Modified inverted selective plane illumination microscopy for sub-micrometer imaging resolution in polydimethylsiloxane soft lithography devices

Moldable, transparent polydimethylsiloxane (PDMS) elastomer microdevices enable a broad range of complex studies of three-dimensional cellular networks in their microenvironment in vitro. However, the uneven distribution of refractive index change, external to PDMS devices and internally in the sample chamber, creates a significant optical path difference (OPD) that distorts the light sheet beam and so restricts diffraction limited performance. We experimentally showed that an OPD of 120 mu m results in the broadening of the lateral point spread function by over 4-fold. In this paper, we demonstrate steps to adapt a commercial inverted selective plane illumination microscope (iSPIM) and remove the OPD so as to achieve sub-micrometer imaging ranging from 0.6 +/- 0.04 mu m to 0.91 +/- 0.03 mu m of a fluorescence biological sample suspended in regular saline (RI approximate to 1.34) enclosed in 1.2 to 2 mm thick micromolded PDMS microdevices. We have proven that the removal of the OPD from the external PDMS layer by refractive index (RI) matching with a readily accessible, inexpensive sucrose solution is critical to achieve a >3-fold imaging resolution improvement. To monitor the RI matching process, a single-mode fiber (SMF) illuminator was integrated into the iSPIM. To remove the OPD inside the PDMS channel, we used an electrically tunable lens (ETL) that par-focuses the light sheet beam with the detection objective lens and so minimised axial distortions to attain sub-micrometer imaging resolution. We termed this new light sheet imaging protocol as modified inverted selective plane illumination microscopy (m-iSPIM). Using the high spatial-temporal 3D imaging of m-iSPIM, we experimentally captured single platelet (approximate to 2 mu m) recruitment to a platelet aggregate (22.5 mu m x 22.5 mu m x 6 mu m) under flow at a 150 mu m depth within a microfluidic channel. m-iSPIM paves the way for the application of light sheet imaging to a wide range of 3D biological models in microfluidic devices which recapitulate features of the physiological microenvironment and elucidate subcellular responses.