Infrared Spectroscopy for Rapid Triage of Cancer Using Blood Derivatives: A Reality Check.

Infrared (IR) spectroscopy of serum/plasma represents an alluring molecular diagnostic tool, especially for cancer, as it can provide a molecular fingerprint of clinical samples based on vibrational modes of chemical bonds. However, despite the superior performance, the routine adoption of this technique for clinical settings has remained elusive. This is due to the potential confounding factors that are often overlooked and pose a significant barrier to clinical translation. In this Perspective, we summarize the concerns associated with various confounding factors, such as fluid sampling, optical effects, hemolysis, abnormal cardiovascular and/or hepatic functions, infections, alcoholism, diet style, age, and gender of a patient or normal control cohort, and improper selection of numerical methods that ultimately would lead to improper spectral diagnosis. We also propose some precautionary measures to overcome the challenges associated with these confounding factors.

Smart optical sensing of multiple antibiotic residues from wastewater effluents with ensured specificity using SERS assisted with multivariate analysis.

Surface-enhanced Raman spectroscopy offers great potential for rapid and highly sensitive detection of pharmaceuticals from environmental sources. Herein, we investigated the feasibility of label-free sensing of antibiotic residues from wastewater effluents with high specificity by combining with multivariate analysis. Highly ordered silver nanoarrays with ∼34 nm roughness have been fabricated using a cost-effective electroless deposition technique. As-fabricated Ag arrays showed superior LSPR effects with an enhancement factor of 8 × 107. Excellent reproducibility has also been noticed with RSD values within 11%, whilst the sensor showed good stability and reusability characteristics for being used as a low-cost and reusable sensor. SERS studies demonstrated that antibiotics-spiked wastewater effluents can be detected with high efficiency in a label-free method. The molecular fingerprint bands of antibiotics such as sulfamethoxazole, sulfadiazine, and ciprofloxacin were well analyzed in effluent, tap, and deionized water. It has been found that antibiotics can be detected near picomolar levels; meanwhile, liquid chromatography-mass spectrometry (LC-MS) exhibited a detection limit within nanomolar concentrations only. Furthermore, the specificity of SERS sensing has been further analyzed using a multivariate analysis method, principal component analysis followed by linear discriminant analysis (PCA-LDA); which showed prominent discrimination to distinguish each antibiotic residue from wastewater effluents. The current study presented the potential of Ag nanoarray sensors for rapid, highly specific, and cost-effective analysis of pharmaceutical products for environmental remediation applications.

Near infrared-emitting multimodal nanosystem for in vitro magnetic hyperthermia of hepatocellular carcinoma and dual imaging of in vivo liver fibrosis.

Prolonged usage of traditional nanomaterials in the biological field has posed several short- and long-term toxicity issues. Over the past few years, smart nanomaterials (SNs) with controlled physical, chemical, and biological features have been synthesized in an effort to allay these challenges. The current study seeks to develop theranostic SNs based on iron oxide to enable simultaneous magnetic hyperthermia and magnetic resonance imaging (MRI), for chronic liver damage like liver fibrosis which is a major risk factor for hepatocellular carcinoma. To accomplish this, superparamagnetic iron oxide nanoparticles (SPIONs) were prepared, coated with a biocompatible and naturally occurring polysaccharide, alginate. The resultant material, ASPIONs were evaluated in terms of physicochemical, magnetic and biological properties. A hydrodynamic diameter of 40 nm and a transverse proton relaxation rate of 117.84 mM-1 s-1 pronounces the use of ASPIONs as an efficient MRI contrast agent. In the presence of alternating current of 300 A, ASPIONs could elevate the temperature to 45 °C or more, with the possibility of hyperthermia based therapeutic approach. Magnetic therapeutic and imaging potential of ASPIONs were further evaluated respectively in vitro and in vivo in HepG2 carcinoma cells and animal models of liver fibrosis, respectively. Finally, to introduce dual imaging capability along with magnetic properties, ASPIONs were conjugated with near infrared (NIR) dye Atto 700 and evaluated its optical imaging efficiency in animal model of liver fibrosis. Histological analysis further confirmed the liver targeting efficacy of the developed SNs for Magnetic theranostics and optical imaging as well as proved its short-term safety, in vivo.

Fluorescent carbon dots tailored iron oxide nano hybrid system forin vivooptical imaging of liver fibrosis.

Hybrid nanoparticles are innovative invention of last decade designed to overcome limitations of single-component nanoparticles by introducing multiple functionalities through combining two or more different nanoparticles. In this study, we are reporting development of magneto-fluorescent hybrid nanoparticles by combining iron oxide and carbon nanoparticles to enablein vivofluorescence imaging which also has all the required characteristic properties to use as Magnetic Resonance Imaging (MRI) contrast agent. In order to achieve dual-functional imaging, alginate and pullulan coated super paramagnetic iron oxide nanoparticles (ASPION and PSPION) and Carbon dots (Cdts) were synthesised separately. ASPIONs and PSPIONs were further chemically conjugated with Cdts and developed dual-functional nanohybrid particles ASPION-Cdts and PSPION-Cdts. Subsequently, evaluation of the materials for its size, functionalisation efficiency, fluorescence and magnetic properties, biocompatibility and cellular uptake efficiency has been carried out. Fluorescence imaging of liver fibrosis was performedin vivoin rodent model of liver fibrosis using the two nanohybrids, which is further confirmed by high fluorescence signal from the harvested liver.

Asialoglycoprotein receptor targeted optical and magnetic resonance imaging and therapy of liver fibrosis using pullulan stabilized multi-functional iron oxide nanoprobe.

Early diagnosis and therapy of liver fibrosis is of utmost importance, especially considering the increased incidence of alcoholic and non-alcoholic liver syndromes. In this work, a systematic study is reported to develop a dual function and biocompatible nanoprobe for liver specific diagnostic and therapeutic applications. A polysaccharide polymer, pullulan stabilized iron oxide nanoparticle (P-SPIONs) enabled high liver specificity via asialogycoprotein receptor mediation. Longitudinal and transverse magnetic relaxation rates of 2.15 and 146.91 mM-1 s-1 respectively and a size of 12 nm, confirmed the T2 weighted magnetic resonance imaging (MRI) efficacy of P-SPIONs. A current of 400A on 5 mg/ml of P-SPIONs raised the temperature above 50 °C, to facilitate effective hyperthermia. Finally, a NIR dye conjugation facilitated targeted dual imaging in liver fibrosis models, in vivo, with favourable histopathological results and recommends its use in early stage diagnosis using MRI and optical imaging, and subsequent therapy using hyperthermia.

Correction: Empirical study on the effects of acquisition parameters for FTIR hyperspectral imaging of brain tissue.

Correction for 'Empirical study on the effects of acquisition parameters for FTIR hyperspectral imaging of brain tissue' by J. Sacharz et al., Anal. Methods, 2020, 12, 4334-4342, DOI: 10.1039/C9AY01200A.

Infrared Microspectroscopy With Multivariate Analysis to Differentiate Oral Hyperplasia From Squamous Cell Carcinoma: A Proof of Concept for Early Diagnosis.

BACKGROUND AND OBJECTIVES: Despite having numerous advances in therapeutics, mortality and morbidity due to oral cancer incidence are still very high. Early detection can improve the chances of survival in most patients. However, diagnosis at early stages can be challenging as premalignant conditions are usually asymptomatic. Currently, histological assessment remains the gold standard for diagnosis. Early diagnosis poses challenges to pathologists due to less severe morphological changes associated with early stages. Therefore, a fast and robust method of detection based on molecular changes is needed for early diagnosis. © 2021 Wiley Periodicals LLC. STUDY DESIGN/MATERIAL AND METHODS: In the present study, Fourier transform infrared (FTIR) spectroscopic imaging has been used to differentiate early-stage oral hyperplasia from adjacent normal (AN) and oral squamous cell carcinoma (OSCC). Hyperplasia is often considered as an initial event in the pathogenesis of oral cancer and OSCC is the most common advanced stage of malignancy. Differentiating normal versus hyperplasia and hyperplasia versus OSCC can remain quite challenging on occasion using conventional staining as the histological assessment is based on morphological changes. RESULTS: Unsupervised hierarchical cluster analysis (UHCA) has been performed on FTIR images of multiple tissues together that provided some degree of classification among tissue groups. The AN epithelium clustered distinctively using UHCA from both hyperplasia and grades 1 and 2 of OSCC. An increase in the content of DNA, denaturation of protein, and altered lipid structures were more clearly elucidated with spectral analysis. CONCLUSION: This study demonstrates a simple strategy to differentiate early-stage oral hyperplasia from AN and OSCC using UHCA. This study also proposes a future alternative method where FTIR imaging can be used as a diagnostic tool for cancer at early stages.

Autofluorescence spectroscopy and multivariate analysis for predicting the induced damages to other organs due to liver fibrosis.

When our liver does not work well, it can induce damage to other organs causing their dysfunction. With this background, we aim to study the effect of liver fibrosis on other organs such as heart, lungs, kidney and spleen by assessing the variations in the inherent emission property of the tissue, using fluorescence spectroscopy. Fluorescence emission spectra from excised organs of liver fibrosis induced rats were collected at excitation wavelengths 320 and 410 nm. Optical redox ratio derived from the spectral data supported by multivariate statistical analysis, principal component analysis followed by linear discriminant analysis (PCA-LDA) distinguished between control and fibrosis induced groups. The two different excitation wavelength provided variations in the endogenous flurophores collagen, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), lipopigments and porphyrins. Additionally, evaluation of redox ratio provided variations in tissue metabolic activity of different organs. The PCA-LDA modelling yielded a sensitivity of 85 to 97% and specificity of 80 to 96% on 320 nm excitation and a sensitivity of 72 to 100% and specificity of 59 to 100% on 410 nm excitation. Fluorescence emission spectral study along with multivariate analysis paved way to identify the biochemical alterations caused to other organs due to the development of liver fibrosis, which could lead to their damage and dysfunction.

Optical diagnosis of oral lichen planus: A clinical study on the use of autofluorescence spectroscopy combined with multivariate analysis.

Oral lichen planus (OLP) is a chronic mucocutaneous inflammatory condition of stratified squamous epithelia. OLP is a potentially malignant condition in oral mucosa. Patients with OLP have an increased risk of developing squamous cell carcinoma. Therefore an early and accurate diagnosis is necessary to avoid further damage to the oral mucosa. Biopsy followed by histopathological examination is the gold standard for the diagnosis of oral mucosal lesions including OLP. But this invasive procedure is traumatic and time consuming with limited statistical confidence level. Autofluorescence spectroscopy (AFS) has recently emerged as a potential tool to evaluate the biochemical changes associated with oral cavity disorders. In this study, we used AFS to differentiate the oral cavity tissue of 20 OLP patients from that of 16 normal volunteers. Spectra from oral mucosa were acquired at 280, 320 and 410 nm excitation wavelengths which correspond to the excitation energy of major endogenous fluorophores. Normalized spectral data at 320 nm excitation showed significant increase in the intensity of collagen peak for OLP. Optical redox ratio and total hemoglobin concentration estimated from the spectral data revealed significant increase and decrease respectively in OLP and normal patients. Principal component analysis followed by linear discriminant analysis (PCA-LDA) provided sensitivity and specificity of 71 and 80%, 80 and 90%, and 72 and 75% respectively for 280, 320 and 410 nm excited spectral datasets. Meanwhile, partial least square discriminant analysis (PLS-DA) provided sensitivity and specificity of 69 and 77%, 78 and 91% and 73 and 78.13% respectively for 280, 320 and 410 nm excited spectral datasets. From the results, it is concluded that AFS is an efficient tool for the non invasive diagnosis of OLP, with 320 nm light identified as the best wavelength for excitation.

Infrared spectral microscopy as a tool to monitor lung fibrosis development in a model system.

Tissue fibrosis is a progressive and destructive disease process that can occur in many different organs including the liver, kidney, skin, and lungs. Fibrosis is typically initiated by inflammation as a result of chronic insults such as infection, chemicals and autoimmune diseases. Current approaches to examine organ fibrosis are limited to radiological and histological analyses. Infrared spectroscopic imaging offers a potential alternative approach to gain insight into biochemical changes associated with fibrosis progression. In this study, we demonstrate that IR imaging of a mouse model of pulmonary fibrosis can identify biochemical changes observed with fibrosis progression and the beginning of resolution using K-means analysis, spectral ratios and multivariate data analysis. This study demonstrates that IR imaging may be a useful approach to understand the biochemical events associated with fibrosis initiation, progression and resolution for both the clinical setting and for assessing novel anti-fibrotic drugs in a model system.

Empirical study on the effects of acquisition parameters for FTIR hyperspectral imaging of brain tissue.

Fourier transform infrared (FTIR) spectroscopic imaging is a powerful technique for molecular imaging of pathologies associated with the nervous systems including multiple sclerosis research. However, there is no standard methodology or standardized protocol for FTIR imaging of tissue sections that maximize the ability to discriminate between the molecular, white and granular layers, which is essential in the investigation of the mechanism of demyelination process. Tissue sections are heterogeneous, complex and delicate, hence the parameters to generate high quality images in minimal time becomes essential in the modern clinical laboratory. This article presents an FTIR spectroscopic imaging study of post-mortem human brain tissue testing the effects of various measurement parameters and data analysis methods on image quality and acquisition time. Hyperspectral images acquired from the same region of a tissue using a range of the most common optical and collection parameters in different combinations were compared. These included magnification (4× and 15×), number of co-added scans (1, 4, 8, 16, 32, 64 and 128 scans) and spectral resolution (4, 8 and 16 cm-1). Images were compared in terms of acquisition time, signal-to-noise (S/N) ratio, and accuracy of the discrimination between three major tissue types in a section from the cerebellum (white matter, granular and molecular layers). In the latter case, unsupervised k-means cluster (KMC) analysis was employed to generate images from the hyperspectral images, which were compared to a reference image. The classification accuracy for tissue class discrimination was highest for the 4× magnifying objective, with 4 cm-1 spectral resolution and 128 co-added scans. The 15× magnifying objective gave the best accuracy for a spectral resolution of 4 cm-1 and 64 scans (96.3%), which was just above what was achieved using the 4× magnifying objective, with 4 cm-1 spectral resolution and 32 and 64 co-added scans (95.4 and 95.6%, respectively). These findings were correlated with a decrease in S/N ratio with increasing number of scans and was generally lower for the 15× objective. However, longer scan times were required using the 15× magnifying objective, which did not justify the very small improvement in the classification of tissue types.

Luminescent Gold Nanorods To Enhance the Near-Infrared Emission of a Photosensitizer for Targeted Cancer Imaging and Dual Therapy: Experimental and Theoretical Approach.

Strong plasmon absorption in the near-infrared (NIR) region renders gold nanorods (GNRs) amenable for biomedical applications, particularly for photothermal therapy. However, these nanostructures have not been explored for their imaging potential because of their weak emission profile. In this study, the weak fluorescence emission of GNRs is tuned to match that of the absorption of a photosensitizer (PS) molecule, and energy transfer from the GNR to PS enhances the emission profile of the GNR-PS combination. GNR complexes generally quench the fluorescence emission of nearby chromophores. However, herein, the complex retains or rather enhances the fluorescence through competition in energy transfer. Excitation-dependent energy transfer has been explained experimentally and theoretically by using DFT calculations, the CIE chromaticity diagram, and power spectrum. The final GNR-PS complex modified for tumor specificity serves as an excellent organ-specific theranostic probe for bioimaging and dual therapy both in vitro and in vivo. Principal component analysis designates photodynamic therapy a better candidate than that of photothermal therapy for long-term efficacy in vivo.

Label-free Identification of Antibody-mediated Rejection in Cardiac Allograft Biopsies Using Infrared Spectroscopic Imaging.

BACKGROUND: Antibody-mediated rejection (AMR) in cardiac allograft recipients remains less well-understood than acute cellular rejection, is associated with worse outcomes, and portends a greater risk of developing chronic allograft vasculopathy. Diffuse immunohistochemical C4d staining of capillary endothelia in formalin-fixed, paraffin-embedded right ventricular endomyocardial biopsies is diagnostic of immunopathologic AMR but serves more as a late-stage marker. Infrared (IR) spectroscopy may be a useful tool in earlier detection of rejection. We performed mid-IR spectroscopy to identify a unique biochemical signature for AMR. METHODS: A total of 30 posttransplant formalin-fixed paraffin-embedded right ventricular tissue biopsies (14 positive for C4d and 16 negative for C4d) and 14 native heart biopsies were sectioned for IR analysis. Infrared images of entire sections were acquired and regions of interest from cardiomyocytes were identified. Extracted spectra were averaged across many pixels within each region of interest. Principal component analysis coupled with linear discriminant analysis and predictive classifiers were applied to the data. RESULTS: Comparison of averaged mid-IR spectra revealed unique features among C4d-positive, C4d-negative, and native heart biopsies. Principal component analysis coupled with linear discriminant analysis and classification models demonstrated that spectral features from the mid-IR fingerprint region of these 3 groups permitted accurate automated classification into each group. CONCLUSIONS: In cardiac allograft biopsies with immunopathologic AMR, IR spectroscopy reveals a biochemical signature unique to AMR compared with that of nonrejecting cardiac allografts and native hearts. Future study will focus on the predictive capabilities of this IR signature.

Biosafety of citrate coated zerovalent iron nanoparticles for Magnetic Resonance Angiography.

Though nanoparticles are being used for several biomedical applications, the safety of the same is still a concern. It is very routine procedure to check the preliminary safety aspects of the particles intended for in vivo applications. The major tests include how the material reacts to a normal cell, how it behaves with the blood cells and also whether any lysis take place in the presence of these materials. Here we present these test data of two novel nanomaterials designed for its use as contrast agent for magnetic resonance imaging and a multimodal contrast agent for targeted liver imaging. On proving the biosafety, the materials were tested for Magnetic Resonance Angiography using normal rats as model. The data of the same were clear identification of the prominent vascular structures and is included as the colour coded MRI image. Lateral and oblique view data are also presented for visualizing other major blood vessels.

Fluorescence spectroscopy as an efficient tool for staging the degree of liver fibrosis: an in vivo comparison with MRI.

The study utilizes autofluorescence spectroscopy (AFS) along with multivariate spectral analysis for differentiating various stages of hepatic fibrosis. AFS has recently emerged as an efficient tool for evaluating the variations in different endogenous flurophores. In this study, the potential of AFS for differentiating the stages of liver fibrosis is assessed and compared with the results of enzyme evaluation, histopathology and the most advanced diagnostic tool, MRI. Using a fiber optic probe, the emission profile of the flurophores such as flavin adenine dinucleotide (FAD), lipofuscin-like lipopigments (lipopigments), porphyrins and the variation in the total hemoglobin concentration are evaluated in vivo on liver fibrosis induced animal models adopting a minimally invasive technique. Significant difference (p < 0.05) in the level of these biomarkers was observed between different stages of liver fibrosis. Normal hepatic tissue could be distinguished from mild and moderate hepatic fibrosis with a sensitivity of 95 to 100% and specificity of 90 to 100% using multivariate spectral analysis. The results are favourable to consider this technique as a potential tool for diagnosing liver fibrosis at an early stage, which is monumental as it otherwise can lead to cirrhosis and liver failure.

Multifunctional hybrid nanoconstruct of zerovalent iron and carbon dots for magnetic resonance angiography and optical imaging: An In vivo study.

In magnetic resonance imaging (MRI), gadolinium (Gd) complexes are very often used as contrast agents to enhance the signal from soft tissue deformities and vascular anomalies, to improve the accuracy of diagnosis. The safety concern of using Gd complexes in renally compromised patients pose limitations on its application. To overcome this scenario, we introduce a nontoxic zerovalent iron based nanoparticle as a novel contrast agent for MR angiography and a hybrid version of the same to serve as a dual function contrast agent for targeted liver imaging. The synthesized zerovalent iron (ZVI) nanoparticles after citrate stabilization (C@ZVI) had an average size of 10 nm and exhibited paramagnetic property which is a prerequisite for a positive MRI contrast agent. The longitudinal magnetic relaxivity, r1 of C@ZVI was 4.93 mM-1s-1 which is much higher than that of clinically used Gd based agent, gadoterate meglumine (3.6 mM-1s-1). For multimodal imaging of the liver, initially the ZVI nanoparticle was tailored with a highly liver specific polysaccharide pullulan, and later with fluorescent carbon dots (Cdts) facilitating both optical and MR imaging. The magnetic relaxivity was retained in P@ZVI-Cdts for T1 contrast imaging with an r1 value of 3.48 mM-1s-1. The in vivo MR angiogram using C@ZVI and the liver targeted MRI and optical imaging using P@ZVI-Cdts were successfully demonstrated proving their potential as MRA contrast agent and a liver specific multimodal imaging agent.

Infrared spectroscopic imaging: Label-free biochemical analysis of stroma and tissue fibrosis.

Infrared spectroscopic tissue imaging is a potentially powerful adjunct tool to current histopathology techniques. By coupling the biochemical signature obtained through infrared spectroscopy to the spatial information offered by microscopy, this technique can selectively analyze the chemical composition of different features of unlabeled, unstained tissue sections. In the past, the tissue features that have received the most interest were parenchymal and epithelial cells, chiefly due to their involvement in dysplasia and progression to carcinoma; however, the field has recently turned its focus toward stroma and areas of fibrotic change. These components of tissue present an untapped source of biochemical information that can shed light on many diverse disease processes, and potentially hold useful predictive markers for these same pathologies. Here we review the recent applications of infrared spectroscopic imaging to stromal and fibrotic regions of diseased tissue, and explore the potential of this technique to advance current capabilities for tissue analysis.

Monitoring the biochemical alterations in hypertension affected salivary gland tissues using Fourier transform infrared hyperspectral imaging.

Fourier transform infrared spectroscopy (FTIR) imaging has been applied to investigate biochemical differences between salivary glands from control and hypertensive rats. Male Sprague-Dawley rats were divided into two groups including a control group and another hypertension group that were treated orally, with N-nitro-l-arginine methyl ester (l-NAME) via drinking water for 3 weeks to develop hypertension. In the control group, rats were treated with only drinking water for 3 weeks. The formalin-fixed paraffin embedded tissue specimens from submandibular and sublingual glands were analysed with a FTIR focal plane array imaging spectrometer and multi-composite images of all tissue sections were analysed simultaneously using Unsupervised Hierarchical Cluster Analysis (UHCA) and the extracted spectra were further analysed using Partial Least Squares Discriminant Analysis (PLS-DA). In general, hypertension affected salivary gland tissues were characterised by higher concentrations of triglycerides as evidenced by an increase in the 1745 cm-1 band. Higher concentrations of carbohydrates and proteins were also observed in the hypertensive group along with a decrease in bands associated with nucleic acids. PLS-DA scores plots provided good differentiation in sublingual gland tissues between control (n = 3734 spectra) and hypertension (n = 4538) and also in submandibular gland tissues between control (n = 5051) and hypertension (n = 4408). We have shown that FTIR imaging can be used to differentiate the macromolecular information between physiological and pathological conditions in tissue biopsy specimens. In the next phase, we will investigate the infrared predictive markers of hypertension in biofluids including serum and saliva using attenuated total refection spectroscopy.

Multifunctional nano manganese ferrite ferrofluid for efficient theranostic application.

Ferrofluid-based manganese (Mn(2+)) substituted superparamagnetic iron oxide nanoparticles stabilized by surface coating with trisodium citrate (MnIOTCs) were synthesized for enhanced hyperthermic activity and use as negative magnetic resonance imaging (MRI) contrast media intended for applications in theranostics. The synthesized MnIOTC materials were characterized based on their physicochemical and biological features. The crystal size and the particle size at the nano level were studied using XRD and TEM. The presence of citrate molecules on the crystal surface of the iron oxide was established by FTIR, TGA, DLS and zeta potential measurements. The superparamagnetic property of MnIOTCs was measured using a vibrating sample magnetometer. Superparamagnetic iron oxide substituted with Mn(2+) with a 3:1 molar concentration of Mn(2+) to Fe(2+) and surface modified with trisodium citrate (MnIO75TC) that exhibited a high T2 relaxivity of 184.6mM(-1)s(-1) and showed excellent signal intensity variation in vitro. Hyperthermia via application of an alternating magnetic field to MnIO75TC in a HeLa cell population induced apoptosis, which was further confirmed by FACS and cLSM observations. The morphological features of the cells were highly disrupted after the hyperthermia experiment, as evidenced from E-SEM images. Biocompatibility evaluation was performed using an alamar blue assay and hemolysis studies, and the results indicated good cytocompatibility and hemocompatibility for the synthesized particles. In the current study, the potential of MnIO75TC as a negative MRI contrast agent and a hyperthermia agent was demonstrated to confirm its utility in the burgeoning field of theranostics.

Rapid and simple method of photobleaching to reduce background autofluorescence in lung tissue sections.

Autofluorescence exhibited by tissues often interferes with immunofluorescence. Using imaging and spectral analysis, we observed remarkable reduction of autofluorescence of formalin fixed paraffin embedded tissues irradiated with light prior to incubation with immunofluorescent dyes. The technique of photobleaching offers significant improvement in the quality and specificity of immunofluorescence. This has the potential for better techniques for disease diagnosis.