Abstract
Abstract Super-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live-cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work, we present a multiplane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multiplane image splitting prism combined with high-speed SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally highly resolved 3D image stack. We demonstrate the performance of multiplane SIM by applying this instrument to imaging the dynamics of mitochondria in living COS-7 cells.
Highlights
Conventional fluorescence microscopy is inherently limited in its spatial resolution due to diffraction
We present a multiplane approach to Super-resolution structured illumination microscopy (SR-SIM) that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multiplane image splitting prism combined with highspeed SIM illumination
SIM is an enabling imaging method, as its increase in spatial resolution and inherent background suppression allow us to visualize the mitochondria and their 3D morphology and dynamic changes thereof, which cannot be observed with conventional wide-field resolution [20]
Summary
Conventional fluorescence microscopy is inherently limited in its spatial resolution due to diffraction. The excitation powers are comparable to conventional widefield imaging, so photo-damage can be minimized, and no special dyes (e.g. dyes that are photo-switchable or inherently blinking) are required for the approximately twofold resolution enhancement. The combination of these features makes SR-SIM a very fast super-resolution imaging technique, with current instruments [8, 9] providing 2D. Descloux et al.: High-speed multiplane structured illumination microscopy of living cells imaging with approximately 100-nm spatial resolution in the lateral direction at video-rate speed and even faster. We present a multiplane imaging approach to SR-SIM that circumvents these issues and achieves high-speed imaging rates previously available only to 2D SR-SIM
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