Abbe’s diffraction limit amounts for the shortest wavelength of visible light to about 200 nm for the lateral resolution. Smaller details can not be resolved. The axial resolution is even worse.
Many efforts are taken to acquire sharper images. One approach is to abandon the advantageous visible light altogether because of its res-olution limit of 200 nm. Reduction of the wavelength used for imaging is pursued inUV(13)[1,36] andX-ray microscopy [170,222]. The higher resolution is paid for by complex optics and the impossibility of imaging living samples, which are harmed by the high-energy radiation.
Electron microscopy [266,326] provides resolutions in the angstrom regime [267], using electrons with a very short de Broglie wave-length [61] for imaging. Only very thin samples (or surfaces) can be imaged and the specimen is placed in vacuum during imaging. There-fore imaging under physiological conditions is not possible. Further-more, the sample preparation is cumbersome and the staining not as versatile as in light microscopy.
(12)“Having ascertained about five and thirty years ago, by comparisons of the perfor-mance of several telescopes of very different apertures that the diameters of star-disks varied inversely as the diameter of the aperture, I examined with a great variety of apertures a vast number of double stars, whose distances seemed to be well determined, and not liable to rapid change, in order to ascertain the separating power of those aper-tures, as expressed in inches of aperture and seconds of distance. I thus determined as a constant, that a one-inch aperture would just separate a double star composed of two stars [. . . ], if their central distance was400.56; – the atmospheric circumstances being moderately favourable. Hence, the separating power of any given aperture,a, will be expressed by the fraction400.56/a.”
(13)Ultra Violet
Surface scanning techniques such as AFM(14) [26], STM(15) [27], SNOM(16) [306,13,242], TERS(17) [10,167,120,301] and SICM(18) [113]
can only image surfaces, but some can reach molecular resolution on biological samples [226].
It is the light microscopy which remains arguably the most impor-tant method for imaging in the biosciences due to the shortcomings of the alternative techniques. It is the only method that allows the non-invasive imaging of thick samples under physiological conditions in three dimensions. Furthermore, there is not only a large array of specific markers available for the study of countless cellular processes, but also many of them are compatible with imaging living samples. Im-munostainings [188] are highly specific and regularly applied to fixated biological samples. Fluorescent proteins [314, 198, 321] like GFP(19)
[285, 44] allow researchers to engineer organisms with self-staining properties. FlAsH(20)[103], SnapTag(21)[169] and HaloTag(22)[204,203]
enable the use of organic dyes in living cells. Intracellular staining with quantum dots [236] is possible by microinjection [76]. Moreover, func-tional studies are possible using dyes or proteins that change their flu-orescence behavior depending on external parameters such as pH or calcium concentration [286,313]. The option of studying dynamic pro-cesses provides an enormous field of applications in both biological [298]
and synthetic samples (such as colloidal systems [56]).
A multitude of methods was therefore developed to surpass the res-olution limit in light microscopy (For reviews see [126,131,152,49,63, 197,130,256,133,128,84,105]):
In a confocal microscope [223], the sample is scanned with a focused beam of light. The fluorescence is recorded with a point detector be-hind a pinhole. The pinhole rejects the out of focus light thereby
en-(14)Atomic Force Microscopy
(15)Scanning Tunneling Microscopy
(16)Scanning Near Field Optical Microscopy
(17)Tip-Enhanced Raman Spectroscopy
(18)Scanning Ion-Conductance Microscopy
(19)Green Fluorescent Protein
(20)Fluorescein Arsenical Helix Binder
(21)Based on the use of the deoxyribonucleic acid repair protein alkyl guanine DNA alkyl transferase
(22)Based on the use of a modified haloalkane dehalogenase
abling axial sectioning. The lateral resolution is increased by a factor of
≈1.4 [105].
4Pi-microscopy [127,135] and I2M(23) [107, 108] increase the effec-tive total aperture by the use of two opposing objeceffec-tives, thereby en-hancing especially the axial resolution. TIRF microscopy(24) [16, 307]
enhances the axial resolution by illuminating only a very thin layer of the sample that is adjacent to the coverslip via an evanescent field of light.
Hyperlenses [323], consisting of materials with a negative refractive index [322], promise to enhance the resolution by recovering the evanes-cent field [238]; but they also require close proximity to the sample, even if their image can be magnified in the far field [202].
Various kinds of structured illumination microscopy [105], some-times combined with TIRF [55,175] or with the two-lens approach, in-cluding I3M(25) [107,108], I5M(26) [109], I5S(27) [284],SWFM(28) [17] and HELM(29) [96] use non-uniform illumination of the specimen to extract high spatial frequencies. Recently, the combination with point scanning was proposed, which led to SPIN(30) and SPADE(31) microscopy [206].
This combination would enable the use of structured illumination to-gether with two-photon excitation [67] or spontaneous Raman scatter-ing [206].
These methods lead to extended resolution microscopy [105], but not to unlimited resolution microscopy in the far field. The diffraction limit is not fundamentally broken by any of these methods. In all of the far-field methods, the attainable resolution is limited to a finite value. They reach a new limit, which is on the order of a factor two below Abbe’s value, but they can not provide a theoretically unlimited resolution.
(23)Image Interference Microscopy
(24)Total Internal Reflection Microscopy
(25)Incoherent Interference Illumination Microscopy
(26)The combination of I2M and I3M
(27)A combination of I5M with laterally structured illumination
(28)Standing Wave Fluorescence Microscopy
(29)Harmonic Excitation Light Microscopy
(30)Scanning Patterned Illumination
(31)Scanning Patterned Detection