Volumetric chemical imaging in vivo by a remote-focusing stimulated Raman scattering microscope.

Hongli Ni,Yuying Tan,Ruyi Gong,Thomas Bifano,Ji-Xin Cheng,Peng Lin,Nicholas A Vickers,Huate Li

doi:10.1364/oe.404869

Abstract

Operable under ambient light and providing chemical selectivity, stimulated Raman scattering (SRS) microscopy opens a new window for imaging molecular events on a human subject, such as filtration of topical drugs through the skin. A typical approach for volumetric SRS imaging is through piezo scanning of an objective lens, which often disturbs the sample and offers a low axial scan rate. To address these challenges, we have developed a deformable mirror-based remote-focusing SRS microscope, which not only enables high-quality volumetric chemical imaging without mechanical scanning of the objective but also corrects the system aberrations simultaneously. Using the remote-focusing SRS microscope, we performed volumetric chemical imaging of living cells and captured in real time the dynamic diffusion of topical chemicals into human sweat pores.

Highlights

Volumetric chemical imaging through an optical microscope can visualize molecular distributions and dynamic processes in a 3-dimensional (3-D) specimen with sub-micrometer resolution, which facilitates the study of cellular functions, developments, and activities in living organisms or tissues [1,2]
The deformable mirror (DM) surface was set to an initial pattern (Ai) to obtain an initial value (Qi) by imaging 1-μm polymethyl methacrylate (PMMA) beads
We fitted the three test values versus their current DM patterns ((Qi+, Ai+αWi), (Qi, Ai), (Qi−, Ai+αWi)) with a quadratic function to find a local maxima value corresponding to its DM pattern, named Ai+1, which will be used as the initial pattern to the iteration of optimization

Summary

Introduction

Volumetric chemical imaging through an optical microscope can visualize molecular distributions and dynamic processes in a 3-dimensional (3-D) specimen with sub-micrometer resolution, which facilitates the study of cellular functions, developments, and activities in living organisms or tissues [1,2]. To image the molecules of interest, fluorescence microscopy is often employed to detect the fluorescent markers labeling specific molecules [5]. The exogenous markers often perturb the functionalities of molecules, and most of them are not allowed for human study [1,2,5,6]. Infrared spectroscopic imaging is label-free but significantly suffers from water absorption in living organisms and offers limited 3-D imaging capacity [7]. Nonlinear microscopy using second and third harmonics is sensitive to specific structures such as collagen or lipid droplets but provides insufficient chemical contrasts [8]

Methods

Results

Discussion

Conclusion