Abstract
BackgroundConfocal microscopy is a widely employed methodology in cellular biology, commonly used for investigating biological organization at the cellular and sub-cellular level. Most basic confocal microscopes are equipped to cleanly discriminate no more than four fluorophores in a given sample, limiting the utility of this method for co-localization, co-expression, and other multi-parameter analyses. In this study, we evaluated the use of red and near-infrared emitting quantum dot staining reagents to expand the multi-parameter capabilities of basic confocal microscopes.ResultsWe modified a three-laser Zeiss Pascal confocal microscope by the addition of two band-pass filters and one long-pass filter for the detection of three different red to near-infrared quantum dot conjugates. We then performed direct comparisons between organic dye- and quantum dot-labeled detection reagents for the detection of subcellular structures. We found that the quality of staining was generally indistinguishable, although quantum dot reagents do have certain limitations, relative to organic dye conjugates. Using the modified Pascal system, three quantum dot conjugates, two organic dye conjugates, and one fluorescent protein, we demonstrated clean discrimination of six distinct fluorescent labels in a single sample.ConclusionOur data demonstrate that nearly any basic confocal microscope can be modified by the simple addition of appropriate emission filters, allowing the detection of red and near-infrared quantum dot conjugates. Additionally, quantum dot- and organic dye-based secondary reagents can be successfully combined in complex intracellular staining experiments. Substantial expansion of the multi-parameter capabilities of basic confocal instruments can be achieved with a financial investment that is minimal in comparison to instrument replacement or upgrade with additional lasers.
Highlights
Confocal microscopy is a widely employed methodology in cellular biology, commonly used for investigating biological organization at the cellular and sub-cellular level
To test the use of Quantum dot (Qdot) in a basic confocal system, we employed a Zeiss LSM5 Pascal system equipped with a 25 mW 405 nm diode laser, a 25 mW argon laser tunable to 458 nm, 488 nm or 514 nm, a 5 mW 543 nm Helium/ Neon laser, and two fluorescence detector channels, which utilize Hamamatsu R6357 photomultiplier tubes (PMTs)
We found that the Qdot-conjugated secondary antibodies yielded data of similar quality to organic dye labeled secondary reagents, we did note certain limitations of the Qdot labels in the course of our study
Summary
Confocal microscopy is a widely employed methodology in cellular biology, commonly used for investigating biological organization at the cellular and sub-cellular level. Most basic confocal microscopes are equipped to cleanly discriminate no more than four fluorophores in a given sample, limiting the utility of this method for co-localization, co-expression, and other multi-parameter analyses. By using a pinhole to exclude scattered light, confocal instruments can be used to optically section biological samples, producing 2- and 3-dimensional images with spatially resolved details at the sub-micron level. Beyond visualizing fluorescently labeled specimens, confocal microscopy has become a powerful tool for biologists in many disciplines for diverse applications,. Most basic confocal microscopes are equipped with 2, 3, or 4 lasers, and are generally configured to detect one fluorophore per laser, giving a maximum detection of four distinct fluorescent labels in a single sample. The number of proteins or cell structures that can be imaged concurrently is quite restricted (reviewed in [1] and [2])
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