Interferometric temporal focusing microscopy using three-photon excitation fluorescence.

Keisuke Toda,Hiroyuki Kawano,Keisuke Isobe,Atsushi Miyawaki,Katsumi Midorikawa,Kana Namiki

doi:10.1364/boe.9.001510

Abstract

Super-resolution microscopy has become a powerful tool for biological research. However, its spatial resolution and imaging depth are limited, largely due to background light. Interferometric temporal focusing (ITF) microscopy, which combines structured illumination microscopy and three-photon excitation fluorescence microscopy, can overcome these limitations. Here, we demonstrate ITF microscopy using three-photon excitation fluorescence, which has a spatial resolution of 106 nm at an imaging depth of 100 µm with an excitation wavelength of 1060 nm.

Highlights

Various types of fluorescence microscopies, such as super-resolution microscopy and multiphoton excitation fluorescence microscopy have become powerful tools for investigating biological phenomena
3.1 Optical sectioning capability To estimate the optical sectioning capability of 3PEF-Interferometric temporal focusing (ITF) microscopy, we measured the signal distribution from one layer of 100-nm fluorescent beads (F8797, Molecular Probes) along the axial direction
It was confirmed that the optical sectioning capability of 3PEF-ITF microscopy was 0.75 times the wavelength of the excitation light (1060 nm)

Summary

Introduction

Various types of fluorescence microscopies, such as super-resolution microscopy and multiphoton excitation fluorescence microscopy have become powerful tools for investigating biological phenomena. With these techniques, there are trade-offs among the spatial resolution, penetration depth, temporal resolution, and imaging area. Superresolution techniques combined with two-photon excitation fluorescence (2PEF) microscopy have led to super-resolution deep imaging [10,11,12,13,14,15,16]. Stimulated emission depletion (STED) microscopy [2], which employs a tightly focused laser as an excitation light source, has been combined with laser scanning 2PEF microscopy [17,18] to provide super-resolution deep imaging [10,11,12]. On the other hand, structured illumination microscopy (SIM) [7,8,9] has been coupled with 2PEF temporal focusing (TF) microscopy [16], which allows depthresolved wide-field two-photon imaging without laser scanning [19,20]

Methods

Results

Conclusion