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Basic Confocal Microscopy - Semantic Scholar
We 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. Our 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.
Over the past 20 years, confocal microscopy has become a centrally important technique for the analysis of biological samples. 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 simply visualizing fluorescently labeled specimens, confocal microscopy has become a powerful tool for biologists in many disciplines for diverse applications, including establishing structure-function relationships at the cellular and tissue level, defining dynamic processes in living specimens, and for detection of close interactions between biological molecules at the subcellular level [ 1 ].
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. There are several different factors that contribute to this limitation, including the fact that the most prevalent fluorescent probes are small organic molecules which have a small Stoke's shift. Thus, with few exceptions, each fluorescent dye in an experiment requires a distinct laser for excitation, and the emission spectrum is slightly red-shifted, relative to the excitation wavelength.
As a result, the number of proteins or cell structures that can be imaged concurrently is quite restricted reviewed in [ 1 ] and [ 2 ]. Quantum dot Qdot -coupled detection reagents offer an opportunity to expand the capabilities of basic confocal instruments. Qdots are semi-conductor nanocrystals consisting of a CdSe core and a surface chemistry treatment which allows the Qdot to be coupled to proteins [ 3 ].
A striking advantage of Qdots over most organic fluorophores involves their long fluorescence half-life and high resistance to photobleaching, allowing them to be imaged extensively with minimal loss of signal [ 3 ]. Qdots have several additional properties which make them attractive for imaging applications, including a wide excitation spectrum, a narrow emission spectrum, and a long Stoke's shift. The physics governing Qdots fluorescence is such that the emission wavelength is determined by the size of the Qdot.
Consequently, larger Qdots have longer emission wavelengths. Importantly, all Qdots share overlapping excitation spectra, with maximal excitation by ultraviolet UV wavelengths, meaning that the Stoke's shift for red and infrared Qdots spans hundreds of nanometers, which clearly distinguishes these fluorophores from organic dyes [ 4 ]. An additional consequence of this Qdots property is that all Qdots can be efficiently excited by a single laser in the UV to blue region of the spectrum [ 3 ].
Commercially produced Qdots reagents are now available with defined emission wavelengths that extend from green to the near-infrared emission wavelengths. The physical properties of Qdots, predominantly their large size diameters in the nanometer range [ 5 ] , dictate that numerous antibodies are coupled to a single Qdot. In contrast, when labeling with organic dyes which are small, relative to an antibody , many dye molecules are coupled to an individual antibody.
Thus, Qdot coupled antibodies are both much larger and have many more ligand binding sites than organic dye coupled antibodies. It is therefore reasonable to expect that these reagents may behave quite differently for applications such as the staining of detergent permeabilized cells. Given that quantum dot reagents with red and near-infrared IR emissions are very bright and cover a region of the spectrum that is under-utilized on many basic confocal systems, staining reagents that are coupled to red and near-IR quantum dots represent a particularly attractive strategy to expand the multi-parameter capabilities of confocal microscopes.
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However, due to the large size of quantum dot conjugates and their multivalency, these reagents perform poorly for one-to-one ligand-to-target binding [ 3 ]. Thus, Qdot-labeled reagents can be expected to behave differently from organic dye-labeled conjugates in fluorescent labeling experiments.
We thus initiated a study to compare the quality of images obtained from Qdot- and organic dye-labeled conjugates for the staining of fixed, detergent permeabilized cells, and to test the suitability of red and near-IR Qdot reagents for performing multi-parameter fluorescence labeling experiments in combination with organic dye-based detection reagents. Together, our data demonstrate that, with simple and inexpensive modifications to a basic confocal microscope, red and near-IR Qdot-labeled conjugates can readily be combined with conventional fluorophores, increasing the number of labeled structures that can be detected and cleanly discriminated in a single experiment.
With this system, three-color detection of organic fluorophores in combinations such as DAPI, Alexa and Alexa nm, nm, and nm excitation or combinations of fluorescent proteins and organic probes, such as CFP, YFP, and Alexa are easily achieved. To expand the multi-parameter capabilities of our LSM5 Pascal, we added additional emission filters to allow combinatorial detection of red- and near infrared-emitting Qdots End users can easily add or change emission filters in the LSM5 Pascal scan head, following brief training by a Zeiss service engineer.
Filter configuration and data acquisition strategy for 6-parameter imaging experiments. Graphs show emission spectra of organic dye and Qdot reagents employed in this study. The spectra are grouped with the laser line used for excitation nm, top; nm, middle; nm, bottom. Colored bands indicate the approximate range of emissions transmitted by the filters see also Table 1. In the middle graph, the red line and red axis indicates the approximate quantum efficiency of the Hamamatsu PMT through the displayed wavelength range. Evaluation of the Pascal's fluorescence detection hardware suggested that fluorescence emission from each of these Qdots could be detected.
Duplicate coverslips were then stained with either an organic dye-labeled secondary antibody Alexa goat anti-mouse IgG 1 or a Qdot-labeled secondary antibody Qdot goat anti-mouse IgG. Fluorescence data were acquired as z-stack images on our modified Zeiss LSM5 Pascal, and data were further processed by digital deconvolution and projection into a 2-dimensional image. As shown in Fig. Alexa and Qdot secondary antibodies produce images of similar quality. Insets show magnification of outlined region. Images in A and B are maximal projections of deconvolved z-stack data.
There are basically three things to consider when you try to get good signal separation into three channels here or 2, which is less problematic : 1 You want a good combination of fluorophores. Keep in mind that signal separation is not only dependent on the spectral properties but also on the relative intensities of the signals. The spectra above show normalized intensities.
In a real situation the relative intensity values at a given wavelength maybe completely different between 2 fluorophores. In general, you will have more channel bleedthrough with lower NA objectives. For example, I doubt that you can get good separation for the combination above with any of the air objectives. The idea is to only use one of the laser line at a time and get a significantly reduced response from the fluorophore whose excitation spectrum is further away form this wavelength.
Our confocal lets you do that without having to do sequential scans. It can switch back and forth between scanning frames with different settings. Methods Cell Biol One thing that people tend to forget is that the most important and hardest part of doing microscopy is mounting the specimen.
Two things are important: 1 You want to preserve the three-dimensional structure of the tissue as well as possible. It has been described that potentially any histochemistry can screw up the structure of the specimen. At least in the case of arthropod wholemount preparations, the only real problem seems to be the mounting medium.
Mounting in glycerol is a bad idea.
It creates torsions and all kinds of anisometric shrinkage arifacts. The agent of choice in my opinion is methyl salicylate wintergreen oil.http://grandaweek.co.uk/oddities-in-the-house.php
A practical guide for fluorescent confocal microscopy
Correction methods for three-dimensional reconstructions from confocal images: I. J Neurosci Methods In theory, most objectives are optimized for spherical corrections for a micron cover slip and if you deviate from that you will lose signal. But that is only important if you have a really, really weak signal.
Usually you want a thinner cover slip to win some working distance. Most companies have cover slips in different thickness ranges. I leave the nerves stn, dvn, avn, agn fairly long and pin the ganglion into a thinly coated sylgard dish.
Sensitive, spectral confocal imaging and topography
After fixing, but before dehydrating in buffer , I cut the sylgard around the ganglion with a scalpel but leave everything in place. I leave that in a vaseline-sealed dry glass container with silica gel on the bottom , to keep the EtOH from drawing water. After h, I transfer the sylgard piece to a sylgard coated glass dish and pin it down. I put a drop of pure methyl salicylate on top. Now I cut the nerves proximal to the pins that hold them. I usually glue 2 coverslips on a microscope slide with nailpolish from the top. The STG goes into the gap between them. I carefully position everything the way I want it and then fill up with mounting medium, remove the cover slip shard, put a cover slip on top, and seal everything with nail polish.
Basically, the image you will get in a.
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Laser intensity: Just to clarify this: There are 2 ways of regulating it.