Single Plane Illumination Microscopy for Microfluidic Device Imaging

Abstract

Three-dimensional imaging of live processes at a cellular level is a challenging task. It requires high-speed acquisition capabilities, low phototoxicity, and low mechanical disturbances. Three-dimensional imaging in microfluidic devices poses additional challenges as a deep penetration of the light source is required, along with a stationary setting, so the flows are not perturbed. Different types of fluorescence microscopy techniques have been used to address these limitations; particularly, confocal microscopy and light sheet fluorescence microscopy (LSFM). This manuscript proposes a novel architecture of a type of LSFM, single-plane illumination microscopy (SPIM). This custom-made microscope includes two mirror galvanometers to scan the sample vertically and reduce shadowing artifacts while avoiding unnecessary movement. In addition, two electro-tunable lenses fine-tune the focus position and reduce the scattering caused by the microfluidic devices. The microscope has been fully set up and characterized, achieving a resolution of 1.50 μm in the x-y plane and 7.93 μm in the z-direction. The proposed architecture has risen to the challenges posed when imaging microfluidic devices and live processes, as it can successfully acquire 3D volumetric images together with time-lapse recordings, and it is thus a suitable microscopic technique for live tracking miniaturized tissue and disease models.

Two-photon microscopy of acoustofluidic trapping for highly sensitive cell analysis

“…While several fluorescence-based microscopy techniques are capable of achieving 3D images with high spatial and temporal resolution, TPEFM stands out in comparison to others such as confocal laser scanning microscopy (CLSM) 26 and single plane illumination microscopy (SPIM). 27,28…”

“…While several fluorescence-based microscopy techniques are capable of achieving 3D images with high spatial and temporal resolution, TPEFM stands out in comparison to others such as confocal laser scanning microscopy (CLSM) 26 and single plane illumination microscopy (SPIM). 27,28 CLSM has traditionally been considered the gold standard for fluorescence-based experiments requiring 3D information or high resolution. By incorporating a point detector in place of a camera and utilizing dual pinhole aperturesone in the detection and one in the illumination paths, along with a galvo-galvo scanner systemthis microscopy type allows the recording of 3D images through restricted detection areas enabled by the pinholes.…”

Whole-cell multi-target single-molecule super-resolution imaging in 3D with microfluidics and a single-objective tilted light sheet

“…Previous single-objective LS designs have been demonstrated to reduce the complexity and limitations of multi-objective systems, but they have been limited by beam thickness 28 , the need for beam scanning 29,30 , and limited effective NA in the detection path and the requirement for post-processing due to an illumination beam that is not aligned to the detection axis 31 . Our approach based on a fully steerable single-objective tilted LS together with engineered PSFs solves all issues above and enables the use of a single high-NA objective lens for light sheet focusing and high photon collection efficiency without steric hindrance or relative drift, optical sectioning of entire adherent cells cultured on regular coverslips, and easy combination with microfluidic chips while avoiding the LS aberrations that occur when imaging through microfluidic chip walls 24,[31][32][33][34][35] . By using LS illumination for Exchange-PAINT imaging, freely diffusing imager strands outside of the focal plane are not excited, resulting in reduced fluorescence background and improved single-molecule localization precision.…”