PHOTON AND SUPER-RESOLUTION MICROSCOPY

Photon and Super-resolution Microscopy: Biosciences Revolution

Photon and Super-resolution Microscopy: Biosciences Revolution

Introduction

Scientists have been using light microscopes for centuries to visualize structures that are too small to see with the naked eye. The quest to peer into the nanoscopic details of living cells has led to ever increasing technical innovations and molecular labelling strategies. Fluorescent microscopy stands out among the techniques for its ability to visualize intracellular targets tagged with spectrally distinct colours. With the advent of genetically encoded fluorescent proteins (FPs), it has even become possible to study protein dynamics by noninvasively visualizing the interior of cells (Betzig, 2006, 1642).

Despite the sweeping impact of live-cell fluorescent microscopy, fluorescence microscopy is fundamentally limited in resolution by the diffraction of light through the optical path. Abbe, Rayleigh, and others identified this constraint as imposing a minimum required spacing between two point sources of light in order for them to be resolved. This spacing is commonly defined as d = ?/(2nsina), where d is the minimum spacing for resolvability, ? the wavelength of light, n the refractive index of the medium surrounding the sample, and a is the angular aperture of the microscope objective. The diffraction limit represents the point-spread function (PSF) or the size of the intensity profile for a spot measured at the full width half maximum. For a typical fluorescent probe, d = ~200 nm. Therefore, proteins smaller than 200 nm appear to be 200 nm in size, and proteins closer to each other than 200 nm cannot be distinguished from each other. Clearly, resolution beyond the diffraction limit is needed to define the structure and function of dynamic cellular nanomachines using light microscopy. In fact, it might even be argued that the limitations imposed by diffraction are almost more disadvantageous to fluorescent microscopy than bright field microscopy because we can label individual molecules with different colours, but we cannot optically separate them with conventional microscopy.

The recent revolution in light microscopy has removed the diffraction barrier as a limiting factor to resolution. With the emergence of super-resolution light microscopy, we can now visualize details of cellular and macromolecular structures that were previously impossible to see. Light microscopy now has the resolution to match the level of detail that molecular labelling techniques provide, and molecular specific resolution of 10-20 nm is now routine. Visualizing nanosensing with 60 nm resolution of dynamically reorganizing molecular ensembles in living cells is now an exciting reality.

Source: (Harke, 2008, 1309)

Photon and Super-Resolution Microscopy

Super-resolution microscopy is defined as any method that improves resolution by at least a factor of two over conventional microscopy. The methods that are used to achieve this are frequently distinguished as either hardware or software based on their approach to shrinking the PSF and increasing resolution. In order to provide a logical basis for pairing technologies with specific biological applications, we will limit the discussion in this review to two hardware-based or ensemble methods and two software-based or single molecule methods. However, as improvements and variations of the technologies are emerging ...