Optics far Beyond the Diffraction Limit: Stimulated Emission Depletion Microscopy
In this chapter we show that stimulated emission depletion (STED) microscopy and its derivative concepts are able to radically overcome the diffraction barrier in far-field fluorescence imaging, thus disclosing fluorescent details on the macromolecular sc
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16. Optics far Beyond the Diffraction Limit: Stimulated Emission Depletion Microscopy
Optics far Bey
It is sometimes argued that confocal and multiphoton excitation provide subdiffraction resolving power in fluorescence microscopy; in reality neither breaks the diffraction barrier. Confocal microscopy can, in principle, attain a 1.4-fold reduction of the effective FWHM of the focal spot [16.2], because the consecutive point-like illumination and detection in a confocal fluorescence microscope yields an effective PSF that is the product of the excitation and detection PSFs. The PSF multiplication doubles the bandwidth of the optical transfer function of the system. However, this bandwidth increase is rarely realized because the larger spatial frequencies are heavily damped, and therefore swamped by noise. If they are not, the resolution can be improved with the help of a linear image deconvolution by a factor of 2, but no more. Hence, this technique is also conceptually diffraction-limited. Two- or three-photon excitation fluorescence microscopy benefits from the quadratic or cubic dependence of the fluorescence signal on the focal intensity. While the nonlinearity provided by multiphoton absorption indeed shifts the generation of the signal to the inner part of the excitation spot slightly, a narrowing of the effective spot is usually not obtained. The reason is that the energy gap of the excitation band of the fluorophore to be imaged is finite. Hence it is bridged by the instant
16.1 Principles of STED Microscopy ................. 1092 16.2 Nanoscale Imaging with STED ................ 1094 References .................................................. 1097 ∆r of the main diffraction maximum of the pointspread function (PSF) in the focal plane of the lens is ∆r = λ/(2n sin α), with λ and n denoting the wavelength of light and the refractive index, respectively [16.1]. If the distance between two objects is smaller than this FWHM, the objects cannot be readily resolved from one another. The diffraction resolution limit is particularly disadvantageous in the life sciences where about 80% of all microscopy applications are carried out with far-field fluorescence systems.
absorption of m photons of 1/m the energy, which inevitably entails a wavelength of the excitation light m times longer than in the single-photon excitation case. The m-times-longer wavelength renders the focal spot m-times larger, which is not compensated by the nonlinear excitation near the √ focal center [16.3]. In fact, the resulting spot scales as m. Thus, multiphoton processes do not improve the resolution in absolute terms, and so it is not surprising that multiphoton processes did not open up a new chapter in far-field optical resolution, despite their common use for more than 15 years [16.4]. Moreover, even in the hypothetic case that the requirement for a longer wavelength is not present, a quadratic or cubic intensity dependence would only expand the optical transfer function by a factor of two or three, respectively. Thus, the diffract
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