Live-cell 3D super-resolution imaging in thick biological samples

Abstract

We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.

Fluorescence nanoscopy. Methods and applications

“…Recent advances that address these problems include precompensation of aberrations through adaptive optics and PSF engineering [57,64,214], two-photon activation and/or excitation [58,59] and the application of light sheet microscopy concepts to PALM [60]. Red-shifted photoconvertible probes will also be demanded for in-depth imaging.…”

“…Several strategies have been put forward to achieve three-dimensional localisation microscopy: biplane detection [53], astigmatic imaging [54], engineered PSFs whose profiles unambiguously change with depth [55][56][57], confined two-photon activation [58][59][60], interferometric PALM (iPALM) [61] and highly inclined and laminated optical sheet illumination [62].…”

“…The activating IR beam can either follow the excitation path so that the same objective is used for activation, excitation and detection [58,59] or be relayed to the sample through an objective for activation and excitation set in an orthogonal configuration to a second, imaging, objective (selective plane illumination regime, SPIM [65]). Confined nonlinear activation is advantageous for nanoscopic depth imaging of whole cells and thicker samples such as spheroids (SPIM configuration [60]). …”

3‐D stimulated emission depletion microscopy with programmable aberration correction

“…Stochastic techniques can also provide axial super-resolution by using engineered point spread functions [14] or interferometry techniques [15] to localise fluorophores in the third dimension, though these are very susceptible to aberrations. Three-dimensional super-resolution live-cell imaging in thick samples has been demonstrated combining far-field individual molecule localization with selective plane illumination microscopy [16]. Here we are particularly interested to apply SRM to study protein structure and function at the immunological synapse (IS) that forms between white blood cells (lymphocytes) and their target cells as part of the immune response.…”

On-chip light sheet illumination enables diagnostic size and concentration measurements of membrane vesicles in biofluids

“…Light sheet illumination presents an attractive alternative to conventional epi-illumination as it combines superior contrast with real-time imaging, as is required to capture the MV's fast Brownian motion [14][15][16] . Although light sheet illumination has mainly been applied to mesoscopic imaging set-ups for developmental biology [17][18][19][20] , some reports demonstrate its usefulness for high-resolution imaging applications as well 15,16,21,22 . As illustrated in Figure 1a, this requires two objective lenses positioned perpendicular to one another in very close proximity, one for creating the light sheet, the other one for imaging.…”