Quantitative confocal microscopy: Beyond a pretty picture

doi:10.1016/B978-0-12-420138-5.00007-0

Methods in Cell Biology

Volume 123, 2014, Pages 113-134

https://doi.org/10.1016/B978-0-12-420138-5.00007-0 Get rights and content

Abstract

Quantitative optical microscopy has become the norm, with the confocal laser-scanning microscope being the workhorse of many imaging laboratories. Generating quantitative data requires a greater emphasis on the accurate operation of the microscope itself, along with proper experimental design and adequate controls. The microscope, which is more accurately an imaging system, cannot be treated as a “black box” with the collected data viewed as infallible. There needs to be regularly scheduled performance testing that will ensure that quality data are being generated. This regular testing also allows for the tracking of metrics that can point to issues before they result in instrument malfunction and downtime. In turn, images must be collected in a manner that is quantitative with maximal signal to noise (which can be difficult depending on the application) without data clipping. Images must then be processed to correct for background intensities, fluorophore cross talk, and uneven field illumination. With advanced techniques such as spectral imaging, Förster resonance energy transfer, and fluorescence-lifetime imaging microscopy, experimental design needs to be carefully planned out and include all appropriate controls. Quantitative confocal imaging in all of these contexts and more will be explored within the chapter.

Section snippets

The Classic Confocal: Blocking Out the Blur

Widefield microscopy can often provide acceptable resolution, reasonable contrast, and fast acquisition rates. However, if the sample thickness is more than 15–20 μm, then in-focus features are obscured by blur from out-of-focus regions of the sample. By imaging through a well-placed pinhole, a confocal microscope blocks the out-of-focus light coming from above and below the plane of focus, thereby reducing blur and producing a sharp image of the sample (Fig. 7.1). For thick samples, this

You Call that Quantitative?

The modern CLSM is designed and built as an expansive quantitative device. Virtually all questions researchers are asking today require a rigorous quantitative method of analysis, even for straightforward intensity or morphometric measurements. In the following sections, specific areas of quantification will be examined along with recommendations for CLSM performance tests and metrics.

Cross talk

For any experiments that are designed to look at interactions between labeled molecules, cross talk must be corrected for. Excitation cross talk results when two or more dye molecules are excited by the same wavelength of laser light. So when imaging a green emission dye, it is possible that a red emission dye is also excited. Emission cross talk is more common and results when fluorophores emit at the same wavelengths or their emission spectra overlap. The ideal way to get rid of cross talk is

Controls: Who Needs Them?

Controls might not be the most fun to prepare or to image; however, they are vital to all experiments. While controls are rarely published, they do provide the foundation for the quantitative experimental results that are published.

Protocol 1: Measuring instrument PSF (Resolution and objective Lens Quality)

Imaging

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Prepare a slide of subresolution microspheres (≤ 0.175 μm for ≥ 0.6 NA lens or 0.5 μm for ≤ 0.6 NA lens; see Section 7.2.1).
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Turn on the microscope and allow the laser to warm up for 1 h.
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Ensure that all DIC optical elements are removed from the light path.
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Clean the objective lens.
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If the lens has a correction collar, ensure that it is properly adjusted.
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If the confocal pinhole is user-adjustable, then use a green fluorescent plastic slide to align it. If it is not user-adjustable, ensure it is aligned

Conclusions

While the confocal microscope is a powerful quantitative tool, it should not be viewed as a “black box.” Instead, both the microscope and the sample need to be approached with a stepwise analytic approach. Starting with, is the CLSM the appropriate microscope to use based on the sample and the hypothesis being tested? Next, is the microscope operating as designed, that is, are the objective lens, illumination system, and detection system within specification? On the majority of nonimaging

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Chapter 7 - Quantitative confocal microscopy: Beyond a pretty picture

Abstract

Section snippets

The Classic Confocal: Blocking Out the Blur

You Call that Quantitative?

Cross talk

Controls: Who Needs Them?

Protocol 1: Measuring instrument PSF (Resolution and objective Lens Quality)

Conclusions

Antibody validation

Biotechniques

Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt

Nature Protocols

Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus

Proceedings of the National Academy of Sciences of the United States of America

Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control

Nature Protocols

International test results for objective lens Quality, Resolution, spectral accuracy and spectral separation for confocal laser scanning microscopes

Microscopy and Microanalysis

Live-cell microscopy—Tips and tools

Journal of Cell Science

Spectral imaging: Principles and applications

Cytometry. Part A : The Journal of the International Society for Analytical Cytology

mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures

PLoS One