A multi-functional microfluidic device compatible with widefield and light sheet microscopy

Abstract

FEP-based microfluidics enable diverse applications in light sheet microscopy.

A vascularized breast cancer spheroid platform for the ranked evaluation of tumor microenvironment-targeted drugs by light sheet fluorescence microscopy

“…Robotic handling of hydrogel-embedded, cleared VTSs seems conceivable yet challenging, as problems like precise, unsupervised sample positioning in the light sheet need to be overcome. A simpler, less expensive solution might be the use of microfluidic systems to transport the processed VTSs, one after another, into the imaging window of the LSFM 69 , 70 . However, multichannel image acquisition by LSFM requires the VTS to be precisely kept in place and in the same orientation over several minutes.…”

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

“…LS illumination 2,7,14,15 , where the sample is illuminated with a thin sheet of light at the image plane, has emerged as a simple and effective method of reducing fluorescence background, phototoxicity, and photobleaching of fluorophores. Several LS approaches involving the use of separate objectives for illumination and detection have been designed for single-molecule imaging in cells, however, they suffer from optical complexity, low numerical aperture (NA) objectives, steric hindrance, the inability to optically section adherent cells, incompatibility with microfluidic systems, or drift between the objectives 14,[16][17][18][19][20][21][22][23][24][25][26][27] . 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 .…”

“…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.…”