Resolution enhancement of 2-photon microscopy using high-refractive index microspheres

Abstract

Intravital microscopy using multiphoton processes is the standard tool for deep tissue imaging inside of biological specimens. Usually, near-infrared and infrared light is used to excite the sample, which enables imaging several mean free path inside a scattering tissues. Using longer wavelengths, however, increases the width of the effective multiphoton Point Spread Function (PSF). Many features inside of cells and tissues are smaller than the diffraction limit, and therefore not possible to distinguish using a large PSF. Microscopy using high refractive index microspheres has shown promise to increase the numerical aperture of an imaging system and enhance the resolution. It has been shown that microspheres can image features ~λ/7 using single photon process fluorescence. In this work, we investigate resolution enhancement for Second Harmonic Generation (SHG) and 2-photon fluorescence microscopy. We used Barium Titanate glass microspheres with diameters ∼20–30 μm and refractive index ∼1.9–2.1. We show microsphere-assisted SHG imaging in bone collagen fibers. Since bone is a very dense tissue constructed of bundles of collagen fibers, it is nontrivial to image individual fibers. We placed microspheres on a dense area of the mouse cranial bone, and achieved imaging of individual fibers. We found that microsphere assisted SHG imaging resolves features of the bone fibers that are not readily visible in conventional SHG imaging. We extended this work to 2-photon microscopy of mitochondria in mouse soleus muscle, and with the help of microsphere resolving power, we were able to trace individual mitochondrion from their ensemble.