Fast TIRF-SIM imaging of dynamic, low-fluorescent biological samples

Abstract

Fluorescence microscopy is the standard imaging technique to investigate the structures and dynamics of living cells. However, increasing the spatial resolution comes at the cost of temporal resolution and vice versa. In addition, the number of images that can be taken in sufficiently high quality is limited by fluorescence bleaching. Hence, super-resolved imaging at several Hertz of low fluorescent biological samples is still a big challenge and, especially in structured illumination microscopy (SIM), is often visible as imaging artifacts. In this paper, we present a TIRF-SIM system based on scan-mirrors and a Michelson interferometer, which generates images at 110 nm spatial resolution and up to 8 Hz temporal resolution. High resolution becomes possible by optimizing the illumination interference contrast, even for low fluorescent, moving samples. We provide a framework and guidelines on how the modulation contrast, which depends on laser coherence, polarization, beam displacement or sample movements, can be mapped over the entire field of view. In addition, we characterize the influence of the signal-to-noise ratio and the Wiener filtering on the quality of reconstructed SIM images, both in real and frequency space. Our results are supported by theoretical descriptions containing the parameters leading to image artifacts. This study aims to help microscopists to better understand and adjust optical parameters for structured illumination, thereby leading to more trustworthy measurements and analyses of biological dynamics.

Super-Resolution Microscopy and Single-Molecule Tracking Reveal Distinct Adaptive Dynamics of MreB and of Cell Wall-Synthesis Enzymes

“…TIRF-SIM can reveal protein dynamics at a spatial resolution of about 120 nm and in the range of few seconds for temporal resolution, while SMT has a temporal resolution in the milliseconds range. We imaged GFP-MreB [or YFP-MreB for SMT, these fusions have been shown to functionally replace wild type MreB in Reimold et al (2013) ] in 0.5 s intervals, using a custom built setup ( Roth et al, 2020 ). In order to avoid overproduction artifacts, the fusion was expressed at very low levels (0.01% xylose) from an ectopic site on the chromosome, such that cells express very few GFP-MreB molecules besides wild type MreB protein ( Supplementary Figure S1A ).…”

Motionless Polarizing Structured Illumination Microscopy

“…Structured illumination microscopy has been widely used for measuring biomedical samples based on fluorescent microscopic techniques [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ]. Although widefield fluorescence microscopy is a sensitive method for detecting labeled proteins, it cannot provide a clear image of a sample because of the image blurring caused by the light from out-of-focus regions.…”

“…To prevent the image blurring, structured illumination microscopy uses a structured illumination pattern to computationally eliminate the out-of-focus light and enhances the fluorescent image quality of the sample. Typically, structured illumination microscopy has been developed as two categories: lateral resolution enhancement, so-called super-resolution structured illumination microscopy (SR-SIM) [ 1 , 2 , 3 , 4 , 5 ]; and 3D optical sectioning structured illumination microscopy (SIM) [ 6 , 7 , 8 , 9 , 10 ]. Conventional optical microscopy only has a limited lateral resolution due to the Abbe diffraction limit, and also lacks the ability of 3D imaging.…”

“…However, SR-SIM enhances lateral resolution by collecting several patterned images and analyzing them in reciprocal space, such as in the Fourier domain [ 1 , 2 , 3 , 4 , 5 ]. On the other hand, SIM can provide 3D imaging of the sample by detecting the visibility (modulation depth) of the illumination pattern [ 6 , 7 , 8 , 9 , 10 , 11 , 12 ].…”

“…The proposed system approaches the system modification with a completely different way, not tried previously. Currently, recent research work relevant to SIM has been implemented as two categories: biomedical applications [ 7 , 8 , 9 , 10 ] and system modifications [ 23 , 24 , 25 , 26 , 27 ]. For the system modification of SIM to enhance the measurement sensitivity and speed, they used two cameras for differential operation [ 23 ], fast temporal phase-shifting [ 24 ], deep-learning [ 25 ], iterative algorithm [ 26 ], and HiLo technique [ 27 ].…”