Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination

Keisuke Isobe,Qiyuan Song,Akira Suda,Takanori Takeda,Hiroyuki Kawano,Katsumi Midorikawa,Atsushi Miyawaki,Akiko Kumagai,Kyohei Mochizuki,Fumihiko Kannari

doi:10.1364/boe.4.002396

Abstract

We demonstrate super-resolution imaging with background fluorescence rejection by interferometric temporal focusing microscopy, in which temporal focusing is combined with structured illumination. The lateral resolution and the optical sectioning capability are simultaneously improved by factors of 1.6 and 1.4, respectively, compared to conventional temporal focusing microscopy. Fluorescent beads (200 nm diameter) that are difficult to distinguish from the background fluorescence in conventional temporal focusing microscopy, are clearly visualized by interferometric temporal focusing microscopy.

Highlights

Multiphoton excited fluorescence (MPEF) microscopy has become a powerful tool for investigating biological phenomena because of its inherent advantages, including threedimensional resolution without a confocal pinhole, high penetration depth with near-infrared#196374 - $15.00 USD Received 26 Aug 2013; revised 20 Sep 2013; accepted 23 Sep 2013; published 10 Oct 2013 light excitation, and reduced out-of-focus photon-induced damage and photobleaching [1,2,3]
We demonstrate super-resolution imaging with background fluorescence rejection by interferometric temporal focusing microscopy, in which temporal focusing is combined with structured illumination
We demonstrate super-resolution imaging with background fluorescence suppression by interferometric Temporal focusing (TF) (ITF) microscopy in which the structured illumination technique is combined with TF microscopy

Summary

Introduction

Multiphoton excited fluorescence (MPEF) microscopy has become a powerful tool for investigating biological phenomena because of its inherent advantages, including threedimensional resolution without a confocal pinhole, high penetration depth with near-infrared#196374 - $15.00 USD Received 26 Aug 2013; revised 20 Sep 2013; accepted 23 Sep 2013; published 10 Oct 2013 light excitation, and reduced out-of-focus photon-induced damage and photobleaching [1,2,3]. Keisuke Isobe,1,2,* Takanori Takeda,1,3 Kyohei Mochizuki,1,3 Qiyuan Song,2,4 Akira Suda,3 Fumihiko Kannari,4 Hiroyuki Kawano,5 Akiko Kumagai,5 Atsushi Miyawaki,2,5 and Katsumi Midorikawa1,2 “Isotropic image in structured illumination microscopy patterned with a spatial light modulator,” Opt. Express 17(17), 14710–14721 (2009).

Results

Conclusion