ABSTRACT
Structured illumination microscopy (SIM) was recently adapted to coherent imaging, named structured oblique-illumination microscopy (SOIM), to improve the contrast and resolution of a light-scattering image. Herein, we present high-resolution laterally isotropic SOIM imaging with 2D hexagonal illuminations. The SOIM is implemented in a SIM fluorescence system based on a spatial-light modulator (SLM). We design an SLM pattern to generate diffraction beams at 0° and ± 60.3° simultaneously to form a 2D hexagonal illumination, and undertake calculations to obtain optimal SLM shifts at 19 phases to yield a reconstructed image correctly. Beams of linear and circular polarizations are used to show the effect of polarization on the resolution improvement. We derive the distributions of the electric field of the resultant hexagonal patterns and work out the formulations of the corresponding coherent-scattering imaging for image reconstruction. The reconstructed images of gold nanoparticles (100 nm) confirm the two-fold improvement of resolution and reveal the effect of polarization on resolving adjacent nanoparticles. To demonstrate biological applications, we present the cellular structures of a label-free fixed HeLa cell with improved contrast and resolution. This work enables one to perform high-resolution dual-mode - fluorescence and light-scattering - imaging in a system, and is expected to broaden the applications of SOIM.
ABSTRACT
Three-dimensional structured illumination microscopy (3D-SIM) is a wide-field technique that can provide doubled resolution and improved image contrast. In this work, we demonstrate a simple approach to 3D-SIM - using three-beam interference with circular polarization to generate the pattern of structured illumination, so that the modulation contrast is routinely maintained at all orientations without a complicated polarization rotator or mechanical motion. We derive the resultant intensity distribution of the interference pattern to confirm the modulation contrast independent of orientation, and compare the result with those using interfering beams of linear polarization. To evaluate the influence of the modulation contrast on imaging, we compare the simulated SIM images of 100-nm beads. Experimental results are presented to confirm the simulations. Our approach requires merely a λ/4-wave plate to alter the interfering beams from linear to circular polarization. This simplicity together with the use of a spatial light modulator to control the interference pattern facilitates the implementation of a 3D-SIM system and should broaden its application.
ABSTRACT
A reflective light-scattering (RLS) microscope with structured illumination (SI) provides subdiffraction resolution and improves the image quality of gold nanoparticles in biological systems. The three-dimensional (3D)-structured pattern is rapidly and precisely controlled with a spatial light modulator and scrambled at the conjugate image plane to increase spatial incoherence. The reconstructed SI-RLS image of 100 nm gold nanoparticles reveals lateral and axial resolutions of approximately 117 and 428 nm. We present a high-resolution image of gold nanoparticles inside a HeLa cell, with improved contrast.
Subject(s)
Gold/chemistry , Metal Nanoparticles/chemistry , Equipment Design , HeLa Cells , Humans , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Lasers , Light , Microscopy/methods , Models, Statistical , Nanoparticles/chemistry , Optics and Photonics , Scattering, RadiationABSTRACT
We developed a structured illumination microscopy (SIM) system that uses a spatial light modulator (SLM) to generate interference illumination patterns at four orientations - 0 degrees, 45 degrees, 90 degrees, and 135 degrees, to reconstruct a high-resolution image. The use of a SLM for pattern alterations is rapid and precise, without mechanical calibration; moreover, our design of SLM patterns allows generating the four illumination patterns of high contrast and nearly equivalent periods to achieve a near isotropic enhancement in lateral resolution. We compare the conventional image of 100-nm beads with those reconstructed from two (0 degrees +90 degrees or 45 degrees +135 degrees) and four (0 degrees +45 degrees +90 degrees +135 degrees) pattern orientations to show the differences in resolution and image, with the support of simulations. The reconstructed images of 200-nm beads at various depths and fine structures of actin filaments near the edge of a HeLa cell are presented to demonstrate the intensity distributions in the axial direction and the prospective application to biological systems.
