Abbe diffraction limit derivation12/4/2023 ![]() The vortex phase plate is most widely used for super-resolution in the lateral direction, while the annular phase plate is used for depletion in the axial direction, leading to super-resolution in the Z dimension (see Figure 3). This shape modification can also be realized with a vortex phase plate (Figure 2 (c) top figure). One way to create a perfect null intensity in the focus is by using an annular phase plate. Over the last decade, different options have been implemented to create such a focal spot, and the development is still ongoing. To create a perfect null intensity in the focus of a high NA lens, it is quite intuitive that the phase front of the incoming beam must be shaped such that the focused light interferes destructively in the focus center. ![]() This doughnut-like shape (Figure 2 (c)) allows efficient and symmetrical confinement of the depleted region. A more delicate solution would be to shape a single STED beam in such a way that it creates a symmetrical high intensity profile around a zero intensity center in the focus. Both options are experimentally challenging, since it requires very precise alignment of multiple beams. However, this requires either multiple STED lasers or splitting a single high intensity STED beam into four separate beams. This limitation could be partially solved by using four offset beams. The offset method as described in the previous section allows resolution increase only in the lateral direction of the offset. See also our Microscopy Today article on Huygens STED deconvolution. Additionally, the Huygens deconvolution algorithms are able to increase the resolution in the image with a factor of 2 in both lateral and axial direction. This theoretical PSF can be used in the deconvolution process, which will significantly reduce the noise in the image, and will increase the contrast of up to 10 times. Since 2012, the Huygens software is able to generate a theoretical PSF based on STED microscope parameters. Luckily, Huygens deconvolution of STED images offers a great solution. Therefore the SNR value in STED images will be much lower compared to their confocal counterpart. Because of the Poisson nature of photon statistics, the signal-to-noise ratio of the resulting image will decrease compared to normal confocal imaging. Unfortunately, this increased optical resolution also leads to a drawback: because many fluorophores are depleted by the depletion laser, this also results in a lower signal (fewer photons) being captured by the detector. STED microscopy allows super-resolution imaging in the 50nm range. As a result, the effective fluorescence spot is reduced to below the diffraction limit, but only in the direction of the offset. In the region where there is no STED field present, the fluorophores are still able to fluoresce. (b) When two diffraction limited STED spots are overlapped with a diffraction limited excitation spot with offset Δx and when the STED intensity is high enough, the fluorophores in the outer region of the excitation spot are effectively depleted to the ground state by stimulated emission. (a) Jablonski diagram illustrating the process of excitation, stimulated emission and fluorescence. This filtering ultimately leads to a size reduction of the effective fluorescent spot as is illustrated in Figure 1b.įigure 1. The photons that arise from stimulated emission are suppressed in the emission path with an appropriate filter, while the fluorescent photons are able to reach the detector. The fluorophores in the overlapping region are thus not switched into an ’off-state’ by the STED beam, but are merely stimulated to emit photons with the same wavelength as the STED laser. The process of stimulated emission effectively depletes the fluorophores in the overlapping region leaving only the flourophores in the non-overlapping region able to fluoresce. ![]() If the stimulated emission rate is high enough, the fluorophores in the overlapping region will emit photons with wavelength λ STED due to stimulated emission rather than emitting fluorescent photons with wavelength λ F (spontaneous emission). ![]() In the region where both lasers overlap, the fluorophores are excited by the excitation laser but are stimulated to emit at the wavelength of the second laser (λ STED ). The idea presented in Hell's first STED paper is based on depleting fluorophores in the outer region of the diffraction limited spot with a second laser, the STED-laser, which has a red-shifted wavelength (λ STED ) and a small spatial offset with respect to the excitation laser. Imaged with Abberior Instruments’ STEDYCON and deconvolved with Huygens. Image description: Primary hippocampal neurons with cytoskeleton proteins labelled (magenta, alpha-Adducin, Abberior STAR 635P and green, ßII spectrin, Alexa 594).
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