Unfortunately, commonly used nonlinear two- and multiphoton excitations suffer from two main drawbacks. The combination of strong light gradients and strong nonlinear response can further shrink the spatial extent of the point-spread function (PSF), as schematically shown in Fig. demonstrated a novel and clever approach for superresolution imaging that is quite different from previous approaches, holding great promise for future application ( 9).Īlthough the most recent superresolution methods listed above rely only on the volumetric shaping of the excitation light (through a near-field aperture/tip or periodic light gradients generated by interference), attempts have also been made to enhance resolution further by using a nonlinear relationship between the excitation and the fluorescence emission ( 10, 11). These include surface-specific two-dimensional aperture ( 2) and aperture-less ( 3) near-field scanning optical microscopy, wide-field three-dimensional image restoration by computational methods ( 4) and three-dimensional nonuniform periodic excitation (and emission) patterns that contain high spatial frequency components as in 4PI ( 5), incoherent interference illumination image interference ( 6), standing-wave total-internal-reflection fluorescence ( 7), and harmonic excitation light ( 8) microscopies. In recent years, we have witnessed, in response to this challenge, inventive solutions that successfully break the diffraction limit of light. For commonly used dyes and high numerical aperture oil immersion objectives, this resolution limit is on the order of 250–300 nm. As was shown by Abbe over 100 years ago, the wave nature of light imposes a fundamental constraint on the attainable spatial resolution known as the “diffraction limit of light” ( 1). However, conventional optical microscopy lacks the required nanometer resolution needed for the task described above. The application of novel spectroscopic methods and advanced image analysis techniques now provides quantitative analytical tools for the study of cellular dynamics. The developments of green (and other color) fluorescent proteins as specific genetically manipulated molecular markers and of other sophisticated fluorescence indicators together with improvements in imaging techniques have revolutionized fluorescence imaging of live cells. It is noninvasive it provides imaging in three dimensions it has high sensitivity down to the single molecule level, and it allows the observation of molecular- and organelle-specific signals. Fluorescence microscopy offers many advantages for probing live cells.
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