Spectral Characteristics of Autofluorescence in Renal Tissue and Methods for Reducing Fluorescence Background in Confocal Laser Scanning Microscopy

Zhang, Yang; Wang, Yan; Cao, Weiwei; Ma, Ketao; Ji, Wei; Han, Zhaobin; Si, Junqiang; Li, Li

doi:10.1007/s10895-018-2217-4

Cited by 13 publications

(8 citation statements)

References 37 publications

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“…With respect to applying super-resolution microscopy for diagnostic purposes, tissue samples are more likely to originate from elderly patients and thus autofluorescence might become a major issue. It has been observed in numerous studies on human [51,52] as well as murine FFPE kidney tissue and its full reduction proved challenging [47,53,54]. Therefore, if a method other than SMLM is used, we suggest not to invest in reducing the background due to its negligible effect on kidney tissue autofluorescence.…”

Section: Effect Of Background Signalmentioning

confidence: 99%

Superresolving the kidney—a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier

Wunderlich

Ströhl

et al. 2020

Anal Bioanal Chem

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Immunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney’s glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world.

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Section: Effect Of Background Signalmentioning

confidence: 99%

Superresolving the kidney—a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier

Wunderlich

Ströhl

et al. 2020

Anal Bioanal Chem

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“…Imaging mass cytometry (IMC) is a technology that supports simultaneous detection of more than 40 protein markers on a single section of formalin-fixed, paraffin-embedded tissue, with the resultant reconstructions achieving 1-μm resolution (13,14). In the kidney, endogenous fluorescence, particularly in tubules, can reduce signal-to-noise ratio and compromise image quantitation in fluorescence-based imaging, making a mass spectrometry-based analytic technique particularly appealing (15). IMC has been used to characterize the tumor microenvironment in solid malignancies, yielding information about the identity, number, and spatial relationship between immune and stromal cells (13,16).…”

Section: Introductionmentioning

confidence: 99%

Development of a 2-dimensional atlas of the human kidney with imaging mass cytometry

Singh¹,

Avigan²,

Kliegel³

et al. 2019

JCI Insight

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“…Such signal-to-noise ratios may occur when examining tissues which exhibit high amounts of autofluorescence. For instance, murine renal tissues have previously been reported to display intense autofluorescence at a broad range of excitation wavelengths, including those for DAPI, FITC, and Texas Red (Sun et al, 2011;Zhang et al, 2018). Furthermore, fixatives such as aldehydes are known to increase autofluorescence of fixed tissues and impede histological observation (Baschong et al, 2001).…”

Section: Ijoq Robustly Analyzes In Silico Disruption Of Ijsmentioning

confidence: 99%

Dynamic Python-Based Method Provides Quantitative Analysis of Intercellular Junction Organization During S. pneumoniae Infection of the Respiratory Epithelium

Rosa

et al. 2022

Front. Cell. Infect. Microbiol.

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Many respiratory pathogens compromise epithelial barrier function during lung infection by disrupting intercellular junctions, such as adherens junctions and tight junctions, that maintain intercellular integrity. This includes Streptococcus pneumoniae, a leading cause of pneumonia, which can successfully breach the epithelial barrier and cause severe infections such as septicemia and meningitis. Fluorescence microscopy analysis on intercellular junction protein manipulation by respiratory pathogens has yielded major advances in our understanding of their pathogenesis. Unfortunately, a lack of automated image analysis tools that can tolerate variability in sample-sample staining has limited the accuracy in evaluating intercellular junction organization quantitatively. We have created an open source, automated Python computer script called “Intercellular Junction Organization Quantification” or IJOQ that can handle a high degree of sample-sample staining variability and robustly measure intercellular junction integrity. In silico validation of IJOQ was successful in analyzing computer generated images containing varying degrees of simulated intercellular junction disruption. Accurate IJOQ analysis was further confirmed using images generated from in vitro and in vivo bacterial infection models. When compared in parallel to a previously published, semi-automated script used to measure intercellular junction organization, IJOQ demonstrated superior analysis for all in vitro and in vivo experiments described herein. These data indicate that IJOQ is an unbiased, easy-to-use tool for fluorescence microscopy analysis and will serve as a valuable, automated resource to rapidly quantify intercellular junction disruption under diverse experimental conditions.

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Spectral Characteristics of Autofluorescence in Renal Tissue and Methods for Reducing Fluorescence Background in Confocal Laser Scanning Microscopy

Cited by 13 publications

References 37 publications

Superresolving the kidney—a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier

Superresolving the kidney—a practical comparison of fluorescence nanoscopy of the glomerular filtration barrier

Development of a 2-dimensional atlas of the human kidney with imaging mass cytometry

Dynamic Python-Based Method Provides Quantitative Analysis of Intercellular Junction Organization During S. pneumoniae Infection of the Respiratory Epithelium

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