Autofluorescence Spectra Research Articles

Optical diagnostics of malignant and benign skin neoplasms

In this work the study of the optical properties of skin tissues using Raman spectroscopy and autofluorescence analysis was performed. The analysis of different skin neoplasms was carried out based on the Raman and autofluorescence spectra stimulated by 785 nm laser. In this study the spectral data was processed by different mathematical methods allowed for effective classification of malignant and benign tumors. The proposed diagnostic methods helped to achieve 82.1-87% differentiation accuracy of skin neoplasms.

Fluorescing fatty acids in rat fatty liver models.

The autofluorescence (AF) of NAD(P)H and flavins has been at the basis of many in-situ studies of liver energy metabolism and functionality. Conversely, few data have been so far reported on fluorescing lipids. In this work we investigated the AF of liver lipid extracts from two fatty liver models, Wistar rats fed with MCD diet for 12 days (Wi-MCD), and obese (fa/fa) Zucker rats. Among the most abundant fatty acids in the lipid extracts, indicated by mass spectrometry, arachidonic acid (AA) exhibited higher quantum yield than the other fluorescing fatty acids (FLFA), and red shifted AF spectrum. This allowed to estimate the AA contribution to the overall emission of lipid extracts by curve fitting analysis. AA prevailed in obese Zucker livers, accounting for the different AF spectral profiles between the two models. AF and mass spectrometry indicated also a different balance between the fluorescing fraction and the overall amount of AA in the two models. The ability of AF to detect directly AA and FLFA was demonstrated, suggesting its supportive role as tool in wide-ranging applications, from the control of animal origin food, to experimental investigations on liver fat accumulation, lipotoxicity and disease progression, with potential translation to the clinics.

Seeing the Invisible: Revealing Atrial Ablation Lesions Using Hyperspectral Imaging Approach.

BackgroundCurrently, there are limited means for high-resolution monitoring of tissue injury during radiofrequency ablation procedures.ObjectiveTo develop the next generation of visualization catheters that can reveal irreversible atrial muscle damage caused by ablation and identify viability gaps between the lesions.MethodsRadiofrequency lesions were placed on the endocardial surfaces of excised human and bovine atria and left ventricles of blood perfused rat hearts. Tissue was illuminated with 365nm light and a series of images were acquired from individual spectral bands within 420-720nm range. By extracting spectral profiles of individual pixels and spectral unmixing, the relative contribution of ablated and unablated spectra to each pixel was then displayed. Results of spectral unmixing were compared to lesion pathology.ResultsRF ablation caused significant changes in the tissue autofluorescence profile. The magnitude of these spectral changes in human left atrium was relatively small (< 10% of peak fluorescence value), yet highly significant. Spectral unmixing of hyperspectral datasets enabled high spatial resolution, in-situ delineation of radiofrequency lesion boundaries without the need for exogenous markers. Lesion dimensions derived from hyperspectral imaging approach strongly correlated with histological outcomes. Presence of blood within the myocardium decreased the amplitude of the autofluorescence spectra while having minimal effect on their overall shapes. As a result, the ability of hyperspectral imaging to delineate ablation lesions in vivo was not affected.ConclusionsHyperspectral imaging greatly increases the contrast between ablated and unablated tissue enabling visualization of viability gaps at clinically relevant locations. Data supports the possibility for developing percutaneous hyperspectral catheters for high-resolution ablation guidance.

Spectral diffuse reflectance and autofluorescence imaging can perform early prediction of blood vessel occlusion in skin flaps

Flap transfer has become a common technique in reconstructive surgery. However, a significant number of compromised skin flaps are not successfully salvaged because the current clinical method for flap assessment relies heavily on the clinician's experience. Vascular occlusion is the major reason for flap failure, thus the accurate and objective early prediction of blood vessel occlusion is vitally important. Our parallel point measurement study has demonstrated the great potential of joint diffuse reflectance and autofluorescence spectroscopy in the early detection and differentiation of venous and arterial occlusion in skin flaps. Unfortunately, the technique of point measurements is not suitable to examine a large skin flap when a high spatial resolution is required. In this study, we attempted to overcome this problem by performing spectral diffuse reflectance and autofluorescence imaging on a rat skin flap model. Both imaging data and reconstructed spectra were used to statistically differentiate control flaps, arterially occluded flaps and venously occluded flaps. Our preliminary results suggest that the technique of joint diffuse reflectance and autofluorescence spectroscopic imaging can achieve high classification accuracy thus could be used to detect and differentiate flaps with venous and arterial occlusion accurately at an early time point in a large skin flap. Typical reconstructed spectra of (a) diffuse reflectance and (b) autofluorescence after normalization.

Identification of protein secondary structures by laser induced autofluorescence: A study of urea and GnHCl induced protein denaturation

In the present study an attempt has been made to interrogate the bulk secondary structures of some selected proteins (BSA, HSA, lysozyme, trypsin and ribonuclease A) under urea and GnHCl denaturation using laser induced autofluorescence. The proteins were treated with different concentrations of urea (3M, 6M, 9M) and GnHCl (2M, 4M, 6M) and the corresponding steady state autofluorescence spectra were recorded at 281nm pulsed laser excitations. The recorded fluorescence spectra of proteins were then interpreted based on the existing PDB structures of the proteins and the Trp solvent accessibility (calculated using “Scratch protein predictor” at 30% threshold). Further, the influence of rigidity and conformation of the indole ring (caused by protein secondary structures) on the intrinsic fluorescence properties of proteins were also evaluated using fluorescence of ANS-HSA complexes, CD spectroscopy as well as with trypsin digestion experiments. The outcomes obtained clearly demonstrated GnHCl preferably disrupt helix as compared to the beta β-sheets whereas, urea found was more effective in disrupting β-sheets as compared to the helices. The other way round the proteins which have shown detectable change in the intrinsic fluorescence at lower concentrations of GnHCl were rich in helices whereas, the proteins which showed detectable change in the intrinsic fluorescence at lower concentrations of urea were rich in β-sheets. Since high salt concentrations like GnHCl and urea interfere in the secondary structure analysis by circular dichroism Spectrometry, the present method of analyzing secondary structures using laser induced autofluorescence will be highly advantageous over existing tools for the same.

Autofluorescence discrimination of metabolic fingerprint in nutritional and genetic fatty liver models

Liver tissue autofluorescence (AF) has been characterized in two models with a different potential to undergo disease progression to steatohepatitis: Wistar rats, administered with a methionine, choline deficient diet (MCD), and Zucker (fa/fa) rats, homozygous for a spontaneous mutation of leptin receptor. AF spectra were recorded from liver tissue cryostatic sections by microspectrofluorometry, under 366nm excitation. Curve fitting analysis was used to estimate the contribution of different endogenous fluorophores (EFs) to the overall AF emission: i) fluorescing fatty acids, a fraction of liver lipids up to now poorly considered and complicated to detect by conventional procedures; ii) lipofuscin-like lipopigments, biomarkers of oxidizing events; iii) NAD(P)H and flavins, biomarkers of energy metabolism and tissue redox state. AF data and biochemical correlates of hepatocellular injury resulted to depend more on rat strain than on intratissue bulk lipid or ROS levels, reflecting a different metabolic ability of the two models to counteract potentially harmful agents. AF analysis can thus be proposed for extensive applications ranging from experimental hepatology to the clinics. AF based diagnostic procedures are expected to help both the prediction of the risk of fatty liver disease progression and the prescreening of marginal organs to be recruited as donors for transplantation. A support is also foreseen in the advancement and personalization of strategies to ameliorate the donor organ preservation outcome and the follow up of therapeutic interventions.

Discrimination and quantification of autofluorescence spectra of human lung cells

To study laser-induced autofluorescence spectroscopy of the human lung cell line, we evaluated the native fluorescence properties of cancer QU-DB and normal MRC-5 human lung cells during continuous exposure to 405 nm laser light. Two emission bands centered at ~470 nm and ~560 nm were observed. These peaks are most likely attributable to mitochondrial fluorescent reduced nicotinamide adenine dinucleotide and riboflavin fluorophores, respectively. This article highlights lung cell autofluorescence characterization and signal discrimination by collective investigation of different spectral features. The absolute intensity, the spectral shape factor or redox ratio, the full width of half-maximum and the full width of quarter maximum was evaluated. Moreover, the intensity ratio, the area under the peak and the area ratio as a contrast factor for normal and cancerous cells were also calculated. Among all these features it seems that the contrast factor precisely and significantly discriminates the spectral differences of normal and cancerous lung cells. On the other hand, the relative quantum yield for both cell types were found by comparing the quantum yield of an unknown compound with known fluorescein sodium as a reference solution.

A novel high-throughput multi-parameter flow cytometry based method for monitoring and rapid characterization of microbiome dynamics in anaerobic systems

A novel multidimensional flow cytometry based method has been demonstrated to monitor and rapidly characterize the dynamics of the complex anaerobic microbiome associated with perturbations in external environmental factors. While community fingerprinting provides an estimate of the meta genomic structure, flow cytometry provides a fingerprint of the community morphology including its autofluorescence spectrum in a high-throughput manner. Using anaerobic microbial consortia perturbed with the controlled addition of various carbon sources, it is possible to quantitatively discriminate between divergent microbiome analogous to community fingerprinting techniques using automated ribosomal intergenic spacer analysis (ARISA). The utility of flow cytometry based method has also been demonstrated in a fully functional industry scale anaerobic digester to distinguish between microbiome composition caused by varying hydraulic retention time (HRT). This approach exploits the rich multidimensional information from flow cytometry for rapid characterization of the dynamics of microbial communities.

Microalgal cytometric analysis in the presence of endogenous autofluorescent pigments

Flow cytometry (FCM) is a well-established tool in the field of aquatic phytoplankton ecology and microalgal biotechnology, which allows for rapid assessment of the viability and physiological state of individual cells in algal populations. However, the autofluorescent spectra of different types of chlorophyll and other algal pigments may overlap with fluorescent dyes and affect the resolution of algae clusters, sensitivity, and signal-to-noise ratio. Dying algal cells continue to exhibit a strong autofluorescent signal, which may affect the evaluation of algal viability.Herein, we tested two different approaches to measure algal fluorescence in the presence of a strong autofluorescent signal: 1) by separating dyes between different excitation lasers in order to reach minimal spectral overlap with the autofluorescent signal using flow and imaging cytometry and 2) through full spectrum analysis, virtual filtering and spectral unmixing of dye combinations and algal pigments' autofluorescence via spectral flow cytometry. For this purpose, we used viability dyes from the SYTOX family and lipophilic dyes. Among the dyes tested, the SYTOX Blue (SB) dye had minimal overlap with chlorophyll fluorescence and can be combined with autofluorescence assessment and lipophilic dyes (validated with Emiliania huxleyi algal monocultures). Imaging cytometry provided a detailed characterization of algal subpopulations stained with a combination of fluorescent dyes. A spectral flow cytometer allowed us to analyze environmental phytoplankton samples stained with fluorescent dyes in the presence of strong and heterogeneous autofluorescence from intrinsic algal pigments. We concluded that the multi-color staining of algal samples can be achieved in the presence of strong and diverse algal autofluorescence using dyes with minimal spectral overlap, a multi-laser approach (flow and imaging cytometry) and/or virtual filter and spectral flow cytometry instrumentation. This can open a new page for analytical and cell sorting algal applications.

Synthesis of highly photostable NIR-emitting quantum dots CdTeSe/CdS/CdZnS/ZnS

The use of near IR (NIR) fluorescent labels is promising in such areas as molecular biology, diagnostics and imaging, because this spectrum region is absorbed minimally by tissues and is significantly shifted from the autofluorescence spectra of biological materials. The development of NIR LED sources is of interest as well. One of the key limitations of most NIR fluorophores is lack of stability to environmental conditions, in particular their low photostability. In this paper we propose a method of synthesis of luminescent core/shell cadmium telluride-selenide nanoparticles with quantum yield of 80% and increased resistance to photodegradation.

Autofluorescence spectroscopy in the differentiation of laryngeal epithelial lesions – preliminary results

Conclusions Autofluorescence spectroscopy may be a supporting tool for differential diagnosis of changes in laryngeal epithelium. Objectives Early detection and differential diagnosis of proliferative changes in the larynx are still a challenge for laryngologists. The aim of the study was to evaluate the autofluorescence spectroscopy technique to in vitro differential diagnosis of pathological changes in the epithelium of the larynx. Methods Forty-two patients aged 34–79 years were included in the study. The fifty-two tissue specimens, including 10 samples of cancerous lesion, 10 adjacent normal tissue, 10 chronic inflammation, eight cyst, three leukoplakia, four polyp, and seven Reinke’s edema, were obtained during laryngological procedures. All tissue samples were independently diagnosed histopathologically. The autofluorescence emission spectra at two excitation wavelengths, 290 nm and 370 nm, were measured for every sample studied. Results The autofluorescence signals of cancerous tissue samples at both excitations exhibited identical emission band shapes of much lower intensities at their maxima as compared to the adjacent healthy tissue samples studied. The autofluorescence spectra intensities of cancerous and normal tissues varied inter-individually. Evident differences in autofluorescence intensities and its band shapes of different pathological laryngeal changes at the 290 nm excitations were demonstrated.

NADH Conformation Assessed using Spectral Phasor Analysis: Illustrating Concepts from Molecular Folding to Metabolic Monitoring

The wide applicability of spectroscopy makes fluorescence measurements on biomolecules an attractive modality for introducing biophysical topics into undergraduate physics laboratory courses. Here we show how the spectroscopic assessment of reduced nicotinamide adenine dinucleotide (NADH) conformation is useful for conveying concepts spanning multiple size scales. While advanced sources of optical contrast for NADH conformation include fluorescence excitation transfer efficiency, excited-state lifetime, and anisotropy decay rate, our recently published studies have shown how the quantification of autofluorescence spectrum shape using spectral phasor analysis is useful for monitoring NADH conformations during metabolic transitions. Here we present the pedagogical use of spectral phasor approaches to assess NADH conformation, with educational modules illustrating concepts ranging from molecular folding to protein binding, in addition to cellular metabolic monitoring. In the first module, we explore the NADH folding-unfolding transition in solution during methanol denaturation. The two-state nature of the transition and the degree of unfolding are evaluated using spectral phasors. A two-state solvent-denaturation model is used to estimate the free energy of transition. Next, we consider the protein binding of NADH to various dehydrogenases, showing that binding to different proteins can be discriminated optically, providing a spectroscopic means of examining ligand-binding behavior. The NADH binding constant to malate dehydrogenase is also measured, with the degree of ligand binding quantified using spectral phasors. Finally at the cellular level, the response of energy metabolism in Saccharomyces cerevisiae to various functional modifiers is monitored. We note that all modules were originally research projects involving undergraduate students, and so results here illustrate the relationship between student-driven, faculty-mentored research and the translation of those outcomes to courses centered around discovery-oriented learning.

The molecular basis of the characteristics of the serum auto-fluorescent spectrum of patients with ovarian cancer and the influence of surgery on it

Objective To explore the molecular basis of the characteristics of the serum auto-fluorescent spectrum in patients with ovarian cancer and the changes that might be induced by surgery. Methods Using fluorospectrophotometer and 300nm excitation light,the serum auto-fluorescent spectrum of 84 patients with ovarian cancer before and after the surgery and 30 healthy people were detected. Meanwhile, the serum tumor signs (CEA, CA199 and CA125), hemoglobin and plasma albumin level of all patients with ovarian cancer were detected. Their correlation with the fluorescence spectral characteristic parameters were analyzed. Results Compared with healthy people, the serum auto-fluorescent spectrum in patients with ovarian cancer exhibited purple-shifted position of λ2 peak and red shift in λ4 peak,had higher peak extent in λ1, λ2, λ4 and λ6 peak, and larger peak area of λ1, λ2, λ3, λ4 and λ6. Compared with those in ovarian cancer patients before surgery, the serum auto-fluorescent spectrum in these patients after operation had red shifts in λ1, λ2 and λ3 peak, lower peak extent in λ1, λ2, λ3 and λ4, and smaller peak area in λ1, λ2, λ3, λ4 and λ6 peak. In ovarian cancer patients, the serum level of CEA was positively correlated with the λ2 peak extent and the peak area of λ2 and λ3, while the serum level of CA125 was positively correlated with the peak extent of λ1-λ4 and λ6 and the peak area of λ1-λ3. The serum level of CA199 was negatively correlated with the λ2 position and positively correlated with the peak extent of λ1-λ6 and the peak area of λ1-λ3 and λ6 in patents with ovarian cancer. Besides, the serum albumin was positively correlated with the λ2 peak position and negatively with the peak extent of λ1-λ6 and the λ1-λ3 peak area, while the level of hemoglobin was positively correlated with λ1 peak position. Conclusions The elevated serum tumor markers and lower albumin (plasma) level lead to the changes of the serum autofluorescence spectra characteristic parametersin in patients with ovarian cancer.These changes can be modestly corrected by surgery. Key words: Ovarian neoplasms; Spectrometry, fluorescence; Serum; Tumor markers, biological; Plasma albumin; Hemoglobin

Spectral characteristics of caries-related autofluorescence spectra and their use for diagnosis of caries stage.

The purpose of the present study was to identify factors useful for diagnosis of the caries stage from laser-induced autofluorescence (AF) spectra. Affected teeth were accurately staged and allocated to four groups: sound, stage II, stage III, or stage IV. A 405-nm laser was used to produce AF spectra. The spectrum factors analyzed were spectrum slope at 550 to 600 nm, spectral area from 500 and 590 nm, and intensity ratio of peaks 625 and 667 nm ( 625/667 nm ). DIAGNOdent was used as control measurement. AF spectra of sound teeth had a peak near 500 nm followed by a smooth decline to 800 nm. As caries progressed, some specimens in stages II to IV showed one or two peak(s) near 625 and 667 nm. Slopes at 550 to 600 nm and areas under the curve at 500 to 590 nm were significantly different ( p 0.001 ) for each stage. Two-peak ratios were also significantly different ( p 0.001 ) except for stage III and stage IV. DIAGNOdent readings for sound and stage II and stage III and IV were not significantly different. Among the studied factors, the spectrum slope at 550 to 600 nm and area under curve at 500 to 590 nm could be useful treatment decision-making tools for carious lesions.

Fluorescence characteristics of human Barrett tissue specimens grafted on chick chorioallantoic membrane.

To improve (pre)malignant lesion identification in Barrett’s esophagus (BE), recent research focuses on new developments in fluorescence imaging and spectroscopy to enhance tissue contrast. Our aim was to validate the chorioallantoic membrane (CAM) model as a preclinical tool to study the fluorescence characteristics such as autofluorescence and exogenously induced fluorescence of human Barrett’s tissue. Therefore, esophageal biopsy specimens from Barrett’s patients were freshly grafted onto the CAM of fertilized hen’s eggs to simulate the in vivo situation. The BE biopsy specimens stayed between 1 and 9 days on the CAM to study the persistence of vitality. Fluorescence spectroscopy was performed using six excitation wavelengths (369, 395, 400, 405, 410, 416 nm). Obtained autofluorescence spectra were compared with in vivo spectra of an earlier study. Exogenous administration of 5-aminolevulinic-acid to the biopsy specimens was followed by fluorescence spectroscopy at several time points. Afterwards, the biopsy specimens were harvested and histologically evaluated. In total, 128 biopsy specimens obtained from 34 patients were grafted on the CAM. Biopsy specimens which stayed on average 1.7 days on the CAM were still vital. Autofluorescence spectra of the specimens correlated well with in vivo spectra. Administered 5-aminolevulinic-acid to the biopsy specimens showed conversion into protoporphyrin-IX. In conclusion, we showed that grafting freshly collected human BE biopsy specimens on the CAM is feasible. Our results suggest that the CAM model might be used to study the fluorescence behavior of human tissue specimens. Therefore, the CAM model might be a preclinical research tool for new photosensitizers.

Microfluorescent analysis for bioindication of ozone on unicellular plant systems

Various unicellular model plant systems, on which autofluorescence, fluorescence after histochemical treatment, and growth rates were investigated, have been proposed as bioindicators of ozone. Analysis is performed using fluorescent microscopy in different modifications, including microspectrofluorimetry and laser scanning confocal microscopy. It is found that low ozone doses (0.005 μLL−1 for 2.5-h exposure or 0.008 μLL−1 for 4-h exposure) do not affect or stimulate autofluorescence of the samples. In addition, vegetative microspores and pollen retain their ability to germinate in an artificial medium, and their growth is even enhanced. Higher ozone concentrations lead to either a decrease in the emission intensity or a shift of peaks in the fluorescence spectrum. In particular, 16-h exposure of vegetative microspores of horsetail to ozone (total dose 0.032 μLL−1) leads to occurrence of a new peak in the wavelength range of 515 to 520 nm in their autofluorescence spectrum. Exposure to high (more than 0.1 μLL−1) doses gives rise to a similar peak in the spectrum of leaf secretory hairs of Raphanus sativus. The spectrum of leaf secretory hairs of Fragaria viridis exhibits a decrease in the emission intensity at wavelengths of 520 to 550 nm. Stress metabolites have been revealed by fluorescence at wavelengths from 460 to 480 nm after specific histochemical reactions for determination of catecholamines or histamine. After exposure in ozone to a total dose of 0.012 μLL−1, a significant increase in the amount of catecholamines and the histamine content was observed for pollen of Populus balsamifera. Higher concentrations of catecholamines (in comparison with control samples) are found in vegetative microspores of Equisetum arvense and pollen of Corylus avellana and Populus balsamifera after exposure in ozone to high doses (0.032 μLL−1), whereas a decrease in the catecholamine concentration was observed for pollen of Tulipa hybridum and Dolichothele albescens after this treatment.

Discrimination of Dental Caries Using Colorimetric Characteristics of Fluorescence Spectrum

The feasibility of colorimetric parameters for the discrimination of the stages of dental caries based on a light-induced autofluorescence spectrum at a 405-nm excitation wavelength was investigated. The fluorescence spectra of 4 groups of tooth samples (10 sound, 10 early-stage decay, 14 established decay, and 10 severe decay), which were classified by the International Caries Detection and Assessment System, were experimentally measured in vitro. The carious lesion samples had an additional fluorescence peak at around 627 nm. The mathematical relation of the fluorescence spectrum and human color perception was established and computed. With increasing severity, the fluorescence color changed from green to yellow according to the colorimetric parameters of the CIE 1931 (x, y) chromaticity coordinates and dominant wavelengths. The results from a one-way ANOVA of the dominant wavelength showed a statistically significant difference among the 4 classified groups. The colorimetric parameters of the light-induced fluorescence spectrum can potentially be applied to evaluate the various carious levels.

High-resolution, label-free imaging of living cells with direct electron-beam-excitation-assisted optical microscopy.

High spatial resolution microscope is desired for deep understanding of cellular functions, in order to develop medical technologies. We demonstrate high-resolution imaging of un-labelled organelles in living cells, in which live cells on a 50 nm thick silicon nitride membrane are imaged by autofluorescence excited with a focused electron beam through the membrane. Electron beam excitation enables ultrahigh spatial resolution imaging of organelles, such as mitochondria, nuclei, and various granules. Since the autofluorescence spectra represent molecular species, this microscopy allows fast and detailed investigations of cellular status in living cells.

Autofluorescence from NADH Conformations Associated with Different Metabolic Pathways Monitored Using Nanosecond-Gated Spectroscopy and Spectral Phasor Analysis

Cellular NADH conformation is increasingly recognized as an endogenous optical biomarker and metabolic indicator. Recently, we reported a real-time approach for tracking metabolism on the basis of the quantification of UV-excited autofluorescence spectrum shape. Here, we use nanosecond-gated spectral acquisition, combined with spectrum-shape quantification, to monitor the long excited-state lifetime autofluorescence (usually associated with protein-bound NADH conformations) separately from the autofluorescence signal as a whole. We observe that the autofluorescence response induced by two NADH-oxidation inhibitors—cyanide and ethanol—are similar in Saccharomyces cerevisiae when monitored using time-integrated detection but easily distinguished using time-gated detection. Results are consistent with the observation of multiple NADH conformations as assessed using spectral phasor analysis. Further, because well-known oxidation inhibitors are used, changes in spectrum shape can be associated with NADH conformations involved in the different metabolic pathways, giving bioanalytic utility to the spectral responses.

Automated measurement of multiple cancer biomarkers using quantum-dot-based microfluidic immunohistochemistry.

We report an automated multiple biomarker measurement method for tissue from cancer patients using quantum dot (QD)-based protein detection combined with reference-based protein quantification and autofluorescence (AF) removal. For multiplexed detection of biomarkers in tissue samples, visualization of QDs on cytokeratin was performed to create a multichannel microfluidic device on sites with dense populations of tumor cells. Three major breast cancer biomarkers (i.e., estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) were labeled using QDs successively on cancer cells in tissue sections. For the automated measurement of biomarkers, a cytokeratin-based biomarker normalization method was used to measure the averaged expression of proteins. A novel AF-removal algorithm was developed, which normalizes the reference AF spectra reconstructed from unknown AF spectra based on random sampling. For accurate quantification of QDs, we automatically and accurately removed the AF signal from 344 spots of QD-labeled tissue samples using 240 reference AF spectra. Using analytical data with 10 tissue samples from breast cancer patients, the measured biomarker intensities were in good agreement with the results of conventional analyses.