorganic ions (nitrogen, sulfur). The results showed that the degradation rate of these two SAs in the 0.5 g/L BiVO4–algae group could reach >96% within 7 d, which was higher than that in the 2 g/L BiVO4 group (93%) and the algae group (28%). The increased degradation efficiency of SAs in BiVO4 and microalgae systems was mainly due to the increased amount of ROS. Meanwhile, more SAs were degraded to inorganic compounds such as NH4+-N, NO3−-N and SO42−-S under ROS stress. It was found that microalgae can absorb the degradation products of antibiotics such as NH4+-N for their own growth, thereby reducing the toxicity of antibiotic by-products. In addition, BiVO4 had no damaging effect on the autofluorescence intensity of the microalgae. Our study provides an efficient and eco-economic approach to remove antibiotics using visible-light irradiation in aquatic environments and provides new insights into the biological removal of other antibiotic contaminants in aquatic environments.

Skin's green autofluorescence at dorsal centremetacarpus may become a novel biomarker for diagnosis of lung cancer.

It is critical to discover novel biomarkers of lung cancer for establishing economical technology for diagnosis of lung cancer. Our study has suggested that the autofluorescence (AF) of the skin may become a novel biomarker of this type: First, development of lung cancer led to a significant increase in the skin's green AF in a mouse model of lung cancer; second, lung cancer patients had significantly higher skin's green AF at certain positions compared with healthy volunteers and pulmonary infection patients; and third, using the skin's green AF intensity at dorsal centremetacarpus as the variable, the areas under curve (AUC) for differentiating lung cancer patients and pulmonary infection patients and for differentiating lung cancer patients and healthy volunteers was 0.871 and 0.813, respectively. Collectively, our study has indicated that the skin's green AF at dorsal centremetacarpus may become a novel biomarker for establishing a ground-breaking diagnostic strategy for lung cancer.

The Contribution of Anterior Segment Abnormalities to Changes in Intraocular Pressure in the DBA/2J Mouse Model of Glaucoma: DBA/2J-Gpnmb +/SjJ Mice as Critical Controls.

The contributions of anterior segment abnormalities to the development of ocular hypertension was determined in the DBA/2J mouse model of glaucoma. Intraocular pressure (IOP) was measured non-invasively. Iris pigment dispersion (IPD) and corneal calcification were measured weekly starting at 20 weeks of age in DBA/2J and DBA/2J-Gpnmb+/SjJ mice. Thickness, surface area, auto-fluorescence intensity, and perimeter length of calcified regions were measured in postmortem corneas using confocal microscopy. DBA/2J mice developed elevated IOP between 9 and 12 months of age, but DBA/2J-Gpnmb+/SjJ mice did not. Corneal calcification was found at all ages observed and at similar frequencies in both strains with 83.3% of DBA/2J eyes and 60.0% of DBA/2J-Gpnmb+/SjJ eyes affected at 12 months (P = 0.11). Calcification increased with age in both DBA/2J (P = 0.049) and DBA/2J-Gpnmb+/SjJ mice (P = 0.04) when assessed qualitatively and based on mixed-effects analysis. No differences in the four objective measures of calcification were observed between strains or ages. At 12 months of age, DBA/2J mice with corneal calcification had greater mean IOP than DBA/2J mice without corneal calcification. IOP was not correlated with the qualitatively assessed measures of calcification. For the subset of eyes with ocular hypertension, which were only found in DBA/2J mice, IOP was negatively correlated with the qualitative degree of calcification, but was not correlated with the four quantitative measures of calcification. Differences in IOP were not observed between DBA/2J-Gpnmb+/SjJ mice with and without calcification at any age. IPD increased with age and demonstrated a moderate correlation with IOP in DBA/2J mice, but was not observed in DBA/2J-Gpnmb+/SjJ mice. In the DBA/2J mouse model of glaucoma, increased IPD is positively correlated with an increase in IOP and corneal calcification is present in the majority of eyes at and after age 9 months. However, while IPD causes ocular hypertension, corneal calcification does not appear to contribute to the elevation of IOP, as the control strain DBA/2J-Gpnmb+/SjJ exhibits corneal calcification similar to DBA/2J mice, but does not develop ocular hypertension. Corneal calcification, therefore, does not appear to be a contributing factor to the development of elevated IOP in DBA/2J mice.

In vivo melanin 3D quantification and z-epidermal distribution by multiphoton FLIM, phasor and Pseudo-FLIM analyses

Characterizing melanins in situ and determining their 3D z-epidermal distribution is paramount for understanding physiological/pathological processes of melanin neosynthesis, transfer, degradation or modulation with external UV exposure or cosmetic/pharmaceutical products. Multiphoton fluorescence intensity- and lifetime-based approaches have been shown to afford melanin detection, but how can one quantify melanin in vivo in 3D from multiphoton fluorescence lifetime (FLIM) data, especially since FLIM imaging requires long image acquisition times not compatible with 3D imaging in a clinical setup? We propose an approach combining (i) multiphoton FLIM, (ii) fast image acquisition times, and (iii) a melanin detection method called Pseudo-FLIM, based on slope analysis of autofluorescence intensity decays from temporally binned data. We compare Pseudo-FLIM to FLIM bi-exponential and phasor analyses of synthetic melanin, melanocytes/keratinocytes coculture and in vivo human skin. Using parameters of global 3D epidermal melanin density and z-epidermal distribution profile, we provide first insights into the in vivo knowledge of 3D melanin modulations with constitutive pigmentation versus ethnicity, with seasonality over 1 year and with topical application of retinoic acid or retinol on human skin. Applications of Pseudo-FLIM based melanin detection encompass physiological, pathological, or environmental factors-induced pigmentation modulations up to whitening, anti-photoaging, or photoprotection products evaluation.

Evaluation of Autofluorescence in Identifying Parathyroid Glands by Measuring Parathyroid Hormone in Fine-Needle Biopsy Washings

BackgroundNear-infrared autofluorescence imaging has potentially great value for assisting endocrine surgeons in identifying parathyroid glands and may dramatically change the surgical strategy of endocrine surgeons in thyroid surgery. This study is designed to objectively evaluate the role of near-infrared autofluorescence imaging in identifying parathyroid glands during thyroid surgery by measuring intraoperative parathyroid hormone in fine-needle aspiration biopsy washings.MethodsThis study was conducted at a tertiary referral teaching hospital in China from February 2020 to June 2020. Patients undergoing total thyroidectomy with or without neck lymph node dissection were consecutively included. The surgeon used near-infrared autofluorescence imaging to identify parathyroid glands during thyroid surgery and confirmed suspicious parathyroid tissues by measuring their intraoperative parathyroid hormone. Nanocarbon was injected into the thyroid gland if the thyroid autofluorescence intensity was too strong. The sensitivity and accuracy of near-infrared autofluorescence imaging and vision for identifying parathyroid glands, and the difference in autofluorescence intensity in various tissues were the main outcomes.ResultsOverall, 238 patients completed the trial. Based on the pathological and aIOPTH results, the sensitivity of near-infrared autofluorescence imaging for detecting parathyroid glands (568 of 596 parathyroid glands; 95.30%)was significantly higher than that of vision (517 of 596 parathyroid glands; 86.74%, P<.001). The accuracy of near-infrared autofluorescence imaging (764 of 841 tissues; 90.84%) was significantly higher than that of vision (567 of 841 tissues; 67.42%, P<.001) when the evaluations of certain tissues were inconsistent. There was a significant difference between the autofluorescence intensity of the parathyroid glands and that of the lymph nodes (74.19 ± 17.82 vs 33.97 ± 10.64, P<.001).ConclusionThe use of near-infrared autofluorescence imaging, along with intraoperative parathyroid hormone and nanocarbon for the identification of parathyroid glands in thyroid surgery may increase the number of confirmed parathyroid glands. Using near-infrared autofluorescence imaging can effectively distinguish lymph nodes and parathyroid glands during lymph node dissection.

Comparison of Parathyroid Autofluorescence Signals in Different Types of Hyperparathyroidism.

There are scant data in the literature regarding whether parathyroid autofluorescence (AF) signal patterns vary based on the etiology of hyperparathyroidism. The aim of this study was to compare AF signals of parathyroid glands across different etiologies of hyperparathyroidism. As a prospective institutional review board-approved study between 2016 and 2019, AF intensities and heterogeneity indexes (HIs) of parathyroid glands in patients who underwent parathyroidectomy using AF were calculated and compared using Chi-square, Kruskal Wallis, Mann Whitney U, and logistic regression tests. Of the total of 183 patients, 127 patients had sporadic classic primary hyperparathyroidism, 30 patients had sporadic normohormonal primary hyperparathyroidism, 10 patients had sporadic normocalcemic primary hyperparathyroidism, 12 patients had tertiary hyperparathyroidism, and 4 patients had familial primary hyperparathyroidism related to multiple endocrine neoplasia (MEN) 2A. There were no statistical differences in AF signals of abnormal parathyroid glands in classic, normohormonal or normocalcemic sporadic hyperparathyroidism. Parathyroid glands in patients with tertiary hyperparathyroidism were similar in intensity, but more homogenous compared to those in sporadic primary hyperparathyroidism. The pattern of AF exhibited by abnormal parathyroid glands was similar across different spectrums of primary hyperparathyroidism, in accordance with observations in the literature. However, parathyroid glands in tertiary hyperparathyroidism were more homogeneous, despite exhibiting a similar intensity of AF compared to those in sporadic primary hyperparathyroidism. These differences should be kept in mind when using the AF pattern as an adjunct to visual assessment in parathyroid exploration.

Luminescence and fahua‐fermentation qualities of an autofluorescent microorganism from Fu brick tea

Recently, the autofluorescence of Fu brick tea was observed during its production and consumption. Notably, the autofluorescence intensity of Fu brick tea varies in different locations. To explore underlying cause of autofluorescence, we detected ten types of microorganisms from Jingwei Fu tea and Xiangyi Fu tea, which contain the same raw materials but undergo different processing environments, leading to different autofluorescence levels. Consequently, we found that Eurotium cristatum and Aspergillus chevalieri are responsible for the autofluorescence of Fu tea. The differences in autofluorescence intensity are due to different densities of the two fungal species. And the fluorescence intensity of these fungi was affected by pH, temperature, and staining concentration. Therefore, different processing environments lead to different autofluorescence intensities of Fu tea. Additionally, we inoculated the two strains into raw dark tea, we observed altered contents of amino acids, catechins, etc.in dark tea, and these changes correlated with the autofluorescence intensity. Novelty impact statement The cause of autofluorescence of Fu brick tea was obtained. We observed E. cristatum and A. chevalieri produced autofluorescence in these teas. Microorganisms caused changes in various biochemical components of the teas.

Autofluorescence imaging of Barrett's esophageal lesions with additional transformation into spatial images of green autofluorescence intensity

Background: Early diagnosis of patients with Barrett's esophagus is required to implement appropriate treatment to prevent neoplastic disease development. In this work, we examined the usefulness of autofluorescence imaging as a method to increase the sensitivity of targeted biopsy under numerical color value control with the additional conversion of autofluorescence images into spatial green autofluorescence intensity images. Methods: 148 patients were included in the study. Autofluorescence imaging was used in each endoscopic examination. The obtained images of lesions were transformed with Image Pro PLUS 5.0.2 software to show the points of lesions with the highest values of numerical color value and the lowest green intensity. The obtained results were analyzed statistically using Statistica 8.0 software. Mann-Whitney U test was used to compare red to green ratio, red fluorescence intensity and green color intensity between the examined groups of lesions. Results: Thanks to targeted biopsy under the control of red to green ratio factor and green autofluorescence intensity, this imaging method's sensitivity was also increased in all studied stages of histopathological dysplasia in Barrett's esophagus. In total analysis, the sensitivity of tri-modal imaging with the analysis of green autofluorescence intensity was almost 97%. The spatial maps of autofluorescence intensity significantly improved the effectiveness of biopsies performed to take tissue samples for a histopathological examination compared to white light endoscopy. The extension of autofluorescence to spatial autofluorescence intensity maps significantly reduced the percentage of false-negative results. Conclusions: The study results indicate that autofluorescence imaging allows for assessing the extent of dysplasia lesions and determining the margin of healthy and pathologically effected tissues. Our team's method to convert autofluorescence images into spatial images of green autofluorescence intensity further increased the sensitivity of the study.

Choroideremia Gene Therapy.

Choroideremia Gene Therapy.

Method for controlled tissue theranostics using a single tunable laser source.

Tissue diseases and related disorders need to be first recognized using diagnostic methods and then later treated by therapeutic methods-a joint procedure called theranostics. One of the main challenges in the field of retinal therapies remains in the success of the treatment, typically improving the local metabolism, by sparing the surrounding tissue and with the immediate information of the laser effect. In our study, we present a concept for real-time controlled tissue theranostics on a proof-of-concept study capable of using a single tunable ps laser source (in terms of irradiance, fluence, and repetition rate), done on ex-vivo human retinal pigment epithelium. We have found autofluorescence intensity and lifetime imaging diagnostics very promising for the recognition and quantification of laser effects ranging from selective non-destructive molecular tissue modification to complete tissue ablation. The main novelty of our work presents the developed algorithm for optimized theranostics based on the model function used to quantify laser-induced tissue changes through the diagnostics descriptors, fluorescence lifetime and fluorescence intensity parameters. This approach, together with the operation of the single adaptable laser source, can serve as a new theranostics method in personalized medicine in the future not only limited to treat retinal diseases.

Autofluorescence Is a Common Trait in Different Oceanic Fungi.

Natural autofluorescence is a widespread phenomenon observed in different types of tissues and organisms. Depending on the origin of the autofluorescence, its intensity can provide insights on the physiological state of an organism. Fungal autofluorescence has been reported in terrestrial and human-derived fungal samples. Yet, despite the recently reported ubiquitous presence and importance of marine fungi in the ocean, the autofluorescence of pelagic fungi has never been examined. Here, we investigated the existence and intensity of autofluorescence in five different pelagic fungal isolates. Preliminary experiments of fungal autofluorescence at different growth stages and nutrient conditions were conducted, reflecting contrasting physiological states of the fungi. In addition, we analysed the effect of natural autofluorescence on co-staining with DAPI. We found that all the marine pelagic fungi that were studied exhibited autofluorescence. The intensity of fungal autofluorescence changed depending on the species and the excitation wavelength used. Furthermore, fungal autofluorescence varied depending on the growth stage and on the concentration of available nutrients. Collectively, our results indicate that marine fungi can be auto-fluorescent, although its intensity depends on the species and growth condition. Hence, oceanic fungal autofluorescence should be considered in future studies when fungal samples are stained with fluorescent probes (i.e., fluorescence in situ hybridization) since this could lead to misinterpretation of results.

Correlation between Autofluorescence Intensity and Histopathological Features in Non-Melanoma Skin Cancer: An Ex Vivo Study.

Simple SummaryNon-melanoma skin cancers (NMSC) are the most prevalent neoplasms worldwide, especially in the Caucasian population. Radical surgical excision is considered the therapeutic gold standard, while incomplete tumor removal is invariably associated with recurrence and the need for reintervention. Autofluorescence (AF) spectroscopy has recently been investigated for early diagnosis of NMSC and assessment of tumor margins. Understanding changes in AF intensity in association with peculiar histological features could improve the diagnostic accuracy of skin fluorescence spectroscopy. The main goal of our work was to investigate the correlation between the intensity of cutaneous AF and the histopathological features of NMSC. The intensity of fluorescence emission in tissues obtained from NMSC samples was approximately 4 times lower than that in healthy conditions. In fact, mean AF intensity for BCC group was 4.5 and 4.4 for SCCs, with further variability being recorded according to histopathologic subtypes. Histopathological features such as hyperkeratosis, neoangiogenesis, fibrosis and elastosis are statistically associated with a decrease in AF intensity. Our data suggest that such tissue alterations could be responsible for the difference in AF emission between neoplastic and healthy tissue. These results support the potential application of AF as a useful non-invasive diagnostic tool for NMSCs. Non-melanoma skin cancer (NMSC) is the most common malignant tumor affecting fair-skinned people. Increasing incidence rates of NMSC have been reported worldwide, which is an important challenge in terms of public health management. Surgical excision with pre-operatively identified margins is one of the most common and effective treatment strategies. Incomplete tumor removal is associated with a very high risk of recurrence and re-excision. Biological tissues can absorb and re-emit specific light wave-lengths, detectable through spectrophotometric devices. Such a phenomenon is known as autofluorescence (AF). AF spectroscopy has been widely explored for non-invasive, early detection of NMSC as well as for evaluation of surgical margins before excision. Fluorescence-aided diagnosis is based on differences in spectral characteristics between healthy and neoplastic skin. Understanding the biological basis of such differences and correlating AF intensity to histological features could improve the diagnostic accuracy of skin fluorescence spectroscopy. The primary objective of the present pre-clinical ex vivo study is to investigate the correlation between the intensity of cutaneous AF and the histopathological features of NMSC. Ninety-eight lesions suggestive for NMSCs were radically excised from 75 patients (46 M; 29 F; mean age: 79 years). After removal, 115 specific reference points on lesions (“cases”; 59 on BBC, 53 on SCC and 3 on other lesions) and on peri-lesional healthy skin (controls; 115 healthy skin) were identified and marked through suture stitches. Such reference points were irradiated at 400–430 nm wavelength, and resulting emission AF spectra were acquired through spectrophotometry. For each case, AFIR (autofluorescence intensity ratio) was measured as the ratio between the number of photons emitted at a wavelength ranging between 450 and 700 nm (peak: 500 nm) in the healthy skin and that was captured in the pathological tissue. At the histological level, hyperkeratosis, neoangiogenesis, cellular atypia, epithelial thickening, fibrosis and elastosis were quantified by light microscopy and were assessed through a previously validated grading system. Statistical correlation between histologic variables and AFIR was calculated through linear regression. Spectrometric evaluation was performed on 230 (115 cases + 115 controls) reference points. The mean AFIR for BCC group was 4.5, while the mean AFIR for SCC group was 4.4 and the fluorescence peaks at 500 nm were approximately 4 times lower (hypo-fluorescent) in BCCs and in SCCs than in healthy skin. Histological variables significantly associated with alteration of AFIR were fibrosis and elastosis (p < 0.05), neoangiogenesis, hyperkeratosis and epithelial thickening. Cellular atypia was not significantly associated with alteration of AFIR. The intensity of fluorescence emission in neoplastic tissues was approximately 4 times lower than that in healthy tissues. Histopathological features such as hyperkeratosis, neoangiogenesis, fibrosis and elastosis are statistically associated with the decrease in AFIR. We hypothesize that such tissue alterations are among the possible biophysical and biochemical bases of difference in emission AF between neoplastic and healthy tissue. The results of the present evaluation highlighted the possible usefulness of autofluorescence as diagnostic, non-invasive and real-time tool for NMSCs.

Can near-infrared autofluorescence imaging be used for intraoperative confirmation of parathyroid tissue?

Whether ex vivo autofluorescence (AF) imaging findings could be quantified to intraoperatively differentiate parathyroid tissue has not been reported. Our aim was to assess the ability of AF imaging to confirm parathyroid tissue during thyroidectomy and parathyroidectomy procedures. This was a retrospective Institutional Review Board-approved study. AF signals of specimens imaged ex vivo before submission to pathology during thyroidectomy and parathyroidectomy procedures were quantified. Using receiver operating characteristic (ROC) curves, optimal values for sensitivity/specificity to differentiate parathyroid tissue were calculated. 166 parathyroid and 217 non-parathyroid specimens were analyzed. With surgical drapes as background, the optimal normalized AF intensity threshold to predict parathyroid tissue on ROC curve analysis was 1.72 (86.1% sensitivity, 84.8% specificity, and area under the curve [AUC]: 0.919). The cutoff for 97% sensitivity was 1.31and for 99.1% specificity was 3.16. With Telfa as background, the optimal threshold to predict parathyroid tissue was 1.46 (88.4% sensitivity, 76.8% specificity, and AUC: 0.896). The threshold for 96.8% sensitivity was 1.18and for 98.8% specificity was 2.44. If thresholds for highest specificity were used, 40% of frozen sections to confirm parathyroid tissue could have been avoided. Quantified brightness analysis of ex vivo AF signals may have utility in intraoperative differentiation of parathyroid tissue for 40% of surgical specimens.