Confocal laser scanning microscopy of whole mouse ovaries: Excellent morphology, apoptosis detection, and spectroscopy

Zucker, Robert M.; Jeffay, Susan

doi:10.1002/cyto.a.20315

Cited by 26 publications

(20 citation statements)

References 49 publications

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“…This equipment has the capacity to obtain unique fluorescence spectra of specific dye molecules that will increase our understanding of biological fluorescence emission process (9)(10)(11)(12)(13)(14)(15)(16). The popularity of this new field in biology has been described in a special issue of Cytometry that includes both review articles and specific application articles (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). To insure that this equipment is functioning properly, new and improved tests have been developed to evaluate spectral performance on these instruments (1,2,5,6).…”

Section: Discussionmentioning

confidence: 99%

“…That will create noisy images which can only be improved by excessive averaging or better sample preparation. In fact due to a perceived lack of fluorescence in PMT 1, we changed our sample preparation protocol of ovaries, and embryos to actually introduce more fluorescence into the tissue by fixing with glutaraldehyde molecules (17,33). The sensitivity of a system will be decreased, as fewer photons enter the PMT.…”

Section: Applications and Examples Of Focalcheck Tm Beadsmentioning

confidence: 99%

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Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

Zucker¹,

Rigby²,

Clements³

et al. 2007

Cytometry Pt A

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Section: Discussionmentioning

confidence: 99%

Section: Applications and Examples Of Focalcheck Tm Beadsmentioning

confidence: 99%

Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

Zucker¹,

Rigby²,

Clements³

et al. 2007

Cytometry Pt A

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“…One of the major problems with using a confocal microscopy to image whole organs and embryos is the penetration of laser light into tissues. Good images begins by optimizing the sample preparation technique (1)(2). In addition, the confocal microscope acquisition variables need to be optimized (i.e.…”

Section: Confocal Microscopymentioning

confidence: 99%

Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that Effect Data Quantification.

Zucker¹,

Rogers²,

Ellis-Hutchings³

2008

Microsc Microanal

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Flow and image cytometers can provide useful fluorescence data that can be used to quantify fluorescence. We have devised QA tests to be used on both a flow cytometer and a confocal microscope to assure that the data is accurate, reproducible and precise.Flow Cytometry: We have provided two simple performance criteria to determine if a flow cytometer is aligned correctly and properly functioning. The system can be assessed for alignment and functionality by measuring the CVs, peak channels and histogram distributions, from uniform single intensity beads. The CV of the bead population is a measure of the alignment of the system and the overall functionality of the system. A low CV indicates a good alignment while a high CV indicates either an alignment problem or a fluidic problem. Increasing the flow rate should increase the CV but the histogram distribution should stay relatively symmetrical. Deviations from a symmetrical pattern indicate a system that is not aligned optimally.The 2 nd test monitors the cleanliness of the system and the amount of background light scatter. This test is related to the alignment of the system. Failure to obtain good values in these tests will compromise the systems ability to detect dim fluorescence from background and will affect the precision of the system. It is important that some of these bead intensities are similar to the intensity of the samples measured.The CV test used to check flow cell blockage and alignment and the sensitivity test will be used to evaluate the machine contamination and ability to measure low level fluorescence. A machine that is set up correctly will pass the CV test and the sensitivity test. These values should be measured daily when using the machine. When the machine is not aligned correctly the data will be effected. Confocal Microscopy:The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional sectioning approaches. However, the imaging of such whole-mounts presents technical problems of its own. One of the major problems with using a confocal microscopy to image whole organs and embryos is the penetration of laser light into tissues. Good images begins by optimizing the sample preparation technique (1-2). In addition, the confocal microscope acquisition variables need to be optimized (i.e. objective lens, averaging, pinhole size, bleaching, PMT voltage, laser excitation source, and spectral registration.) as it will affect the accuracy of the data (3-6).

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“…For the systematic analysis of confocal performance a wide range of tests have been developed, mostly relying on inexpensive samples and yet providing a wealth of information on the state of the instrument and individual components [7,8,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]. Although there are many differences in the design of the commercially available confocal microscopes - and each of them available with a multitude of possible configurations - the following parameters are commonly checked for quality control purposes: …”

Section: Introductionmentioning

confidence: 99%

ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance

Hng

Dormann

2013

PLoS ONE

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Laser scanning confocal microscopy has become an invaluable tool in biomedical research but regular quality testing is vital to maintain the system’s performance for diagnostic and research purposes.Although many methods have been devised over the years to characterise specific aspects of a confocal microscope like measuring the optical point spread function or the field illumination, only very few analysis tools are available. Our aim was to develop a comprehensive quality assurance framework ranging from image acquisition to automated analysis and documentation. We created standardised test data to assess the performance of the lasers, the objective lenses and other key components required for optimum confocal operation.The ConfocalCheck software presented here analyses the data fully automatically. It creates numerous visual outputs indicating potential issues requiring further investigation. By storing results in a web browser compatible file format the software greatly simplifies record keeping allowing the operator to quickly compare old and new data and to spot developing trends.We demonstrate that the systematic monitoring of confocal performance is essential in a core facility environment and how the quantitative measurements obtained can be used for the detailed characterisation of system components as well as for comparisons across multiple instruments.

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Confocal laser scanning microscopy of whole mouse ovaries: Excellent morphology, apoptosis detection, and spectroscopy

Cited by 26 publications

References 49 publications

Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that Effect Data Quantification.

ConfocalCheck - A Software Tool for the Automated Monitoring of Confocal Microscope Performance

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