Advances in 3D single particle localization microscopy

Abstract

The spatial resolution of conventional optical microscopy is limited by diffraction to transverse and axial resolutions of about 250 nm, but localization of point sources, such as single molecules or fluorescent beads, can be achieved with a precision of 10 nm or better in each direction. Traditional approaches to localization microscopy in two dimensions enable high precision only for a thin in-focus layer that is typically much less than the depth of a cell. This precludes, for example, super-resolution microscopy of extended three-dimensional biological structures or mapping of blood velocity throughout a useful depth of vasculature. Several techniques have been reported recently for localization microscopy in three dimensions over an extended depth range. We describe the principles of operation and typical applications of the most promising 3D localization microscopy techniques and provide a comparison of the attainable precision for each technique in terms of the Cramér-Rao lower bound for high-resolution imaging.

Three-dimensional volumetric deconvolution in coherent optics and holography

“…To acquire all the sample frequencies, the 3D OTF distribution can be used as a tool to "scan" over the 3D Fourier spectrum of the sample, which can be realized, for example, by a tomography approach [6]. 3DD can enhance the z resolution of reconstructed objects by improving the depth of focus and the lateral resolution of the imaged samples [7,8]. In this study, we consider an optical system that does not employ a complicated setup, such as tomography, and where the scattered wavefront is measured in one plane (single-shot measurement).…”

Real-Time Multidepth Multiphoton Microscopy Using Pixel-to-Pixel Focus-Switching

“…Although these platforms provide high-quality images with sufficient biological information, only one depth can be imaged at the same time. Multifocal-plane microscopy and point-spread function (PSF) engineering are two typical methods to image fluorescent particles at different focal planes simultaneously, and several implementations have been established [22][23][24][25]. However, these systems either require multiple detectors for one imaging modality or are unable to collect signal from scattering specimens.…”

Analyzing engineered point spread functions using phasor-based single-molecule localization microscopy

“…The shape of the PSF quickly deteriorates, however, if the emitter is out of focus (~100s of nm), leading to both a limited available axial range and inaccessibility of the absolute axial position [32]. Therefore, a variety of methods have been developed to modulate the shape of the PSF depending on the emitter's axial position [33]. Historically, the first method (astigmatism; AS) introduced a cylindrical lens in the emission pathway to create ellipsoid PSFs if the emitters are out of focus [34,35].…”