Ever-increasing resolution

Abstract

Overcoming the limitations of spatial and temporal resolution to image within a cell is no easy feat. Kelly Rae Chi examines the latest diffraction-busting technologies.For many years it was a source of frustration for biologists that the internal components of a cell were practically invisible to them. Researchers believed that the wavelength of light determined a fundamental limit to the resolution of optical microscopes.However, it now seems that the wavelength of light was not such a limiting factor after all. Super-resolution technology allows researchers to see details that are difficult or impossible to image with conventional light microscopes -at resolutions of 100 nanometres or better."There's a huge explosion of interest and progress, " says W. E. Moerner, a professor of chemistry and applied physics at Stanford University in California. "That makes it very exciting to watch and to participate in. "Although the theory behind super-resolution is advancing, and many different techniques are now validated, commercialization of these new microscopes is to some extent in its infancyso much so that many research teams still tackle super-resolution using their own lenses, lasers and algorithms. Upping commercializationThe past year or so has been particularly active in terms of commercialization. Throughout 2008, Applied Precision of Issaquah, Washington, tested prototypes of its DeltaVision OMX system. The final version is available this year and is already installed in 11 laboratories. It uses three-dimensional structured-illumination microscopy, or 3D-SIM, which illuminates a sample with a series of light patterns that look like bar codes. The low-resolution light patterns reflect off the fine structure of the sample to create moiré fringes. By applying bar codes in different orientations and processing the reflections using computer algorithms, the microscope generates a high-resolution image of the underlying structure.Carl Zeiss, headquartered in Oberkochen, Germany, is also using the SIM method in its system, ELYRA S.1, to be launched in January. And the company is launching ELYRA P.1, which is based on photo activated localization microscopy (PALM). This uses photo activatable proteins that are scattered throughout the sample. Taking multiple frames of the photoactivated dots, and combining them into a high-resolution image, the ELYRA P.1 produces a resolution of around 20 nm. Zeiss also offers a single system, the ELYRA PS.1, that combines laser scanning microscopy, PALM and SIM.Leica Microsystems in Wetzlar, Germany, is using a variation of stimulated emission depletion (STED) microscopy in its newest super-resolution system. The technique uses a laser beam to excite fluorescent dyes, or fluorophores, within the sample. As with a normal laser scanning microscope, the size of the excited spot determines the resolution of the microscope. In STED microscopy, to improve resolution and narrow the focus of the beam, an excitatory laser pulse is immediately followed by a ring-shaped depletion laser pulse ...

Assembly and Dynamics of Myofibrils

“…These FRET results supported the interpretations of FRAP experiments during myofibrillogenesis [46] and the premyofibril model (Figure 1). The current development of microscopes that break the diffraction limit of light [72] should reveal further insights into the disposition of protein networks in myofibrils and the changes they undergo in the processes of assembly and disassembly.…”

A Plasmodesmata-Localized Protein Mediates Crosstalk between Cell-to-Cell Communication and Innate Immunity inArabidopsis

“…Addressing these questions would help substantiate the functional significance of the association of PDLP5 with the central region of PD. Future investigation aided either by dual immunogold labeling or superresolution fluorescent microscopy techniques, which have recently been developed to allow nanoscale imaging of cells (Bates et al, 2008;Chi, 2009;Lippincott-Schwartz and Patterson, 2009), might be helpful to discern between those possibilities.…”

An Evaluation of the Faster STORM Method for Super-resolution Microscopy

“…Stochastic super-resolution (SR) microscopy methods enable localization imaging of florescent biological samples at lateral resolutions approaching 20 nm, thus circumventing the diffraction limits of conventional florescence microscopy [1], [2]. In traditional methods such as the stochastic optical reconstruction microscopy (STORM) or photo activated localization microscopy (PALM), this is achieved by stochastically activating and subsequently imaging a non-overlapping subset of the fluorophores at a given time [4], [3].…”