Autofluorescence Method Research Articles

The Spatial Profile of Macular Pigments Is Related to the Topological Characteristics of the Foveal Avascular Zone.

Macular pigments are preferentially concentrated in the central fovea, an area devoid of vasculature. We hypothesized that there may be a link between the macular pigment profile and the size and structural characteristics of the foveal avascular zone (FAZ). Two-wavelength autofluorescence method was used to quantify macular pigment optical density (MPOD) and the radius at half peak of MPOD, which was defined as the retinal eccentricity where the MPOD value was 50% of the peak value. Volumetric spectral-domain optical coherence tomography (OCT) images of the macula were obtained from 32 subjects. The equivalent radius of the FAZ was determined using data generated from OCT angiography. Generalized estimating equations were used to test the hypothesis that there are interrelationships among the central foveal thickness, peak MPOD, the radius at half peak of MPOD and the equivalent radius of the FAZ. The equivalent radius of the FAZ was highly correlated with the radius at half peak of MPOD (P < .001). The equivalent radius of the FAZ was a significant predictor for central foveal thickness (P < .001). The significant predictor for peak MPOD was central foveal thickness (P = .004). Eyes with larger FAZs were more likely to have a secondary peak in their MPOD spatial profile in a zone ranging from 0.5 to 1.0 degrees from the foveal center. The spatial distribution of macular pigment is related to the size of the FAZ, in addition to the central foveal thickness. It is possible that xanthophyll pigment accumulation in the macula serves functions, such as attenuation of shorter wavelengths of light, that would have been provided by the light-filtering characteristics of blood vessels.

Effect of nitrogen source and acclimatization on specific growth rates of microalgae determined by a high-throughput in vivo microplate autofluorescence method

Specific growth rates (SGR) of freshwater algae species (Chlorella vulgaris, Auxenochlorella protothecoides, and Chlorella sorokiniana) and the marine species Nannochloropsis oculata on various nitrogen sources (ammonium carbonate, ammonium chloride, sodium nitrate, and urea) could be determined by in vivo chlorophyll-a autofluorescence. These preferences could be determined before large pH changes occurred in the media, with no significant difference (P > 0.05) between buffered and non-buffered media. In all algal species, acclimatization was observed with no significant difference (P > 0.05) between SGRs of the second and third cultivations. ANOVA of SGRs in the acclimatized second and third cultivations revealed preferences for nitrogen sources among most of the algae; C. vulgaris preferred sodium nitrate over other nitrogen sources, A. protothecoides adapted to urea after no growth in the first cultivation, and the SGRs of N. oculata showed an aversion for sodium nitrate over other nitrogen sources (P < 0.05).

Аутофлуоресценция, клеточное дыхание и современные возможности его неинвазивного исследования (обзор литературы)

This review is devoted to modern non-invasive studies of cellular respiration. Characteristic fluorescence biological tissue under the influence of ultraviolet radiation, which can reflect the process of biological oxidation, is presented. The erythron importance to ensure a functional condition of an organism and part of blood cells in the respiratory chain, gas transportation and information functions is considered. The authors described the biomarkers of cell respiration and showed the complexity of modern methods of research of biological oxidation and the necessity of using low-cost techniques based on microscopic analysis. The method of laser biospectrophotometry is characterized. The evaluation method of tissue auto-fluorescence, its biological basis, the possibility of dual-channel spectrophotometer facilities – domestic and foreign, is developed and presented in this review. The importance of fiber-optic television spectrophotometry in the noninvasive determination of cellular respiration in living organisms is proved. The authors gave a footnote to works, confirming the practical diagnostic significance of the definition of auto-fluorescence.

Laser polarization autofluorescence of endogenous porphyrins of optically anisotropic biological tissues and fluids in diagnostics of necrotic and pathological changes of human organs.

This research presents the results of investigation of laser polarization fluorescence of biological layers (histological sections, cytological smears). The polarization structural properties of autofluorescent images of human biological tissues layers and fluids were found and investigated. A model describing the formation of polarizationally heterogeneous images of optically anisotropic biological layers is suggested. On this basis, the practical method of polarization-variable autofluorescence is analytically substantiated and experimentally tested. The efficiency of applying this method to various tasks of medical diagnostics is analyzed: objectification of histological conclusions, defining and differentiating of various forms of cancer (dysplasia--microinvasive cancer) of the cervix uteri, and forensic medical express-differentiation of cause of death. The objective criteria (statistical moments) of differentiation of autofluorescent images of histological sections of myocardium biopsy and endometrium and cytological smears of its mucous tunic are defined. The operational characteristics (sensitivity, specificity, accuracy) of this method are determined concerning the positions of probative medicine, and the clinical efficiency of the technique is demonstrated.

Enhanced visibility of colonic neoplasms using formulaic ratio imaging of native fluorescence

Colonoscopy is the preferred method for colon cancer screening, but can miss polyps and flat neoplasms with low color contrast. The objective was to develop a new autofluorescence method that improves image contrast of colonic neoplasms. We selected the three strongest native fluorescence signals and developed a novel method where fluorescence images are processed in a ratiometric formula to represent the likely cellular and structural changes associated with neoplasia. Native fluorescence images of fresh surgical specimens of the colon containing normal mucosa, polypoid and flat adenomas as well as adenocarcinoma were recorded using a prototype multi-spectral imager. Sixteen patients, with a mean age of 62 years (range 28-81) undergoing elective resection for colonic neoplasms were enrolled. High contrast images were seen with fluorescence from tryptophan (Tryp), flavin adenine dinucleotide (FAD) and collagen. When the image intensity of Tryp was divided pixel by pixel, by the intensities of FAD and collagen, the resulting formulaic ratio (FR) images were of exceptionally high contrast. The FR images of adenomas and adenocarcinomas had increased Weber contrast. FR imaging is a novel imaging process that represents the likely metabolic and structural changes in colonic neoplasia that produces images with remarkably high contrast.

Comparison of macular pigment optical density measured by autofluorescence and reflectometry: The LIMPIA study

AbstractPurpose To compare macular pigment optical density (MPOD) measured by two techniques (autofluorescence, reflectometry) in healthy subjects at high risk for age‐related macular degeneration (AMD).Methods The Limpia Study is a double‐blind, placebo controlled, prospective randomized clinical trial performed in 120 subjects with at least one parent affected by neovascular AMD. Subjects having used supplements containing lutein and/or zeaxanthin in the preceding year were not included. MPOD was measured using two methods: the two‐wavelength autofluorescence method with a modified scanning laser ophthalmoscope (Heidelberg Retinal AnalyzerResults At baseline, mean MPOD within 0.51°, measured with the modified HRA method, was 0.5 (standard deviation 0.2). Maximal MPOD, measured with Visucam, was 0.4 (standard deviation 0.1). Parameters from the modified HRA (optical density within 0.5°, 1°, 2° and 6°) and from the Visucam method (volume, area, mean and maximum optical density) were weakly correlated. The best correlations were observed for MPOD within 2° and 6° with volume from Visucam(r=0.21 and r=0.22, respectively), and mean MPOD from Visucam (r=0.21 and r=0.18).Conclusion The two methods propose different parameters to evaluate macular pigment, which overall correlated weakly in this population of middle‐aged healthy subjects at high risk for AMD. Further research is needed to characterize the differences between the methods and identify the best parameters and techniques to measure macular pigment. Commercial interest

An efficient autofluorescence method for screening Limonium bicolor mutants for abnormal salt gland density and salt secretion

With the spread of saline soils worldwide, it has become increasingly important to understand salt-tolerant mechanisms and to develop halophytes with increased salt tolerance. Limonium bicolor is a typical recretohalophyte and has a typical salt excretory structure in the epidermis called the salt gland. A method that can be used to screen a large population of L. bicolor mutants for altered salt gland density and altered salt secretion is needed but is currently unavailable. Leaves of 1-month-old L. bicolor seedlings were processed by three traditional methods [epidermal peel, nail impression, and clearing/differential interference contrast microscope (clearing/DIC) method] and a fluorescence method (fluorescence microscopic examination of cleared leaves). With the fluorescence method, the autofluorescence of salt glands under UV excitation (330–380nm) was easily distinguished with the least labor and time. The fluorescence method was used to screen ~10,000 seedlings (which grew from gamma-irradiated seeds). Four mutants with reduced salt gland density and 15 with increased salt gland density were obtained. Both kinds of mutants will be useful for the isolation of genes involved in salt gland development and salt secretion in L. bicolor and other halophytes. The fluorescence method was also successfully used to observe the salt glands of Aegialitis rotundifolia and the stomata and trichomes of Arabidopsis. The fluorescence method described here will be useful for examining plant epidermal structures that have autofluorescence under UV or other wavelengths.

Increased advanced glycation end-products (AGEs) assessed by skin autofluorescence in schizophrenia

BackgroundOxidative stress has been intensively studied as a key biochemical system in the pathophysiology of schizophrenia. However, little is known about the implication of oxidative stress in the development of physical illnesses in schizophrenia patients, who are characterized by high cardiovascular risk and decreased life expectancy. Advanced glycation end-products (AGEs) are considered to be markers of oxidative stress and are linked to the development of atherosclerosis. MethodsWe investigated AGE levels determined by a noninvasive skin autofluorescence (skin AF) method (AGE-Reader™) in schizophrenia patients. Skin AF was assessed in 55 schizophrenia patients without diabetes or renal disease and 55 healthy controls matched for age, gender and smoking status. Nineteen of the 55 schizophrenia patients had a severe form of the disease (Kraepelinian schizophrenia). ResultsSkin AF was significantly higher in schizophrenia patients compared to controls (2.46 ± 0.52 and 1.90 ± 0.21, respectively, p < 0.0001). Kraepelinian schizophrenia patients had significantly higher skin AF than non-Kraepelinian schizophrenia patients (p = 0.05). ConclusionsThis is the first study to demonstrate high AGE levels assessed by a noninvasive method in schizophrenia patients.

Ethnic and gender differences in advanced glycation end products measured by skin auto-fluorescence

Background Advanced glycation end products (AGEs) have been shown to be a predictor of cardiovascular risk in Caucasian subjects. In this study we examine whether the existing reference values are useable for non-Caucasian ethnicities. Furthermore, we assessed whether gender and smoking affect AGEs.Methods AGEs were determined by a non-invasive method of skin auto-fluorescence (AF). AF was measured in 200 Arabs, 99 South Asians, 35 Filipinos and 14 subjects of other/mixed ethnicity in the Qatar Metabolomics Study on Diabetes (QMDiab). Using multivariate linear regression analysis and adjusting for age and type 2 diabetes, we assessed whether ethnicity, gender and smoking were associated with AF.Results The mean AF was 2.27 arbitrary units (AU) (SD: 0.63). Arabs and Filipinos had a significant higher AF than the South Asian population (0.25 arbitrary units (AU) (95% CI: 0.11‒0.39), p = 0.001 and 0.34 (95% CI: 0.13‒0.55), p = 0.001 respectively). Also, AF was significantly higher in females (0.41 AU (95% CI: 0.29‒0.53), p < 0.001). AF associated with smoking (0.21 AU (95% CI: 0.01‒0.41), p = 0.04) and increased with the number of pack-years smoked (p = 0.02).Conclusions This study suggests that the existing reference values should take ethnicity, gender and smoking into account. Larger studies in specific ethnicities are necessary to create ethnic- and gender-specific reference values.

Association of age and macular pigment optical density using dual-wavelength autofluorescence imaging

BackgroundSeveral lines of evidence suggest that macular pigment may play a protective role against age-related macular degeneration, but the influence of age on macular pigment density levels remains unclear. This study was designed to investigate the relationship between age and the normal distribution of macular pigment optical density (MPOD) values surrounding the fovea.MethodsConsecutive healthy subjects with no evidence of ocular disease were enrolled in this study. After inclusion, MPOD values were measured at specific eccentricities (0.5, 1, and 2 degrees) from the foveal center using a dual-wavelength autofluorescence method employing a modified confocal scanning laser ophthalmoscope. Whenever both eyes were eligible, one was randomly selected for analysis. The correlation between age and MPOD values was investigated using regression analysis.ResultsThirty subjects (30 eyes) were included (mean age 48.6 ± 16.4 [range 23–77] years). Significant differences were found between MPOD values measured at 0.5, 1, and 2 degrees from the center of the fovea (0.49 ± 0.12 density units, 0.37 ± 0.11 density units, and 0.13 ± 0.05 density units, respectively, P < 0.05). Significant correlations between age and MPOD values at 0.5 and 1 degree were found (P ≤ 0.02). Values measured at 2 degrees did not correlate significantly with age (P = 0.06).ConclusionIn healthy subjects, MPOD values were highest near the foveal center. These values appeared to increase during adulthood (peak at 45–50 years), followed by a gradual reduction after 60 years of age.

Relationship between tissue glycation measured by autofluorescence and pulse wave velocity in young and elderly non-diabetic populations

Advanced glycation end-products (AGEs) and pulse wave velocity (PWV) are pivotal indices of the processes of arterial ageing and damage accumulation. The aim of the present study was to investigate the impact of AGEs, as measured by a non-invasive skin autofluorescence method, on arterial stiffness, estimated by PWV, in two different age groups of non-diabetic subjects. A total of 116 non-diabetic subjects were classified into two groups, with 55 subjects in the group aged < 65 years and 61 in the group aged ≥ 65 years. AGEs were measured by skin autofluorescence while carotid-femoral PWV was assessed by tonometry. A significant (positive) association was observed between PWV and AGE skin autofluorescence in the younger age group (r = 0.51; P < 0.0001). However, this association was no longer significant after further adjustments for age and other factors on multiple regression analyses. In contrast, this correlation was not found in the elderly group (r = 0.098; P = 0.454). Younger non-diabetic subjects exhibit a different correlation profile between AGEs accumulated in skin and cfPWV as an index of arterial stiffness compared with elderly subjects. AGEs were significantly associated with cfPWV in younger individuals, but not in the elderly. A further study with a larger number of subjects is proposed to confirm the contribution of AGEs, the formation of which is manageable, as a determinant of arterial stiffness in younger subjects.

Association of Macular Pigment Density with Plasma Omega-3 Fatty Acids: The PIMAVOSA Study

To assess the correlation between macular pigment optical density and plasma levels of lutein, zeaxanthin, and fatty acids, especially omega-3 polyunsaturated fatty acids (PUFAs). The PIMAVOSA study is an observational study of 107 healthy volunteers, aged 20 to 60 years and born in southwest France, without histories of ocular disease. Macular pigment optical density (MPOD) was measured using the two-wavelength autofluorescence method with a modified scanning laser ophthalmoscope. Plasma measurements (lutein, zeaxanthin, and fatty acids) were performed from fasting blood samples collected on the day of the eye examination. MPOD within 6° correlated with plasma levels of lutein and zeaxanthin (r = 0.35, P < 0.001, and r = 0.30, P < 0.005, respectively). MPOD also significantly correlated with total plasma omega-3 PUFAs (r = 0.22, P < 0.05). Among the different omega-3 PUFAs, docosapentaenoic acid (DPA) had the highest correlation with MPOD (r = 0.31, P < 0.001), whereas correlation with eicosapentaenoic acid (EPA) was moderate (r = 0.21, P < 0.05) and did not reach statistical significance for docosahexaenoic acid (r = 0.14, P = 0.14). In the present study, macular pigment density was associated not only with plasma lutein and zeaxanthin but also with omega-3 long-chain PUFAs, particularly with EPA and DPA. Further studies will be needed to confirm these findings and to identify the underlying mechanisms.

Circulating endothelial progenitor cells and large artery structure and function in young subjects with uncomplicated Type 1 Diabetes

BackgroundCarotid intima-media thickness (IMT), indices of large artery stiffness and measures of endothelium function may be used as markers of early atherosclerosis in type 1 diabetes mellitus (T1DM). The aim of the present study was to compare the indices of large artery structure and function as well as endothelial function and regenerating capacity between adolescents with T1DM and healthy control of similar age. In addition, the associations of different vascular measures with endothelial progenitor cells (EPCs), glyco-metabolic control and serum levels of advanced glycation endproducts (AGEs), soluble receptors for AGEs (sRAGE) and adiponectin were evaluated.MethodsSixteen uncomplicated young T1DM patients (mean age 18 ± 2 years, history of disease 11 ± 5 years, HbA1c 7.7 ± 1.1%) and 26 controls (mean age 19 ± 2 years) were studied. A radiofrequency-based ultrasound system (Esaote MyLab 70) was used to measure carotid IMT and wave speed (WS, index of local stiffness), applanation tonometry (PulsePen) was applied to obtain central pulse pressure (PP) and augmentation index (AIx), and carotid-femoral pulse wave velocity (PWV, Complior) was used as index of aortic stiffness. Peripheral endothelium-dependent vasodilation was determined as reactive hyperemia index (RHI, EndoPAT). Circulating EPCs, glycometabolic profile, AGEs (autofluorescence method), sRAGE and adiponectin were also measured.ResultsAfter adjusting for age, sex and blood pressure, T1DM adolescents had significantly higher carotid IMT (456 ± 7 vs. 395 ± 63 μm, p < 0.005), carotid WS (p < 0.005), PWV (p = 0.01), AIx (p < 0.0001) and central PP (p < 0.01) and lower EPCs (p = 0.02) as compared to controls. RHI was reduced only in diabetic patients with HbA1c ≥7.5% (p < 0.05). In the overall population, EPCs were an independent determinant of carotid IMT (together with adiponectin), while fasting plasma glucose was an independent determinant of carotid WS, AIx and central PP.ConclusionsOur findings suggest that young subjects with relatively long-lasting T1DM have a generalized preclinical involvement of large artery structure and function, as well as a blunted endothelium regenerating capacity. Hyperglycemia and suboptimal chronic glycemic control seem to deteriorate the functional arterial characteristics, such as large arteries stiffness, wave reflection and peripheral endothelium-dependent vasodilation, whereas an impaired endothelium regenerating capacity and adiponectin levels seem to influence arterial structure.

Characterization of Adenocarcinoma’s Autofluorescence Properties Using Multiexcitation Analysis Method

Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 General purpose of this research is to get an early cancer detection method based on the properties of optical analysis between normal and adenocarsinoma tissue using the multiexcitation autofluorescence method. Observation of autofluorescence properties was done on the biopsy sample of adenocarcinoma tissues, GR mice transplanted by adenocarsinoma, and cell culture SM 1. Excitation on tissue was done by using &nbsp; the lamp Light Emitting Diode (LED) at some visible light wavelength range. This research obtained that the value of Intensity Auto fluorescence (IAF) at range red wavelength of cells and adenocarsinoma tissues tend to lower compared to the cells normal tissues if its were excited by blue LED. On the contrary, the value of IAF at infra red wavelength from cells and carcinoma tissues tend to higher compared to the cells and normal tissues if its were excited by red LED.

Macular pigment density at the site of altered fundus autofluorescence

The purpose of our study was to determine whether abnormalities of increased or decreased fundus autofluorescence (FAF) are associated with local changes in macular pigment (MP) optical density in patients with age-related maculopathy (ARM) and macular degeneration (ARMD). FAF imaging and MP measurement was performed through dilated pupils using a modified confocal scanning laser ophthalmoscope (HRA, Heidelberg Engineering, Germany) according to a standard protocol. Two-wavelength autofluorescence method was employed for determination of local macular pigment optical density (LMPOD). Image analysis and measurement of LMPOD at the area of altered FAF was performed using Heidelberg Eye Explorer Software. Mean values of LMPOD at the site of FAF abnormality were compared to an adjacent location with normal background FAF of the same image. Sixty-three eyes of 63 patients (28 male, 35 female, mean age 75.8 ± 8.8 years) were included in this analysis. Group 1 comprised 31 cases with focal increased FAF. Mean LMPOD in the area of increased FAF was 0.073 ± 0.083 compared to 0.075 ± 0.074 in the adjacent area of normal FAF. Group 2 comprised 32 cases of focal decreased FAF. Mean LMPOD in the area of decreased FAF was -0.004 ± 0.088 compared 0.053 ± 0.075 in the adjacent area of normal FAF. The site of increased FAF showed no significant difference in LMPOD (p = 0.927) compared to adjacent areas of normal FAF, while areas of decreased FAF revealed significantly lower LMPOD (p = 0.001) compared to adjacent areas of normal FAF. Focal increases of FAF due to ARM or ARMD did not lead to change in LMPOD. Presumably, retinal layers containing MP are unaffected by these processes. For lesions exhibiting focal decreased FAF, a reduction of LMPOD cannot be excluded. Further studies are needed to investigate MP in the course of disease.

Effectiveness of autofluorescence to identify suspicious oral lesions—a prospective, blinded clinical trial

Regular screening through white light inspection of the entire oral mucosa is the most important examination method to identify precancerous lesions and early oral carcinoma. Additionally, the physiologic autofluorescence of the oral mucosa has been described as a novel screening method for the detection of mucosal lesions that are not visible by white light. This study aimed to evaluate the sensitivity and specificity of the autofluorescence examination. Seventy-eight patients were examined in this study. All of them suffered from suspicious oral mucosal lesions. Two different investigation methods were applied: the standard examination by white light and an examination by a novel light source of 400nm that evoked a green light emission (>500nm) in normal mucosa. It was proposed that malignant oral mucosal lesions show different autofluorescence characteristics than the green autofluorescence of healthy mucosa. Red autofluorescence indicated SCC with a sensitivity of 20% and a specificity of 98%. The results showed that dysplasia and carcinoma could be identified with a sensitivity of 96% and a specificity of 18% by using the autofluorescence method. The sensitivity decreased according to the grade of mucosal keratosis and was influenced by the localisation of the lesion. In conclusion, benign as well as malignant oral lesions could not be distinguished by a diminished autofluorescence signal. A red autofluorescence signal, however, could indicate cancerous processes of the oral mucosa.

Möglichkeiten der objektiven Messung der optischen Dichte der Makula

Determination of the optical density of the macular pigment may be used for an assessment of the efficacy of oral lutein supplementation in patients with dry AMD. Beside subjective methods like heterochrome flicker photometry, objective methods like the 2-wavelength auto-fluorescence method and the 1-wavelength reflection method are in clinical use today. Both methods show comparable results. For a long-term assessment of the efficacy of oral lutein supplementation, different parameters like mean and maximal optical density as well as the integral over optical density on all pixels ("volume") should be used. The parameter volume often increases also in cases in which other parameters do not change anymore. The 1-wavelength reflection method is used for the newly initiated LUTEGA study. This study will investigate the long-term effects of a fixed lutein/omega-3-fatty acid combination on the optical density in patients with dry AMD.

Simple and objective method for routine detection of the macular pigment xanthophyll

A new simple method for two-dimensional determination of optical density of macular pigment xanthophyll (ODx) in clinical routine is based on a single blue-reflection fundus image. Individual different vignetting is corrected by a shading function. For its construction, nodes are automatically found in structureless image regions. The influence of stray light in elderly crystalline lenses is compensated by a correction function that depends on age. The reproducibility of parameters in a one-wavelength reflection method determined for three subjects (47, 61, and 78 years old) was: maxODx = 6.3%, meanODx = 4.6%, volume = 6%, and area = 6% already before stray-light correction. ODx was comparable in pseudophakic and in an eye with a crystalline lens of the same 11 subjects after stray-light correction. Significant correlation in ODx was found between the one-wavelength reflection method and the two-wavelength autofluorescence method for pseudophakic and cataract eyes of 19 patients suffering from dry age-related macular degeneration (AMD) (R(2) = 0.855). In pseudophakic eyes, maxODx was significantly lower for dry AMD (n = 45) (ODx = 0.491±0.102 ODU) than in eyes with healthy fundus (n = 22) (ODx = 0.615±0.103 ODU) (p = 0.000033). Also in eyes with crystalline lens, maxODx was lower in AMD (n = 125) (ODx = 0.610±0.093 ODU) than in healthy subjects (n = 45) (ODx = 0.674±0.098 ODU) (p = 0.00019). No dependence on age was found in the pseudophakic eyes both of healthy subjects and AMD patients.

Comparison between Macular Pigment Optical Density Measurements Using Two-Wavelength Autofluorescence and Heterochromatic Flicker Photometry Techniques

The association of macular pigment (MP) with age-related macular degeneration has been extensively studied in recent years, and interest in a rapid, objective, and accurate technique to measure MP optical density (MPOD) has increased. The purpose of this study was to compare the MPOD values at specific loci around the fovea using the heterochromatic flicker photometry (HFP) and the two-wavelength autofluorescence (AF) methods in a young, healthy population. Ten patients (20 eyes) were enrolled. Subjects with any ocular or systemic disease were excluded. All patients underwent HFP and AF examination. The AF examination was performed using a modified scanning laser ophthalmoscope. Values for both devices were measured at four eccentricities around the fovea (0.25 degrees , 0.5 degrees, 1.0 degrees, and 1.75 degrees). Each eye was tested three times with each Statistical analysis was based on paired t-test and linear regression analysis. At all retinal eccentricities, the HFP values were consistently lower than the HRA values (P < 0.001). There was, however, a significant correlation at almost all locations. The strongest correlation between two methods was found at 1.75 degrees from the center of the fovea (r = 0.73). The modified-HRA AF method for MPOD generated results that were highly correlated with the standard HPF method but consistently higher at all eccentricities. These findings suggest that HRA can be reliably used in patients unable to perform HPF, which is important for wider clinical application of MP testing.

Dead or alive? Autofluorescence distinguishes heat-fixed from viable cells

Purpose: A proof-of-concept study to evaluate a new autofluorescence method to differentiate necrotic thermally fixed cells from viable tissue following thermal ablation.Methods: A conductive interstitial thermal therapy (CITT) device was used to ablate swine mammary tissue and rabbit VX-2 carcinomas in vivo. The ablated regions and 10-mm margins were resected 24 h following treatment, embedded in HistOmer® and sectioned at 3 mm. The fresh sections were evaluated for gross viability with triphenyl tetrazolium chloride, 1 h post-resection. Representative non-viable and viable areas were then processed and embedded into paraffin, and sectioned at 5 µm. Standard H&E staining and proliferating cell nuclear antigen (PCNA) immunohistochemistry were compared against autofluorescence intensity, at 488-nm wavelength, for cellular viability.Results: Heat-fixed cells in non-viable regions exhibit increased autofluorescence intensity compared to viable tissue (area under receiver operating characteristics (ROC) curve = 0.96; Mann-Whitney P < 0.0001). An autofluorescence intensity-based classification rule achieved 92% sensitivity with 100% specificity for distinguishing non-viable from viable samples. In contrast, PCNA staining did not reliably distinguish heat-fixed, dead cells from viable cells.Conclusions: Examination of H&E-stained sections using autofluorescence intensity-based classification is a reliable and readily available method to accurately identify heat-fixed cells in ablated surgical margins.