Structured illumination multimodal 3D-resolved quantitative phase and fluorescence sub-diffraction microscopy.

Joseph A Izatt,Will J Eldridge,Shwetadwip Chowdhury,Adam Wax

doi:10.1364/boe.8.002496

Abstract

Sub-diffraction resolution imaging has played a pivotal role in biological research by visualizing key, but previously unresolvable, sub-cellular structures. Unfortunately, applications of far-field sub-diffraction resolution are currently divided between fluorescent and coherent-diffraction regimes, and a multimodal sub-diffraction technique that bridges this gap has not yet been demonstrated. Here we report that structured illumination (SI) allows multimodal sub-diffraction imaging of both coherent quantitative-phase (QP) and fluorescence. Due to SI's conventionally fluorescent applications, we first demonstrate the principle of SI-enabled three-dimensional (3D) QP sub-diffraction imaging with calibration microspheres. Image analysis confirmed enhanced lateral and axial resolutions over diffraction-limited QP imaging, and established striking parallels between coherent SI and conventional optical diffraction tomography. We next introduce an optical system utilizing SI to achieve 3D sub-diffraction, multimodal QP/fluorescent visualization of A549 biological cells fluorescently tagged for F-actin. Our results suggest that SI has a unique utility in studying biological phenomena with significant molecular, biophysical, and biochemical components.

Highlights

Optical microscopy has played a crucial role in advancing the frontiers of biological sciences by allowing high-resolution, non-invasive visualization of important biological samples
We refer the reader to the work by Gustafsson et al [34], which beautifully illustrates the framework behind structured illumination (SI) for 3D superresolution fluorescent imaging – we introduce an analogous approach for QP imaging
SI’s capability to enable 2D lateral QP subdiffraction resolution was previously demonstrated using low imaging numerical aperture (NA) [29,30,31,32]. We extend these results to demonstrate SI-enabled subdiffraction resolution QP imaging at high NA

Summary

Introduction

Optical microscopy has played a crucial role in advancing the frontiers of biological sciences by allowing high-resolution, non-invasive visualization of important biological samples. Developments and advances in optical design and manufacturing have made available high-resolution objectives with unprecedented numerical aperture (NA), microscopy faces a fundamental physical diffraction limit that can preclude visualization of important sub-cellular features in biological samples [1, 2]. Sub-diffraction imaging techniques introduced far operate in two main regimes: 1) imaging via spatially-coherent diffraction, or 2) imaging via spatially-incoherent fluorescence. Synthetic aperture (SA) is a popular choice for imaging in the first regime, and operates by using oblique illuminations to spatiotemporally encode a wider frequency support into the final image than directly allowed by the microscope’s physical aperture [5,6,7]. Applications of SA have resulted in both high-resolution imaging, where (Sparrow) resolutions of < 100 nm have been achieved [8], and high-throughput imaging, where gigapixel-scale images with resolutions > 5x over the diffraction limit have been obtained [9,10]

Methods

Results

Conclusion