Saturated excitation microscopy using differential excitation for efficient detection of nonlinear fluorescence signals

Yasunori Nawa,Katsumasa Fujita,Ryosuke Oketani,Hitoshi Hashimoto,Atsushi Kasai,Yasuo Yonemaru,Nicholas I Smith

doi:10.1063/1.5039567

Yasunori Nawa, Katsumasa Fujita + Show 5 more

Open Access

https://doi.org/10.1063/1.5039567

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Journal: APL Photonics	Publication Date: Aug 1, 2018
Citations: 19	License type: cc-by

Affiliation: Osaka University, University of Fukui

Abstract

We report a method to increase the efficiency of detecting nonlinear fluorescence signals in saturated excitation (SAX) microscopy. With this method, we compare fluorescence signals obtained under different degrees of saturated excitation to extract the nonlinear fluorescent signal induced by saturated excitation. Compared to conventional SAX microscopy using the harmonic demodulation technique, the detection efficiency of the fluorescence signal can be increased up to 8 and 32 times in imaging using the second-order and the third-order nonlinear fluorescence signals, respectively. We combined this approach with pulsed excitation, which is effective to reduce photobleaching effects, and achieved super-resolution imaging using third-order nonlinear fluorescence signals induced by saturated excitation of an organic dye. The resolution improvement was confirmed in the observations of fluorescent beads, actin-filaments in HeLa cells, and a spine in mouse brain tissue.

Highlights

Optical microscopy is a technique suitable for biological investigation because it enables visualization analysis inside specimens with advantageous features such as non-contact, low damage, and high precision of the submicron order
In saturated excitation (SAX) microscopy, the spatial resolution is enhanced in three dimensions by detecting the nonlinear fluorescence signal induced by saturation, which is localized within the focal spot
Compared to SAX microscopy using harmonic demodulation, the differential excitation technique can detect a larger amount of nonlinear signals that contribute to the resolution improvement

Summary

INTRODUCTION

Optical microscopy is a technique suitable for biological investigation because it enables visualization analysis inside specimens with advantageous features such as non-contact, low damage, and high precision of the submicron order. In the first demonstration of SAX microscopy, the harmonic demodulation technique was utilized to extract the nonlinear fluorescence response during the laser scanning process, which is useful to avoid post-processing of image data but required the installation of a light modulator and a lock-in amplifier. Presented a method to extract the nonlinear signals from fluorescence measurement with differential excitation, where two or more fluorescence images are obtained at different excitation intensities in order to calculate nonlinear signals induced by saturated excitation.[12] Differential-excitation SAX (dSAX) microscopy was demonstrated with two-photon excitation microscopy[11] and with a commercial laser scanning microscope.[13] In this report, we demonstrate that the technique is effective to improve the SNR in SAX microscopy. We examined the technique experimentally and demonstrate its effectiveness in the observation of biological cells and tissues and achieved fluorescence imaging with the 3rd-order nonlinear fluorescence signals induced by saturated excitation of fluorescent dyes

SAX MICROSCOPY USING DIFFERENTIAL EXCITATION

FLUORESCENCE DETECTION PROPERTY

FLUORESCENCE EXCITATION BY PULSED LASER LIGHT

IMPROVEMENT OF RESOLUTION AND SNR

OBSERVATION OF BIOLOGICAL SAMPLES

CONCLUSION