Quantitative analysis of myocardial tissue with digital autofluorescence microscopy

Abstract

Background:The opportunity offered by whole slide scanners of automated histological analysis implies an ever increasing importance of digital pathology. To go beyond the importance of conventional pathology, however, digital pathology may need a basic histological starting point similar to that of hematoxylin and eosin staining in conventional pathology. This study presents an automated fluorescence-based microscopy approach providing highly detailed morphological data from unstained microsections. This data may provide a basic histological starting point from which further digital analysis including staining may benefit.Methods:This study explores the inherent tissue fluorescence, also known as autofluorescence, as a mean to quantitate cardiac tissue components in histological microsections. Data acquisition using a commercially available whole slide scanner and an image-based quantitation algorithm are presented.Results:It is shown that the autofluorescence intensity of unstained microsections at two different wavelengths is a suitable starting point for automated digital analysis of myocytes, fibrous tissue, lipofuscin, and the extracellular compartment. The output of the method is absolute quantitation along with accurate outlines of above-mentioned components. The digital quantitations are verified by comparison to point grid quantitations performed on the microsections after Van Gieson staining.Conclusion:The presented method is amply described as a prestain multicomponent quantitation and outlining tool for histological sections of cardiac tissue. The main perspective is the opportunity for combination with digital analysis of stained microsections, for which the method may provide an accurate digital framework.

An Overview of the Role of Lipofuscin in Age-Related Neurodegeneration

“…Based on its high lipid content, classical methods of isolation and quantitation of LF employed organic solvent extraction or density gradient ultracentrifugation protocols (Siakotos, 1974 ; Taubold et al, 1975 ; Ottis et al, 2012 ). However, due to its fluorescent properties, most recent methods for LF detection and quantification are based on the use of fluorescence microscopy (Moore et al, 1995 ; Jung et al, 2010 ; Zheng et al, 2010 ; Jensen et al, 2016 ). Provided that LF presents a very broad fluorescence spectrum, fluorescence images of tissue preparations can be acquired over a broad range of wavelengths.…”

Management of autofluorescence in formaldehyde-fixed myocardium: choosing the right treatment

“…One possible explanation for the increased AF could be the reduction of intracellular non-fluorescent NAD(P)+ to fluorescent NAD(P)H by NaBH 4. 32, 33 With the development of label-free imaging methods and their demonstrated needs for cardiovascular research, AF contrast has been widely used by various microscopic imaging techniques to detect intracellular and extracellular structures in the myocardium, such as widefield, 34 confocal, 35 multiphoton 36 and light-sheet 37 . Our finding suggests the potential application of NaBH 4 as an AF enhancer in label-free imaging.…”

Catheter-based optical approaches for cardiovascular medicine: progress, challenges and new directions

“…Many approaches for optical imaging of cardiovascular tissues are based on tissue autofluorescence or labeling of tissue constituents with fluorescent markers [65,66]. Endogenous fluorophores in cardiac tissue are primarily proteins such as collagen and elastin, lipid-containing intracellular granules, lipofuscin, and the coenzyme 1,4-dihydronicotinamide adenine dinucleotide (NADH) [67]. Exogenous fluorophores such as fluorescein, can be tailored to target specific cells or cellular components of the cardiovascular system.…”