Femtosecond diode-based time lens laser for multiphoton microscopy.

Y Lange Simmons,Tarah A Welton,Kenneth J Underwood,Brendan M Heffernan,Emily A Gibson,Juliet T Gopinath,Omkar D Supekar

doi:10.1364/boe.430549

Abstract

We demonstrate a near-infrared, femtosecond, diode laser-based source with kW peak power for two-photon microscopy. At a wavelength of 976 nm, the system produces sub-ps pulses operating at a repetition rate of 10 MHz with kilowatt class peak powers suitable for deep tissue two-photon microscopy. The system, integrated with a laser-scanning microscope, images to a depth of 900 µm in a fixed sample of PLP-eGFP labeled mouse brain tissue. This represents a significant development that will lead to more efficient, compact, and accessible laser sources for biomedical imaging.

Highlights

Pulsed lasers in the near-infrared find use in groundbreaking scientific and medical research, including biological imaging [1,2], laser surgery [3], and the study of Alzheimer’s and Parkinson’s disease [4,5,6]
To prove our system is viable for use in two photon microscopy and can achieve results comparable to Ti:sapphire lasers in terms of tissue penetration depth, the system was integrated with a twophoton microscope
The signal to noise ratio is limited by peak power and repetition rate in comparison to a Ti:Sapphire, images taken at depth in tissue are comparable to those acquired with a Ti:Sapphire system (Fig. S2) and clearly demonstrate our system’s utility for multiphoton microscopy

Summary

Introduction

Pulsed lasers in the near-infrared find use in groundbreaking scientific and medical research, including biological imaging [1,2], laser surgery [3], and the study of Alzheimer’s and Parkinson’s disease [4,5,6]. The wavelength of the laser, 976 nm, is near the peak for two-photon response of eGFP, GCaMP, and YFP (requiring labeling or genetic modification) [27] It is a suitable pump wavelength for coherent anti-Stokes Raman Scattering (CARS) microscopy (a label-free process) [28,29]. We demonstrate fluorescent imaging at depths of 900 μm in the neo-cortex

Laser design

Pulse characterization

Two photon microscopy demonstration

Conclusion