Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser

At a Glance

Metadata	Details
Publication Date	2017-06-01
Journal	Light Science & Applications
Authors	Evan P. Perillo, Jeremy W. Jarrett, Yen‐Liang Liu, Ahmed M. Hassan, Daniel C. Fernée
Institutions	The University of Texas at Austin, Macquarie University
Citations	64
Analysis	Full AI Review Included

Technical Documentation & Analysis: Diamond Raman Laser for Deep Multiphoton Imaging

Executive Summary

This research demonstrates a significant advancement in deep-tissue multiphoton microscopy using a low-cost, high-performance femtosecond diamond Raman laser (DRL), directly applicable to 6CCVD’s core optical diamond market.

Low-Cost Ultrashort Excitation: Details the construction of a custom, synchronized dual-output laser system utilizing a CVD diamond crystal, achieving performance comparable to expensive commercial Optical Parametric Oscillators (OPOs) at approximately one-tenth the cost.
Broadened Spectral Coverage: The system utilizes three effective wavelengths (1055 nm, 1240 nm, and effective 1140 nm 2C2P) to achieve near-complete spectral coverage for far-red fluorophores (1000-1300 nm).
Deep In Vivo Imaging: The longer excitation wavelengths (up to 1240 nm) enabled deep-tissue imaging, demonstrating penetration depths of nearly 1 mm (960 µm) in the cortical vasculature of a live mouse, representing a 20% depth increase over single-color 1055 nm excitation.
High Efficiency for Far-Red Probes: Two-color two-photon (2C2P) excitation at 1140 nm provides an average 90% increase in signal strength for desirable far-red fluorescent proteins (e.g., tdKatushka2) compared to standard 1055 nm excitation.
Novel Excitation Mode: The first reported demonstration of two-color three-photon (2C3P) excitation microscopy, opening new avenues for enhanced contrast, reduced background noise, and super-resolution studies.
Material Criticality: Success relies on a high-quality, <111> oriented, CVD-grown diamond crystal optimized for Stimulated Raman Scattering (SRS) efficiency and low dispersion.

Technical Specifications

The table below summarizes the critical performance parameters and material specifications derived from the experimental setup.

Parameter	Value	Unit	Context
Raman Gain Material	CVD Diamond	8 mm long	Required for Stimulated Raman Scattering (SRS)
Crystal Orientation	<111>	N/A	Aligned for optimal horizontal pump polarization gain
Pump Wavelength ($\lambda$₁)	1055	nm	Ytterbium fiber amplifier output
Stokes Wavelength ($\lambda$₂)	1240	nm	Diamond Raman Laser output
Effective 2C2P Wavelength ($\lambda$₃)	1140	nm	Calculated two-color excitation peak
Excitation Range	1000 - 1300	nm	Comprehensive coverage for far-red fluorophores
Pump Output Power	>500	mW	Minimum required power at 1055 nm (up to 3 W raw)
Stokes Output Power	>300	mW	Minimum required power at 1240 nm
Pump Pulse Width ($\lambda$₁)	120	fs	Measured minimum pulse-width at objective focus
Stokes Pulse Width ($\lambda$₂)	100	fs	Compressed pulse-width (closer to transform limit)
Effective 2C2P Pulse Width	220	fs	Full-width-half-max (FWHM) cross-correlation
Deep Imaging Depth (2C2P)	960 (1)	µm (mm)	In vivo depth achieved in mouse cortex
2C3P Power Dependence	3.04 $\pm$ 0.03	N/A	Confirms three-photon absorption process

Key Methodologies

The two-color excitation system integrates a custom-built ytterbium fiber amplifier with a diamond Raman laser, requiring precise control over material selection, optical alignment, and pulse synchronization.

DRL Material and Cavity Setup

Diamond Selection: A Chemical Vapor Deposition (CVD) grown diamond crystal, 8 mm in length, was used as the Raman gain medium.
Crystal Alignment: The <111> crystal axis was precisely aligned with the horizontal pump polarization to maximize stimulated Raman gain.
Optical Coating: The diamond was custom Anti-Reflection (AR) coated specifically for the Stokes emission wavelength (1240 nm).
Cavity Configuration: A passive, compact ring cavity design ($<1$ m footprint) was implemented, utilizing curved mirrors (200 mm radius of curvature) to focus the pump light into the diamond.

Laser Pumping and Synchronization

Pump Source: A high-power Yb³⁺ fiber amplifier (3 W output, 1055 nm) was selected to provide femtosecond pulses (120 fs) to the DRL.
Mode Matching: The pump light was expanded and focused to achieve a $20$ µm mode-radius within the diamond center.
Phase Locking: A high-precision flexure stage with integrated piezo drive allowed matching the cavity round trip time to the pump repetition rate (80 MHz) for optimal overlap between pump and Stokes pulses.
Pulse Compression: Output Stokes pulses (initially 400 fs due to diamond dispersion) were externally compressed down to 100 fs using a pair of equilateral prisms (P1, P2).

Imaging and Characterization

Pulse Characterization: In situ autocorrelation, performed through the microscope objective (25$\times$, 1.0 NA), was used to measure and optimize the exact pulse-width at the focal plane for both the 1055 nm and 1240 nm beams.
Two-Color Excitation: 2C2P excitation was achieved by spatiotemporal overlap of the synchronized 1055 nm ($\lambda$₁) and 1240 nm ($\lambda$₂) beams via a shortpass dichroic mirror (DM1, edge 1180 nm) and a motorized delay line ($\tau$).
Non-Linear Observation: Two-color three-photon (2C3P) excitation was confirmed for Hoechst 33342 staining by measuring a power dependence slope of $3.04$ on a log-log scale.

6CCVD Solutions & Capabilities

This research validates the use of high-quality CVD diamond as the core component for generating low-cost, high-power femtosecond pulses vital for next-generation deep-tissue imaging. 6CCVD is uniquely positioned to supply the materials required to replicate and advance this research.

Applicable Materials

The foundation of this system is the high-purity, low-loss, orientation-specific diamond crystal.

Optical Grade Single Crystal Diamond (SCD): To replicate the highly efficient Stimulated Raman Scattering (SRS) demonstrated, high-purity SCD is essential. 6CCVD offers optical-grade SCD, minimizing nitrogen defects and ensuring low absorption in the NIR excitation window (1000-1300 nm).
Custom Crystal Orientation: The research emphasizes the need for $<111>$ crystal orientation for maximized Raman gain efficiency. 6CCVD provides custom crystal orientations, guaranteeing optimal performance for specialized non-linear optical applications.
Substrates and Thickness: While the paper used 8 mm long material, future scaling requires thicker or larger plates. 6CCVD supplies SCD materials up to 500 µm thickness and substrates up to 10 mm, suitable for high-power thermal management and cavity integration.

Customization Potential

The experimental success relies on precise dimensions and specific optical coatings—areas where 6CCVD excels.

Requirement in Paper	6CCVD Customization Service	Value Proposition
Specific Dimensions (8 mm length)	Custom Plate/Wafer Dimensions	We offer precision laser cutting and shaping of SCD and PCD plates up to 125 mm, allowing for exact cavity length requirements.
1240 nm AR-coating	Custom Optical Metalization	6CCVD provides in-house metalization and custom optical coatings optimized for specific NIR wavelengths (e.g., 1240 nm) and broad spectral ranges.
Ultra-Low Surface Roughness	High-Precision Polishing	We guarantee ultra-low surface roughness (Ra < 1 nm for SCD and < 5 nm for inch-size PCD), minimizing scattering losses crucial for deep tissue imaging and DRL cavity stability.
Heat Dissipation / Mounting	Metalization Layers	Metalization services (Au, Pt, Pd, Ti, W, Cu) can be applied for robust mounting, electrical contacts, and enhanced thermal management critical for high-power fs laser components.

Engineering Support

This work demonstrates the viability of diamond SRS for complex non-linear optics, including 2C2P and 2C3P microscopy.

Expert Material Consultation: 6CCVD’s in-house PhD team can assist researchers and technical engineers with material selection for similar ultrafast laser development or deep multiphoton imaging projects, ensuring the SCD properties (orientation, defect density, polishing grade) are optimized for Stimulated Raman Scattering (SRS) efficiency and minimizing Third-Order Dispersion (TOD).
Application Advancement: We support the development of next-generation, compact, and low-cost laser sources that leverage diamond’s superior thermal conductivity and broad Raman shift capabilities compared to traditional OPO systems.

For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.

View Original Abstract

Two-color multiphoton microscopy through wavelength mixing of synchronized lasers has been shown to increase the spectral window of excitable fluorophores without the need for wavelength tuning. However, most currently available dual output laser sources rely on the costly and complicated optical parametric generation approach. In this report, we detail a relatively simple and low cost diamond Raman laser pumped by a ytterbium fiber amplifier emitting at 1055 nm, which generates a first Stokes emission centered at 1240 nm with a pulse width of 100 fs. The two excitation wavelengths of 1055 and 1240 nm, along with the effective two-color excitation wavelength of 1140 nm, provide an almost complete coverage of fluorophores excitable within the range of 1000-1300 nm. When compared with 1055 nm excitation, two-color excitation at 1140 nm offers a 90% increase in signal for many far-red emitting fluorescent proteins (for example, tdKatushka2). We demonstrate multicolor imaging of tdKa-tushka2 and Hoechst 33342 via simultaneous two-color two-photon, and two-color three-photon microscopy in engineered 3D multicellular spheroids. We further discuss potential benefits and applications for two-color three-photon excitation. In addition, we show that this laser system is capable of <i>in vivo</i> imaging in mouse cortex to nearly 1 mm in depth with two-color excitation.

Tech Support

Original Source

DOI: https://doi.org/10.1038/lsa.2017.95