Label-Free Super-Resolution Microscopy By Means of Transient Absorption Saturation

Abstract

In recent decades, super-resolution optical microscopy has become an attractive research topic that typically has been developed based on fluorescent labelling that becomes the essential tool for breaking diffraction-limited in biological imaging. However, deep tissue and label-free imaging remain challenging, especially for tick and highly scattering biological specimens [1]. With the development of ultrashort pulse laser sources, non-linear optical (NLO) light-matter interaction has acquired a central role in optical microscopy for label-free imaging due to efficient signal generation in the non-linear process [2]. The advantages of NLO approaches include the reduction of scattering due to the use of near-infrared radiation, which enables high penetration depth and reduces the aberrations introduced by the specimen [1]. Non-linear interaction comprises the generation of sum frequencies, high-harmonic generation (SHG/ THG), Raman scattering, two-photon fluorescence and others [3]. Transient absorption, also known as pump-probe microscopy is a non-linear optical imaging technique that can probe the dynamics of the excited states. [1]. In this work, we perform super-resolution label-free imaging using our custom-built near-infrared pump-probe microscope. Two femtosecond pulsed laser beams, generated by an OPO pumped by a Ti:sapphire laser (Chameleon Ultra II and compact OPO Coherent), are coupled into a commercial laser scanning confocal microscope (Nikon A1 MP). We explore the saturation of transient absorption using a doughnut-shaped beam in the STED-like approach to saturate the absorption [3] and forward SHG and SFG processes to achieve label-free imaging at an improved spatial resolution. [1] Astratov V. (2019). “Label-Free Super-Resolution Microscopy”. Springer Nature.[2] Sheppard. C.J.R. (2020). Multiphoton Microscopy: a personal historical review with some future prediction. Biomed. Opt.[3]Zanini, G. et al. 2019. Label-Free Optical Nanoscopy of Single-Layer Graphene. ACS Nano.