Practical Confocal Microscopy and the Evaluation of System Performance

Zucker, Robert M.; Price, Owen

doi:10.1006/meth.1999.0812

Cited by 55 publications

(109 citation statements)

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“…Regular system checks should be common practice and measure e.g. the point spread function (PSF), the laser power, the illumination homogeneity of the field of view, the detector sensitivity as signal‐to‐noise‐ratio (SNR) and the coefficient of variation (Cole et al, 2011, 2013; Gelman and Rietdorf, 2010; Hibbs, 2006; Stack et al, 2011; Theer et al, 2014; Zucker and Price, 2001, 1999). When performed regularly, standardized test series often help identify damaged and misaligned microscope components, even before they significantly impair the quality of user data.…”

Section: How To Design Set Up and Run Your Imaging Core Facilitymentioning

confidence: 99%

Advanced light microscopy core facilities: Balancing service, science and career

Ferrando‐May

Hartmann

Reymann

et al. 2016

Microsc. Res. Tech.

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Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM‐CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM‐CF operations elaborated by the workgroups of the German network of ALM‐CFs, German Bio‐Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM‐CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences. Microsc. Res. Tech. 79:463–479, 2016. © 2016 THE AUTHORS MICROSCOPY RESEARCH AND TECHNIQUE PUBLISHED BY WILEY PERIODICALS, INC.

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Section: How To Design Set Up and Run Your Imaging Core Facilitymentioning

confidence: 99%

Advanced light microscopy core facilities: Balancing service, science and career

Ferrando‐May

Hartmann

Reymann

et al. 2016

Microsc. Res. Tech.

View full text Add to dashboard Cite

show abstract

“…objective lens, averaging, pinhole size, bleaching, PMT voltage, laser excitation source, and spectral registration). In addition, the CLSM has to be checked and evaluated to ensure that it is delivering its proper performance (24)(25)(26)(27)(28)(29). Image quality is primarily dependent on the sample preparation.…”

Section: Discussionmentioning

confidence: 99%

“…The confocal microscope was tested for field illumination alignment, optical efficiency, colocalization, and axial resolution prior to embryo, fetal tissue, and insect observation (24)(25)(26). The lens was inspected and cleaned prior to use.…”

Section: Laser Scanning Confocal Microscopymentioning

confidence: 99%

“…The CLSM was evaluated to ensure it was in proper working order as previously described (24)(25)(26)(27)(28)(29). The tissues were optically sectioned into 20-50 sections, averaged four times, and the emission pinhole was equivalent to an airy disk of 1.…”

Section: Image Acquisitionmentioning

confidence: 99%

“…Basically, a balance between PMT voltage, frame averaging and airy disc, and laser power has to be determined for each sample. Frame averaging will be necessary if the staining intensity is low, necessitating the use of high PMT voltages (24)(25)(26). The lasers in the microscope have to be aligned properly to obtain correct images.…”

Section: Microscopementioning

confidence: 99%

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Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis

Zucker

2006

Cytometry Pt A

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Background:After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three‐dimensional structures. LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, which both correlate to apoptosis activity. LT has been shown to be an indicator of apoptotic cell death which is correlated to other standard apoptotic assays.Methods:The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Mosquitoes were fixed with MEOH and stained with propidium iodide. Next the tissues were dehydrated with MEOH and cleared with BABB.Results:Tissues as thick as 500 μm can be visualized after clearing with BABB. LT staining revealed apoptotic regions in mammalian limbs, fetuses, and embryos. Morphological observation of insect tissue consisted of combining autofluorescence with either nucleic acid staining (either propidium iodide or ethidium bromide).Conclusions:The use of BABB matches the RI of the tissue within the suspending medium. It helps in increasing the penetration of laser light in a confocal microscope by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissues. LT is a probe that stains the acid regions of tissues and cells and has been correlated to apoptosis. Morphological features of a tissue or organism (embryo, mosquito larvae) can be elucidated by fixation aldehydes, autofluorescence, and red‐emitting probes. This sample preparation procedure with optimization of confocal laser scanning microscopy allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were ∼500‐μm thick. © 2006 International Society for Analytical Cytology

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Algorithms for quantitation of protein expression variation in normal versus tumor tissue as a prognostic factor in cancer: Met oncogene expression, and breast cancer as a model

et al. 2000

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Background Immunohistochemistry and immunofluorescence (IF) assays frequently rely on subjective observer evaluation for grading. The aim of our study was to develop an objective quantitative index based on confocal laser scanning microscopy (CLSM) and image analysis of an IF assay to determine alteration in protein expression levels in normal versus tumor tissue. The relative levels of Met expression, a prognostic factor in breast cancer, were used as a model for evaluating image analysis algorithms. Methods Primary human breast cancer biopsies were collected. Sections containing tumor and adjacent uninvolved normal regions were immunostained for Met and digital images were acquired by CLSM. Subsequently, the digital data were manipulated using several different algorithms to calculate prognostic indexes. The results were correlated with the clinical outcome to determine the prognostic value of these indexes. Results Different algorithms were used to obtain quantitative indexes to evaluate the relative levels of Met expression. We report a statistical correlation between patient prognosis and relative Met level in normal versus tumor tissue as determined by three distinct algorithms using Kaplan‐Meier analysis (log‐rank): calculations based on intensity levels differences DV (P = 0.002), DIntensity (P = 0.014), and entropy divergence (Dentropy; P = 0.0023). Conclusions Using adjacent normal tissue as an internal reference, a quantitative index of tumor Met level divergence can be objectively determined to have a prognostic value. Moreover, this methodology can be used for other proteins in a variety of different diseases. Cytometry 41:155–165, 2000 © 2000 Wiley‐Liss, Inc.

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Practical Confocal Microscopy and the Evaluation of System Performance

Cited by 55 publications

References 3 publications

Advanced light microscopy core facilities: Balancing service, science and career

Advanced light microscopy core facilities: Balancing service, science and career

Whole insect and mammalian embryo imaging with confocal microscopy: Morphology and apoptosis

Algorithms for quantitation of protein expression variation in normal versus tumor tissue as a prognostic factor in cancer: Met oncogene expression, and breast cancer as a model

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