Here, we introduce an easy-to-implement photometric method named Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER) to determine the axial position of single molecules in a TIRF microscope. In 1987 Axelrod revisited the work of Lukosz in the context of TIRF microscopy 31, and from then on, the knowledge to obtain axial positions from a quantitative use of TIRF microscopy was fully available. They could estimate average cell-substrate separation distances of 49вЂ‰nm for focal contacts, and of 69вЂ‰nm for close contacts 30. were the first ones to obtain axial positions from photometric readings of TIRF-illuminated 3T3 fibroblast cells excited at two different angles of incidence, and a theoretical calibration based on the model of Lukosz 27, 28, 29. These initial approaches were based exclusively on the axial dependency of the excitation intensity. Total internal reflection fluorescence (TIRF) microscopy was pioneered by Axelrod in the early 1980s, demonstrating various applications including a scheme to obtain sub-diffraction axial resolution analysing the TIRF intensity as a function of the incidence angle 26. The use of the evanescent illumination field of total internal reflection to obtain sub-diffraction axial information in optical microscopy goes back to the 1950s and 1960s 24, 25. More recently, MINFLUX 22 was demonstrated to deliver sub-10-nm resolution in three dimensions 23, but this is at the cost of elevated technical complexity. However, combining this nanosecond time-resolved method with other nanoscopy methods in order to obtain 3D imaging with sub-10-nm resolution is not straightforward 21. MIET decodes the z-position of fluorophores through lifetime imaging making use of the distance-dependent energy transfer from excited fluorophores to a metal film 18 or a graphene sheet 19, 20. To date, sub-10-nm axial localization of single molecules was only achieved by two methods, metal-induced energy transfer (MIET) and MINFLUX. Isotropic stimulated emission depletion microscopy has been shown to deliver nearly isotropic resolution in the range of 30вЂ“40вЂ‰nm 13, 14, whereas 4-Pi photo-activated localization microscopy/stochastic optical reconstruction microscopy (STORM) has reached 10вЂ“20-nm resolution in 3D 15, 16, 17.

By exploiting the 4-Pi configuration 12 it is possible to reach an axial resolution below 35вЂ‰nm, but at the cost of increased technical complexity. Axial resolution of methods using a single objective lens is typically two- to fivefold worse 7, 8, including recent advances considering the experimentally determined microscope point-spread-functions 9, intensity-based approaches that rely on supercritical angle fluorescence 10 or photometric analysis of the defocused images of single molecules 11. Whereas in theory all super-resolution methods are able to reach nanometric resolution given a sufficiently high fluorescence photons flux, in practice most methods reach a lateral resolution limit of 10вЂ“20вЂ‰nm.

Super-resolution microscopy, also known as far-field fluorescence nanoscopy, has set the conceptual pathway to achieve this goal 1, 2, 3, 4, 5, 6.

Imaging the three-dimensional organization of biological structures down to the size of their structural proteins, ~4вЂ“10вЂ‰nm, can open up exciting opportunities in the life sciences. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10вЂ‰nm axial localization precision. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution.

Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-moleculeвЂ™s image can be determined with much higher accuracy than the size of that image itself.

Paint storm studio 2.4