scholarly journals Fast, Accurate Polarization and Polarity Imaging with Polarized Structured Illumination

2019 ◽  
Author(s):  
Karl Zhanghao ◽  
Wenhui Liu ◽  
Meiqi Li ◽  
Xingye Chen ◽  
Chunyan Shan ◽  
...  
Keyword(s):  
Live Cells ◽  
Polarization Modulation ◽  
Optical Transfer Function ◽  
Wide Field ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Polarization Imaging ◽  
Order Of Magnitude ◽  
Ratiometric Imaging ◽  
Optical Transfer

AbstractThe orientation and wobbling behavior of the fluorescent dipoles are of great significance in revealing the structure and state of cells. Due to the poor optical sectioning capability of wide-field microscopy, the polarization modulation signals are susceptible to the neighboring fluorophores. The missing cone of wide field optical transfer function induces vast out-of-focus background, resulting in biased polarization orientation and decrease polarization factor. Here, we apply polarized structured illumination to achieve polarization modulation imaging with optical sectioning, and simultaneously measure the lipid polarity with two-color ratiometric imaging. Our results demonstrate a significant increase in measurement accuracy of not only the dipole orientations but also the wobbling behavior of the ensemble dipole. Compared to the conventional confocal polarization imaging, our method obtains an order-of-magnitude faster imaging speed, capturing the fast dynamics of subcellular structures in live cells.

scholarly journals Tilt illumination for structured illumination imaging

2021 ◽  
Author(s):  
Xin Jin ◽  
Xuemei Ding ◽  
Jiubin Tan ◽  
Cheng Shen ◽  
Xuyang Zhou ◽  
...  
Keyword(s):  
Transfer Function ◽  
Super Resolution ◽  
Lateral Resolution ◽  
Optical Transfer Function ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Sample Spectrum ◽  
Resolution Imaging ◽  
Optical Transfer ◽  
Illumination Mode

Abstract To achieve super-resolution imaging, the information in higher frequency of the observed sample is collected by illuminating with a structure beam for a limited optical transfer function (OTF). In this paper, tilt illumination mode is introduced to structured illumination microscopy (SIM) for enhancing lateral resolution. More sample spectrum more than traditional SIM, can be obtained by detector. Thus, SIM with tilt illumination can be improved at the aspect of lateral imaging resolution.

scholarly journals Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction

2018 ◽  
Author(s):  
Jakub Pospíšil ◽  
Tomáš Lukeš ◽  
Justin Bendesky ◽  
Karel Fliegel ◽  
Kathrin Spendier ◽  
...  
Keyword(s):  
High Resolution ◽  
Open Source ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Live Cells ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
Raw Data ◽  
Bayesian Image Reconstruction

AbstractBackgroundStructured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution).FindingsFive complete and freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods, and with newer Bayesian restoration approaches which we are developing.ConclusionVarious methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments is not typically published. Publicly available, high quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data was processed with SIMToolbox, an open source and freely available software solution for SIM.

Optical transfer function engineering for a tunable 3D structured illumination microscope

2019 ◽  
Vol 44 (7) ◽  
pp. 1560
Author(s):  
Hasti Shabani ◽  
Ana Doblas ◽  
Genaro Saavedra ◽  
Chrysanthe Preza
Keyword(s):  
Transfer Function ◽  
Optical Transfer Function ◽  
Structured Illumination ◽  
Optical Transfer

scholarly journals Multicolor structured illumination microscopy and quantitative control of polychromatic coherent light with a digital micromirror device

2020 ◽  
Author(s):  
Peter T Brown ◽  
Rory Kruithoff ◽  
Gregory J Seedorf ◽  
Douglas P Shepherd
Keyword(s):  
Closed Form Solution ◽  
Forward Model ◽  
Live Cells ◽  
Optical Transfer Function ◽  
Structured Illumination ◽  
Coherent Light ◽  
Structured Illumination Microscopy ◽  
Excitation Light ◽  
Promising Alternative ◽  
Diffractive Optic

Structured illumination microscopy (SIM) is a broadly applicable super-resolution microscopy technique which does not impose photophysics requirements on fluorescent samples. Multicolor SIM implementations typically rely on liquid crystal on silicon (LCoS) spatial light modulators (SLM's) for precise patterning of the excitation light, but digital micromirror devices (DMD's) are a promising alternative, owing to their lower cost, increased imaging rate, and simplified experimental timings. Given these advantages, why do existing DMD SIM implementations either rely on incoherent projection, resulting in an order of magnitude lower signal-to-noise, or utilize coherent light at only a single wavelength? The primary obstacle to realizing a multicolor coherent DMD SIM microscope is the lack of an efficient approach for dealing with the blazed grating effect. To address this challenge, we developed quantitative tools applicable to a single DMD acting as a polychromatic diffractive optic. These include a closed form solution of the blaze and diffraction conditions, a forward model of DMD diffraction, and a forward model of coherent pattern projection. We applied these to identify experimentally feasible configurations using a single DMD as a polychromatic diffractive optic for combinations of three and four common fluorophore wavelengths. Based on these advances, we constructed a DMD SIM microscope for coherent light which we used to validate these models, develop a high-resolution optical transfer function measurement technique, and demonstrate SIM resolution enhancement for calibration samples, fixed cells, and live cells. This low-cost setup opens the door to applying DMD's in polychromatic applications which were previously restricted to LCoS SLM's.

scholarly journals Concepts for structured illumination microscopy with extended axial resolution through mirrored illumination

2019 ◽  
Author(s):  
James D. Manton ◽  
Florian Ströhl ◽  
Reto Fiolka ◽  
Clemens F. Kaminski ◽  
Eric J. Rees
Keyword(s):  
Confocal Microscopy ◽  
Numerical Aperture ◽  
Wide Field ◽  
Structured Illumination ◽  
Axial Resolution ◽  
Resolution Limit ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
The Cost ◽  
Interferometric Detection

AbstractWide-field fluorescence microscopy, while much faster than confocal microscopy, suffers from a lack of optical sectioning and poor axial resolution. 3D structured illumination microscopy (SIM) has been demonstrated to provide optical sectioning and to double the resolution limit both laterally and axially, but even with this the axial resolution is still worse than the lateral resolution of unmodified wide-field microscopy. Interferometric schemes using two high numerical aperture objectives, such as 4Pi confocal and I5M microscopy, have improved the axial resolution beyond that of the lateral, but at the cost of a significantly more complex optical setup. Here, we investigate a simpler dual-objective scheme which we propose can be easily added to an existing 3D-SIM microscope, providing lateral and axial resolutions in excess of 125 nm with conventional fluorophores and without the need for interferometric detection.

scholarly journals Comparison of Two- and Three-Beam Interference Pattern Generation in Structured Illumination Microscopy

Photonics ◽  
2021 ◽  
Vol 8 (12) ◽  
pp. 526
Author(s):  
Jiuling Liao ◽  
Lina Liu ◽  
Tingai Chen ◽  
Xianyuan Xia ◽  
Hui Li ◽  
...  
Keyword(s):  
Fringe Pattern ◽  
Three Dimensional ◽  
Pattern Generation ◽  
Wide Field ◽  
Structured Illumination ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
Imaging Results ◽  
Dynamic Target ◽  
Beam Patterns

Structured illumination microscopy (SIM) provides wide-field optical sectioning in the focal plane by modulating the imaging information using fringe pattern illumination. For generating the fringe pattern illumination, a digital micro-mirror device (DMD) is commonly used due to its flexibility and fast refresh rate. However, the benefit of different pattern generation, for example, the two-beam interference mode and the three-beam interference mode, has not been clearly investigated. In this study, we systematically analyze the optical sectioning provided by the two-beam inference mode and the three-beam interference mode of DMD. The theoretical analysis and imaging results show that the two-beam interference mode is suitable for fast imaging of the superficial dynamic target due to reduced number of phase shifts needed to form the image, and the three-beam interference mode is ideal for imaging three-dimensional volume due to its superior optical sectioning by the improved modulation of the illumination patterns. These results, we believe, will provide better guidance for the use of DMD for SIM imaging and also for the choice of beam patterns in SIM application in the future.

scholarly journals Successful optimization of reconstruction parameters in structured illumination microscopy – a practical guide

2018 ◽  
Author(s):  
C. Karras ◽  
M. Smedh ◽  
R. Förster ◽  
H. Deschout ◽  
J. Fernandez-Rodriguez ◽  
...  
Keyword(s):  
Wiener Filter ◽  
Super Resolution ◽  
Optical Transfer Function ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Filter Parameter ◽  
Two Samples ◽  
Optical Transfer ◽  
The Impact ◽  
Zero Frequency

AbstractThe impact of the different reconstruction parameters in super-resolution structured illumination microscopy (SIM) onto artifacts is carefully analyzed. They comprise the Wiener filter parameter, an apodization function, zero-frequency suppression and modifications of the optical transfer function. A detailed investigation of the reconstructed image spectrum is concluded to be suitable for identifying artifacts. For this purpose, two samples, an artificial test slide and a more realistic biological system, were used to characterize the artifact classes and their correlation with the image spectra as well as the reconstruction parameters. In addition, a guideline for efficient parameter optimization is suggested and the implementation of the parameters in selected up-to-date processing packages (proprietary and open-source) is depicted.

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