Machine learning assisted interferometric structured illumination microscopy for dynamic biological imaging

Abstract

Structured Illumination Microscopy, SIM, is one of the most powerful optical imaging methods available to visualize biological environments at subcellular resolution. Its limitations stem from a difficulty of imaging in multiple color channels at once, which reduces imaging speed. Furthermore, there is substantial experimental complexity in setting up SIM systems, preventing a widespread adoption. Here, we present Machine-learning Assisted, Interferometric Structured Illumination Microscopy, MAI-SIM, as an easy-to-implement method for live cell super-resolution imaging at high speed and in multiple colors. The instrument is based on an interferometer design in which illumination patterns are generated, rotated, and stepped in phase through movement of a single galvanometric mirror element. The design is robust, flexible, and works for all wavelengths. We complement the unique properties of the microscope with an open source machine-learning toolbox that permits real-time reconstructions to be performed, providing instant visualization of super-resolved images from live biological samples.

Fully-Automated Multicolour Structured Illumination Module for Super-resolution Microscopy

“…Recently integrated solutions realize SIM illumination by means of photonic chips [17], metamaterials [18] or optoacoustic Bragg cells [19]. There are several ways to implement even classical two-beam SIM, such as using a Michelson interferometer [20] or a fiber array [21]. Besides the use of classical diffraction grating in a conjugate image plane, the use of a spatial light modulator allows a flexible and accurate, non-mechanical manipulation of the phases and diffraction orders.…”

Ion Monitoring at Nanoscale Sites of Interorganelle Membrane Contact in Living Cells

“…Recent advancements in super-resolution fluorescence microscopy techniques like the structured illumination microscope (SIM), which can overcome technical challenges with high spatial resolution (80-120 nm) and high time resolution (approximately 100 fps), have provided technical options for nanoscale FRET detection and measurement in living cells. [26,27] To address the need for analysis spatiotemporal structures at the nanoscale in living systems, we combined the use of a BODIPY-rhodamine dyad FRET probe, BDP-RhB, with SIM imaging to develop a system with high spatial and temporal resolution to detect H þ exchange at MLC sites. By monitoring the fluorescence switching of FRET when lysosomes carrying the probe interacted with mitochondria, real-time changes in the H þ microenvironment during MLC formation and dissociation were found to be detectable.…”

“…Recent advancements in super‐resolution fluorescence microscopy techniques like the structured illumination microscope (SIM), which can overcome technical challenges with high spatial resolution (80–120 nm) and high time resolution (approximately 100 fps), have provided technical options for nanoscale FRET detection and measurement in living cells. [ 26,27 ]…”

Surpassing Light Inhomogeneities in Structured-Illumination Microscopy with FlexSIM

“…Among super-resolution fluorescence microscopy techniques [8][9][10], it is one of those that offer the best tradeoff between spatial and temporal resolution [11][12][13]. Moreover, it does not require specific sample preparations, offers high photon efficiency, and supports multicolor imaging [3,14,15]. SIM is a prime example of computational microscopy that combines optics and numerical reconstruction so as to surpass the diffraction limit.…”

“…Among super-resolution fluorescence microscopy techniques [8–10], it is one of those that offer the best tradeoff between spatial and temporal resolution [11–13]. Moreover, it does not require specific sample preparations, offers high photon efficiency, and supports multicolor imaging [3, 14, 15].…”