Correcting for objective sample refractive index mismatch in extended field of view selective plane illumination microscopy

TL;DR

This paper presents a computational model to analyze and correct for refractive index mismatch effects in extended field of view SPIM, improving image quality in large sample imaging.

Contribution

We developed and validated a computational model combining ray tracing and field propagation to optimize sample chamber positioning in remote focusing SPIM.

Findings

Optimizing sample chamber position reduces aberrations caused by refractive index mismatch.

Model predictions align with experimental light sheet profiles.

Proper alignment enhances image quality in large sample SPIM.

Abstract

Selective plane illumination microscopy (SPIM) is an optical sectioning imaging approach based on orthogonal light pathways for excitation and detection. The excitation pathway has an inverse relation between the optical sectioning strength and the effective field of view (FOV). Multiple approaches exist to extend the effective FOV, and here we focus on remote focusing to axially scan the light sheet, synchronized with a CMOS camera's rolling shutter. A typical axially scanned SPIM configuration for imaging large samples utilizes a tunable optic for remote focusing, paired with air objectives focused into higher refractive index media. To quantitatively explore the effect of remote focus choices and sample space refractive index mismatch on light sheet intensity distributions, we developed a computational model integrating ray tracing and field propagation. We validate our model's performance against experimental light sheet profiles for various SPIM configurations. Our findings indicate that optimizing the position of the sample chamber relative to the excitation optics can enhance image quality by balancing aberrations induced by refractive index mismatch. We validate this prediction using a homebuilt, large sample axially scanned SPIM configuration and calibration samples.