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by Russell Ulbrich AL

Super-Resolution Microscopy Helps Extend Optical Observation Beyond Its Theoretical Boundaries

Scientists have long needed to view struc- tures and processes that cannot be resolved via traditional optical microscopy. Indeed, much of what is imaged in today’s life sci- ence research laboratories is fast-changing cellular activity hidden deep within specimens, making such phenomena especially difficult to resolve.

Ernst Abbe’s work on diffraction-limited optics (published in 1873) revealed that the size of the smallest resolvable feature that can be imaged by an optical microscope is inversely proportional to the light-gathering ability of its objective and proportional to the wavelength of observed light. Light with wavelength λ trav- eling in a medium with refractive index n and converging to a spot with angle θ will make a spot with radius:

the diffraction limit of traditional optical mi- croscopy systems. However, most life science researchers cannot jump to high-resolution techniques that abandon light as a means of imaging. These methodologies, such as elec- tron microscopy and atomic force microscopy, either are not capable of sensing the phenom- ena under study or simply cannot maintain a living specimen through sample preparation and observation. Below are some more prac- tical options being selected in laboratories today.

Imaging at the coverslip The deeper within the sample one images,

the more the limits of diffraction and scatter become a hindrance. Imaging in the near field is one solution for enhancing the resolving power of an optical system. Techniques such as total internal reflection fluorescence (TIRF)

The denominator of the equation refers to the light-gathering potential, or numerical aperture (2× NA), of the optics. In practical terms, the Abbe limit is often assumed to be around d = λ/2.8. Imaging with visible light through typical objectives with an NA of 1 yields an Abbe limit of about 250 nm, which is not sufficient to support observation of some complex intracellular structures and live cellular dynamics. Many fluorescence micros- copy techniques, including confocal imaging, multiphoton microscopy and other methods designed specifically to address the challenge of resolution, often require compromises in cost, complexity and sample requirements.

Researchers have numerous options for ad- dressing their need for resolution beyond


microscopy use the evanescent wave that oc- curs when excitation light passes through a high-refractive index (RI) medium and strikes a lower-RI medium at a critical angle of incidence. At this angle, light is internally reflected before it enters the second medium and produces a strong lateral evanescent electromagnetic field in the lower-RI medium containing the sample, which in turn causes local fluorescent excitation. The result is a very thin (~100 nm) band of excitation at the coverslip/specimen interface. Unwanted background and out-of- focus signals are dramatically reduced with TIRF microscopy, enhancing the researcher’s ability to view very dim and fast-changing events. Though limited to observation at the coverslip, TIRF microscopy provides excellent high-contrast imaging of membrane dynamics and single-molecule events, and continues to be a valuable tool in research.

Table 1 – Comparison of various types of super-resolution imaging

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