test-samples-for-optimizing-storm-super-resolution-microscopy

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

National Physical Laboratory

The troponin-tropomyosin-linked regulation of striated muscle contraction occurs through allosteric control by both Ca(2+) and myosin. The thin filament fluctuates between two extreme states: the inactive "off" state and the active "on" state. Intermediate states have been proposed from structural studies and transient kinetic measurements. However, in contrast to the well-characterised, on and off states, the mechanochemical properties of the intermediate states are much less well understood because of the instability of those states. In the present study, we have characterized a myosin-induced intermediate that is stabilized by cross-linking myosin motor domains (S1) to actin filaments (with a maximum of one S1 molecule for 50 actin monomers). A single S1 molecule is known to interact with two adjacent actin monomers. A detailed analysis revealed that thin filaments containing S1 molecules cross-linked to just one actin monomer (actin(1)-S1 complexes) are regulated with a 79% inhibition of the ATPase in the absence of Ca(2+). In contrast, filaments containing S1 molecules cross-linked at two positions, to two adjacent actin monomers (actin(2)-S1 complexes) totally lose their regulation in a highly cooperative manner. This loss of regulation was due both to an enhancement of the ATPase activity without calcium and an inhibition of the ATPase with calcium. Filaments containing actin(2)-S1 complexes, with significant ATPase activity in the absence of calcium (about 50%), did not move on a myosin-coated surface unless calcium was present. This partial uncoupling between the ATPase activity and in vitro motility in the absence of calcium demonstrates that the mechanical steps require actin-myosin contacts, which take place only in the on state and not in the off or intermediate states. These data provide new insights concerning the difference in cooperativity of Ca(2+) regulation that exists between the biochemical and mechanical cycles of the actin-myosin motor.

Characterization of three regulatory states of the striated muscle thin filament.

Recent technological advances in lasers and optical detectors have enabled a variety of new, single molecule technologies to be developed. Using intense and highly collimated laser light sources in addition to super-sensitive cameras, the fluorescence of single fluorophores can now be imaged in aqueous solution. Also, laser optical tweezers have enabled the piconewton forces produced by pair of interacting biomolecules to be measured directly. However, for a researcher new to the field to begin to use such techniques in their own research might seem a daunting prospect. Most of the equipment that is in use is custom-built. However, most of the equipment is essence fairly simple and the aim of this article is to provide an entry point to the field for a newcomer. It focuses mainly on those practical aspects which are not particularly well covered in the literature, and aims to provide an overview of the field as a whole with references and web links to more detailed sources elsewhere. Indeed, the opportunity to publish an article such as this on the Internet affords many new opportunities (and more space!) for presenting scientific ideas and information. For example, we have illustrated the nature of optical trap data with an interactive Java simulation; provided links to relevant web sites and technical documents, and included a large number of colour figures and plots. Our group's research focuses on molecular motors, and the bias of this article reflects this. It turns out that molecular motors have been a paradigm (or prototype) for single molecule research and the field has seen a rapid development in the techniques. It is hoped that the methods described here will be broadly applicable to other biological systems.

Single molecule measurements and biological motors.

The effect of additives on the photostability of semiconductor nanoparticles was studied using CdSe, CdSe/ZnSe and CdSe/ZnS particles of various sizes and composition. The additives included phosphine oxides, amines, and antioxidants. Strong initial enhancement of band edge emission by hexadecylamine was observed for the uncoated particles but stability over a week period in light and air was shown to be poor. Inorganic coatings rendered the nanoparticles initially insensitive to additive but provided little extra stability in terms of photoluminescence. Antioxidant was shown to be effective at reducing the rate of photooxidative degradation for all particles in chloroform but not in toluene.

Stability and quantum yield effects of small molecule additives on solutions of semiconductor nanoparticles.

To obtain accurate and consistent measurements from circular dichroism (CD) instruments over time and from different laboratories, it is important that they are properly calibrated. The characteristics of the available reference materials are not ideal to ensure proper calibration as they typically only give peaks in one or two spectral regions, and often have issues concerning purity and stability. Currently either camphor sulfonic acid or ammonium camphor sulfonate are used. The latter can be an unstable, slightly hygroscopic secondary standard compound with only one characterized CD band. The former is the very hygroscopic primary standard for which only one enantiomer is readily available. We have synthesized a new reference material for CD, Na[Co(EDDS)].H(2)O (EDDS = N,N-ethylenediaminedisuccinic acid) which addresses these problems. It is extremely stable and available in both enantiomeric forms. The CD spectrum of Na[Co(EDDS)].H(2)O has nine distinct peaks between 180 and 599 nm. It thus fulfils the principal requirements for CD calibration chemical standards and has the potential to be used to ensure good practice in the measurement of CD data, providing two spectra of equal magnitude and opposite sign for a given concentration and path length. We have carried out an interlaboratory comparison using this material and show how it can be used to improve CD comparability between laboratories. A fitting algorithm has been developed to assess CD spectropolarimeter performance between 750 and 178 nm. This could be the basis of a formal quality control process once criteria for performance have been decided.

A new reference material for UV-visible circular dichroism spectroscopy.

As part of a programme to develop a metrological framework for single molecule measurements in biology, we have investigated the applications of single molecule imaging to genomics. Specifically, we have developed a technique for measuring the frequencies of single nucleotide polymorphisms (SNPs) in complex or pooled samples of DNA. We believe that this technique has applications to statistical genotyping-the identification of correlations between SNP frequencies and particular phenotypes-and other areas where it is desirable to track the frequencies of SNPs in complex DNA populations.

Single molecule genotyping by TIRF microscopy.

Immunoassays allow the specific detection and quantitation of biological molecules in complex samples at physiologically relevant concentrations. However, there are concerns over the comparability of such techniques when the same assay is performed by different operators or laboratories. An international intercomparison study was performed to assess the uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA.

An international comparability study to determine the sources of uncertainty associated with a non-competitive sandwich fluorescent ELISA.

Immunoassays are biochemical tests applied to measure even very small amounts of substance using the highly specific binding between an antibody and its antigen. They have a wide range of applications. The measurement however, might be associated with substantial uncertainty; this can have significant consequences for any diagnosis, or clinical decision. An international comparability study was thus performed to assess the sources of uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA.

Bayesian analysis of an international ELISA comparability study.

Single-molecule enzymology has a longer history than is often supposed, with the first measurements being made as early as 1961. However, the development of new technologies has meant that most of the progress has been made in the last two decades. I review the development of single-molecule enzymology, focussing on five key papers which are milestones in the field. In particular, I discuss the 1961 paper by Boris Rotman, which made inventive use of what now seems like primitive technology, and continues to be influential to this day.

Single enzyme studies: a historical perspective.

Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (<50 nm) and thus negligible in traditional diffraction limited imaging, we show that object colocalization by multi-color super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).

Correcting chromatic offset in multicolor super-resolution localization microscopy.

Localisation bas&eacute;e Super-r&eacute;solution microscopy techniques n&eacute;cessitent des m&eacute;thodes de correction pr&eacute;cise d&eacute;rive parce que la r&eacute;solution spatiale atteinte avoisine les deux les limites de performances m&eacute;caniques et optiques des microscopes lumi&egrave;re modernes. Les m&eacute;thodes d&#39;imagerie multicolores n&eacute;cessitent des corrections outre celles traitant de la d&eacute;rive due &agrave; la m&eacute;thode statique, mais chromatique, spatialement d&eacute;pendant de d&eacute;calage entre les images. Nous pr&eacute;sentons des simulations informatiques pour quantifier cet effet, qui est caus&eacute; principalement par les objectifs de la haute-NA utilis&eacute;s dans la microscopie de Super-r&eacute;solution. Bien que la compensation chromatique dans des syst&egrave;mes bien corrig&eacute;es n&#39;est qu&#39;une fraction d&#39;une longueur d&#39;onde optique en amplitude (&lt; 50 nm) et donc n&eacute;gligeable dans traditionnelle diffraction limit&eacute;e d&#39;imagerie, nous montrons que co-localisation d&#39;objet par les m&eacute;thodes de Super-r&eacute;solution multicolore est impossible sans une correction d&#39;image appropri&eacute;e. Les donn&eacute;es simul&eacute;es sont en excellent accord avec les exp&eacute;riences utilisant des perles fluorescentes excit&eacute; et localis&eacute;e &agrave; plusieurs longueurs d&#39;onde. Enfin, nous pr&eacute;sentons un protocole d&#39;&eacute;talonnage rigoureux et pratique pour corriger la compensation optique chromatique et d&eacute;montrer son efficacit&eacute; pour l&#39;imagerie de transferrine receptor protein colocalisation dans les cellules HeLa au microscope deux couleurs directes stochastique de la reconstruction optique (dSTORM).

Alex E. Knight

Analytical Science Division

National Physical Laboratory

Test Samples for Optimizing STORM Super-Resolution Microscopy