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

Session

Multiparameter and Multimodal Imaging for High Dimensional Data Acquisition

Authors

Behjat Sadat Kariman (1, 2), Marco Scotto (1), Alberto Diaspro (1, 2), Paolo Bianchini (1)

Affiliations

1. Nanoscopy and NIC@IIT, CHT Erzelli, Istituto Italiano Tecnologia, Genova, Italy
2. Department of Physics, University of Genova, Genova, Italy

Keywords

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.

References

[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).