Fluorescence microscopy has become the work horse imaging tool for biologists studying cellular structure and mechanism at the submicron scale. While numerous discoveries have been made over decades of work, there is a limit to how close two objects can be from one another and still be resolved. This problem, governed by the diffraction of visible light through an objective, was first described 100 years ago by Ernst Abbé. In practicality, for standard confocal microscopy experiments, objects closer than 250 nm in the lateral plane, and 500 nm in the axial direction cannot be discerned, and this limits the microscopist from a wealth of potential biological information.
In the past decade however, significant breakthroughs have been made in fluorescence microscopy. It is now possible to resolve cellular components in the range of 10 to 30 nanometers in the lateral, and 50-60 nm in the axial planes. Techniques such as Stimulated Emission Depletion (STED) and Stochastic Optical Reconstruction Microscopy (STORM) for example, have conquered the diffraction barrier and are now deepening what can be visualized at the microscopic level. Researchers Eric Betzig, Stefan W. Hell, and William E. Moerner recently received the 2014 Nobel Prize in chemistry for these breakthroughs. The methods they invented use photo modulation of dyes that can be selectively converted from fluorescent to non-fluorescent (dark) states. This, in effect, produces non-overlapping fluorescent signals in the excitation field, and the resulting lack of compounding interference patterns allows for high resolution observations to be made. In addition to STED and STORM, a variety of additional methods have been developed that exploit reversible saturable optically linear fluorescence (RESOLFT) concepts and are generally referred to as single molecule localization techniques. Examples include direct stochastic optical reconstruction microscopy (dSTORM), photo-activated localization microscopy (PALM), fluorescence photo-activation localization microscopy (fPALM), ground state depletion followed by individual molecule return (GSDIM), and super resolution optical fluctuation imaging (SOFI).
Owing to the increasing utility of these innovative methods, Jackson ImmunoResearch offers a wide selection of labeled secondary antibodies with dyes known to be robust in both STED, STORM, and related superresolution microscopy methods.
Antibody Conjugate Fluors for STED
A stimulated emission depletion experiment produces superresolution images by narrowly confining the fluorescing region of a sample. To do so, it utilizes two overlapping lasers. The first, called a depletion laser (STED laser), is shaped like a donut with a very small (~30 nm) zero intensity node at its center. Exterior to the node is light of a particular wave length that drives excited electrons back into the ground state before fluorescence can occur. At the node of the STED laser however, molecules are free to fluoresce by the overlapping excitation laser. The combination of these two lasers produce a very small region of single molecule fluorescence and thus high resolution images can be obtained without overlapping interference patters. To achieve superresolution, dyes suited to STED experiments must have a high emission cross section with the STED laser wavelength and efficiently achieve a high saturation. This requires very intense illumination which ensures that the emission of all molecules to be “turned off” by the STED laser are dominated by stimulated emission. Optimal dyes for STED should have a low propensity for photo-bleaching, have high quantum yields and contrast, and contain sufficient density of labeling in close proximity to the target. In this regard, Jackson ImmunoResearch offers secondary antibodies conjugated to dyes over a broad spectral range that have been successfully employed in STED: Alexa Fluor® 488, FITC, Alexa Fluor® 647 and DyLight 594.
Reference: Farahani, J.N., et al., Stimulated Emission Depletion (STED) Microscopy: from Theory to Practice. Microscopy:Science, Technology, Applications and Education (2010) 1539-1547
Antibody Conjugate Fluors for Single Molecule Localization Experiments
Single molecule localization methods that rely on RESOLFT principles can also use a variety of dyes. While it can be beneficial to contain both activation and emission dyes on the same antibody, as in STORM, many dyes can “self-switch”, and result in high quality images. This was demonstrated in 2008 by Heilemann et al. and was referred to as direct STORM (dSTORM). In this case, a relatively low intensity excitation laser illuminates the sample and randomly activates a small number of well resolved antibody conjugated dyes to the fluorescent state. The laser then drives fluorescence from the dyes, and eventually switches them to the dark state. The generation of precise molecular localization data generally depends on the number of collected photons which decreases the standard deviation of the point spread function and allows for molecular assignment. Additionally, the laser power required for dSTORM can be ~ 200 times higher than if an activator dye is used but still ranges within the micro to milliwatt range. Ultimately, the best dyes for single molecule localization are typically very bright and result in enough photons to reliably produce tight Gaussian distributions. Jackson offers many proven dyes in a broad spectral range such as Alexa Fluor® 488, Fluorescein/FITC, Cy3, Alexa Fluor® 647, Cy5, and Alexa Fluor® 790 for use in these types of experiments.
Reference: Dempsey G.T., et al., Evaluation of fluorophores for optimal performance in localization-based super resolution imaging Nature Methods 8 (2012) 1027-1036.
Super-Resolution Microscopy Dyes appended to Jackson ImmunoResearch Secondary Antibodies
|STED||Excitation (nm)||Emission (nm)|
|Alexa Fluor® 488||493||519|
|Fluorescein / FITC||492||520|
|Alexa Fluor® 594||591||614|
Reference: Stimulated Emission Depletion (STED) Microscopy: from Theory to practice. Farahani, J. N. et al, Microscopy: Science Technology, Applications and Education. (2010) 1539-1547.
|STORM||Excitation (nm)||Emission (nm)|
|Alexa Fluor® 488||493||519|
|Fluorescein / FITC||492||520|
|Alexa Fluor® 647||651||667|
|Alexa Fluor® 790||792||803|
Reference: Dempsy, G. T. et al, Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nature Methods 8 (2010) 1027-1037.