Multimodal and Label-Free Super-Resolution Imaging By Means of Pump-Probe Microscopy

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Multiparameter and Multimodal Imaging for High Dimensional Data Acquisition
Behjat Sadat Kariman (1, 2), Marco Scotto (1), Alberto Diaspro (1, 2), Paolo Bianchini (1)
1. Nanoscopy and [email protected], CHT Erzelli, Istituto Italiano Tecnologia, Genova, Italy
2. Department of Physics, University of Genova, Genova, Italy

Pump-probe, non-linear optical microscopy, label-free imaging, 2PE, nanoscopy.

Abstract text

In recent decades, the development of ultrashort pulse laser sources has led non-linear optical (NLO) light-matter interaction to acquire a central role in 3D label-free imaging due to efficient signal generation in the non-linear process [1-2]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 [2]. Non-linear interaction comprises the generation of sum frequencies, high-harmonic generation (SHG/ THG), Raman scattering, two-photon fluorescence, and others [1]. Transient absorption, also known as pump-probe microscopy, is a non-linear optical imaging technique that can probe the dynamics of the excited states [2]. Two types of non-linear interaction, including transient absorption and stimulated Raman scattering (SRS), can be exploited with a pump-probe approach to provide novel contrast mechanisms in non-fluorescent species [2]. Utilizing the combination of ground-state depletion with saturation can help in reaching high- and super-resolution label-free microscopy. Studying complex biological systems typically demands high speed, sensitivity, spectral and multi-contrast capability, and spatial and temporal resolution [2-3]. In this context, we aim to develop a multimodal microscope by combining conceptions of non-linear processes and multiphoton techniques to obtain multi-contrast imaging. In this work, we perform super-resolution label-free imaging using our custom-built near-infrared pump-probe microscope. Three 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 [4] and SHG, SFG, and stimulated Raman scattering processes to achieve label-free imaging at an improved spatial resolution and enhanced information content.


[1] C.J.R. Sheppard. "Multiphoton Microscopy: a personal historical review with some future prediction," Biomed. Opt, (2020).

[2] V. Astratov. "Label-Free Super-Resolution Microscopy," Springer Nature, (2019).

[3] K.P. Herdzik et al. "Multimodal Spectral Focusing CARS and SFG Microscopy with a tailored Coherent Continuum from a microstructured fiber, " Applied Physics B, (2020).

[4] G.Zanini et al. "Label-Free Optical Nanoscopy of Single-Layer Graphene," ACS Nano, (2019).