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Quantitative analysis of myocardial tissue with digital autofluorescence microscopy.

Jensen T, Holten-Rossing H, Svendsen IM, Jacobsen C, Vainer B - J Pathol Inform (2016)

Bottom Line: This data may provide a basic histological starting point from which further digital analysis including staining may benefit.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus

Examples from the additional study of the effect of microsection thickness and fixation time. Panel a is a thin (2 μm) microsection cut from the sample fixated for 1 day and Panel c is a thick microsection (8 μm) from the sample fixated for 4 days. Panel b and d are the corresponding outline masks of the digital quantitations of fibrous tissue. Panels a and b are representative of thin microsections fixated for 4 days and Panels c and d are representative of thick microsections fixated for 1 day
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Figure 9: Examples from the additional study of the effect of microsection thickness and fixation time. Panel a is a thin (2 μm) microsection cut from the sample fixated for 1 day and Panel c is a thick microsection (8 μm) from the sample fixated for 4 days. Panel b and d are the corresponding outline masks of the digital quantitations of fibrous tissue. Panels a and b are representative of thin microsections fixated for 4 days and Panels c and d are representative of thick microsections fixated for 1 day

Mentions: Microsections cut at 2 and 8 μm from samples fixated for 1 and 4 days, respectively, were digitally analyzed and quantified with point grid quantification as described above. Long fixation time as well as thicker microsections resulted in an overall higher autofluorescence intensity in both the blue and the red area. At both fixation times and both microsection thicknesses the digital quantitation algorithm was applied successfully with satisfying correlations to the point grid quantitations. The outer boundaries of this additional experiment were the thin microsections fixated for 1 day and the thick microsections fixated for 4 days, which had the lowest and highest impacts, respectively, of thickness and fixation time on autofluorescence. Close-up examples of these boundary samples with VG-staining and corresponding fibrous tissue quantitation are shown in Figure 9. Panels 9a-b is a thin microsection fixated for 1 day and 9c-d is a thick microsection fixated for 4 days. The areas include transitions from solid fibrous tissue to hardly discernible smaller fibrils. As expected, the borders between stained and unstained components are unclear in the thick microsection (9c) compared to the thin microsection (9a). The fibrous tissue outline of the thin microsection (9b) is by inspection in clear correspondence with the VG-staining, whereas the outline of the thick section (9d) is in less obvious correspondence. This ambiguity was also present in thick microsections fixated for 1 day, but not in thin microsections fixated for 4 days. It should be noted that larger tissue component presentations were clearly outlined in the thick microsections.


Quantitative analysis of myocardial tissue with digital autofluorescence microscopy.

Jensen T, Holten-Rossing H, Svendsen IM, Jacobsen C, Vainer B - J Pathol Inform (2016)

Examples from the additional study of the effect of microsection thickness and fixation time. Panel a is a thin (2 μm) microsection cut from the sample fixated for 1 day and Panel c is a thick microsection (8 μm) from the sample fixated for 4 days. Panel b and d are the corresponding outline masks of the digital quantitations of fibrous tissue. Panels a and b are representative of thin microsections fixated for 4 days and Panels c and d are representative of thick microsections fixated for 1 day
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837794&req=5

Figure 9: Examples from the additional study of the effect of microsection thickness and fixation time. Panel a is a thin (2 μm) microsection cut from the sample fixated for 1 day and Panel c is a thick microsection (8 μm) from the sample fixated for 4 days. Panel b and d are the corresponding outline masks of the digital quantitations of fibrous tissue. Panels a and b are representative of thin microsections fixated for 4 days and Panels c and d are representative of thick microsections fixated for 1 day
Mentions: Microsections cut at 2 and 8 μm from samples fixated for 1 and 4 days, respectively, were digitally analyzed and quantified with point grid quantification as described above. Long fixation time as well as thicker microsections resulted in an overall higher autofluorescence intensity in both the blue and the red area. At both fixation times and both microsection thicknesses the digital quantitation algorithm was applied successfully with satisfying correlations to the point grid quantitations. The outer boundaries of this additional experiment were the thin microsections fixated for 1 day and the thick microsections fixated for 4 days, which had the lowest and highest impacts, respectively, of thickness and fixation time on autofluorescence. Close-up examples of these boundary samples with VG-staining and corresponding fibrous tissue quantitation are shown in Figure 9. Panels 9a-b is a thin microsection fixated for 1 day and 9c-d is a thick microsection fixated for 4 days. The areas include transitions from solid fibrous tissue to hardly discernible smaller fibrils. As expected, the borders between stained and unstained components are unclear in the thick microsection (9c) compared to the thin microsection (9a). The fibrous tissue outline of the thin microsection (9b) is by inspection in clear correspondence with the VG-staining, whereas the outline of the thick section (9d) is in less obvious correspondence. This ambiguity was also present in thick microsections fixated for 1 day, but not in thin microsections fixated for 4 days. It should be noted that larger tissue component presentations were clearly outlined in the thick microsections.

Bottom Line: This data may provide a basic histological starting point from which further digital analysis including staining may benefit.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus