Sub-diffraction imaging on standard microscopes through Photobleaching Microscopy with non-linear Processing

Abstract

SummaryVisualization of organelles and molecules at nanometer resolution is revolutionizing the biological sciences. However, such technology is still limited for many cell biologists. We present here a novel approach using photobleaching microscopy with non-linear processing (PiMP) for sub-diffraction imaging. Bleaching of fluorophores both within the single-molecule regime and beyond allows visualization of stochastic representations of sub-populations of fluorophores by imaging the same region over time. Our method is based on enhancing the probable positions of the fluorophores underlying the images. The random nature of the bleached fluorophores is assessed by calculating the deviation of the local actual bleached fluorescence intensity to the average bleach expectation as given by the overall decay of intensity. Subtracting measured from estimated decay images yields differential images. Non-linear enhancement of maxima in these diffraction-limited differential images approximates the positions of the underlying structure. Summing many such processed differential images yields a super-resolution PiMP image. PiMP allows multi-color, three-dimensional sub-diffraction imaging of cells and tissues using common fluorophores and can be implemented on standard wide-field or confocal systems.

From microscopy to nanoscopy via visible light

“…However, a new method is to record the bleaching process of the fluorophores and making them sparse by using a subtraction calculation (bleaching/blinking-assisted localization microscopy; Figure 9c and 9d). 90,91 On first inspection, these stochastic switching and readout mode techniques are not reliable for dynamic applications. Hence, recent endeavors have mainly focused on fast algorithms that seek to reconstruct the image with less frames.…”

A Joint Richardson-Lucy Deconvolution Algorithm for the Reconstruction of Multifocal Structured Illumination Microscopy Data

“…Since the first paper on stimulated emission depletion microscopy, STED, was published [1], a multitude of super-resolution techniques have arisen, all capable of 'breaking the diffraction limit' [2][3][4][5][6][7][8][9][10]. One of the most prominent methods in this field is structured illumination microscopy, SIM [7].…”

Conical diffraction illumination opens the way for low phototoxicity super-resolution imaging

“…3,4 STED-like techniques use a secondary high-power laser line to restrict fluorescence emission to a sub-diffraction area of the sample. 5 Point-localization microscopy [6][7][8] and photobleaching microscopy with nonlinear processing 9 require repeated cycles of fluorophore activation, acquisition or bleaching to reconstruct SR representations of the sub-diffraction fluorophore distribution. It is therefore most notable for the case where living samples may be targeted by these SR microscopy techniques, that it will be the illumination regime that has perhaps the most significant consequences, including an increased propensity for phototoxic processes.…”