Two-frequency CARS imaging by switching fiber laser excitation

This is the peer reviewed version of the following article: Rentchler EC, Xie R, Hui R, JohnsonCK. Two-frequency CARS imaging by switching fiber laser excitation. Microsc Res Tech. 2018;81:413–418. https://doi.org/10. 1002/jemt.22993, which has been published in final form at https://doi.org/10.1002/jemt.22993. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.To fully exploit the power of coherent Raman imaging, techniques are needed to image more than one vibrational frequency simultaneously. We describe a method for switching between two vibrational frequencies based on a single fiber‐laser source. Stokes pulses were generated by soliton self‐frequency shifting in a photonic crystal fiber. Pump and Stokes pulses were stretched to enhance vibrational resolution by spectral focusing. Stokes pulses were switched between two wavelengths on the millisecond time scale by a liquid‐crystal retarder. Proof‐of‐principle is demonstrated by coherent anti‐Stokes Raman imaging of polystyrene beads embedded in a poly(methyl methacrylate) (PMMA) matrix. The Stokes shift was switched between 3,050 cm−1, where polystyrene has a Raman transition, and 2,950 cm−1, where both polystyrene and PMMA have Raman resonances. The method can be extended to multiple vibrational modes

Real-time nanomicroscopy via three-dimensional single-particle tracking.

We developed a new method for real-time, three-dimensional tracking of fluorescent particles. The instrument is based on a laser-scanning confocal microscope where the focus of the laser beam is scanned or orbited around the particle. Two confocal pinholes are used to simultaneously monitor regions immediately above and below the particle and a feedback loop is used to keep the orbit centered on the particle. For moderate count rates, this system can track particles with 15 nm spatial resolution in the lateral dimensions and 50 nm in the axial dimension at a temporal resolution of 32 ms. To investigate the interaction of the tracked particles with cellular components, we have combined our orbital tracking microscope with a dual-color, wide-field setup. Dual-color fluorescence wide-field images are recorded simultaneously in the same image plane as the particle being tracked. The functionality of the system was demonstrated by tracking fluorescent-labeled artificial viruses in tubulin-eGFP expressing HUH7 cells. The resulting trajectories can be used to investigate the microtubule network with super resolution

Real-time nanomicroscopy via three-dimensional single-particle tracking.

We developed a new method for real-time, three-dimensional tracking of fluorescent particles. The instrument is based on a laser-scanning confocal microscope where the focus of the laser beam is scanned or orbited around the particle. Two confocal pinholes are used to simultaneously monitor regions immediately above and below the particle and a feedback loop is used to keep the orbit centered on the particle. For moderate count rates, this system can track particles with 15 nm spatial resolution in the lateral dimensions and 50 nm in the axial dimension at a temporal resolution of 32 ms. To investigate the interaction of the tracked particles with cellular components, we have combined our orbital tracking microscope with a dual-color, wide-field setup. Dual-color fluorescence wide-field images are recorded simultaneously in the same image plane as the particle being tracked. The functionality of the system was demonstrated by tracking fluorescent-labeled artificial viruses in tubulin-eGFP expressing HUH7 cells. The resulting trajectories can be used to investigate the microtubule network with super resolution

Triple-frequency symmetric subtraction scheme for nonresonant background suppression in coherent anti-Stokes Raman scattering (CARS) microscopy

10.1364/OE.18.015714Optics Express181515714-1572