Two-Color, Two-Photon Imaging at Long Excitation Wavelengths Using a Diamond Raman Laser
At a Glance
Section titled âAt a Glanceâ| Metadata | Details |
|---|---|
| Publication Date | 2016-08-01 |
| Journal | Microscopy and Microanalysis |
| Authors | Johanna TrÀgÄrdh, Michelle Murtagh, Gillian Robb, Maddy Parsons, Jipeng Lin |
| Institutions | Kingâs College London, University of Strathclyde |
| Citations | 8 |
| Analysis | Full AI Review Included |
Diamond Raman Laser for Two-Photon Microscopy: Technical Analysis and 6CCVD Solutions
Section titled âDiamond Raman Laser for Two-Photon Microscopy: Technical Analysis and 6CCVD SolutionsâExecutive Summary
Section titled âExecutive SummaryâThis document analyzes the application of a CVD-grown diamond crystal as the core gain medium in a synchronously-pumped Raman laser system designed for advanced two-photon microscopy (TPEF).
- Core Achievement: Successful demonstration of two-color, two-photon imaging using the second-Stokes output from a diamond Raman laser (DRL), significantly extending the usable wavelength range of a standard Ti:Sapphire pump laser.
- Material Requirement: The system relies on a high-quality, CVD-grown Type IIa Single Crystal Diamond (SCD) to facilitate efficient Raman shifting.
- Wavelength Extension: The DRL utilized the diamondâs large Raman shift (1332 cm-1) to generate second-Stokes emission tunable from 1080 nm to 1200 nm.
- Application Impact: This extended wavelength range enables efficient two-photon excitation of critical red-emitting fluorophores (e.g., RFP, Texas Red, Alexa 568), which is crucial for deep-tissue imaging and multi-label experiments.
- Dual-Color Capability: The setup uniquely allows simultaneous dual-color imaging by combining the Raman-shifted output (red excitation) with the residual pump beam (green excitation).
- Performance Metrics: The second-Stokes output achieved powers up to 80 mW with pulse durations of 1.0 ps, confirming suitability for nonlinear biological imaging.
Technical Specifications
Section titled âTechnical SpecificationsâThe following table summarizes the critical material and performance parameters extracted from the research paper concerning the diamond Raman laser system.
| Parameter | Value | Unit | Context |
|---|---|---|---|
| Diamond Material Type | Type IIa, CVD-grown | N/A | Core Raman gain medium |
| Diamond Crystal Length | 9 | mm | Used in the ring cavity |
| Diamond Raman Shift | 1332 | cm-1 | Determines wavelength extension capability |
| AR Coating Reflectivity | 1-3 | % | Over 796-1200 nm range |
| Pump Laser Type | Ti:Sapphire (Chameleon Ultra II) | N/A | Synchronous pumping source |
| Pump Wavelength Range | 840 to 900 | nm | Used for second-Stokes generation |
| Second-Stokes Tuning Range | 1080 to 1200 | nm | Extended TPEF excitation range |
| Second-Stokes Output Power | Up to 80 | mW | Suitable for biological imaging |
| Second-Stokes Pulse Duration | 1.0 | ps | Measured at 840 nm and 890 nm pump |
| Pulse Repetition Frequency | 80 | MHz | System operating frequency |
| Confirmed Process Order | 1.7 ± 0.1 | N/A | Confirms second-order (two-photon) excitation |
Key Methodologies
Section titled âKey MethodologiesâThe following steps outline the critical parameters and procedures used in the construction and operation of the diamond Raman laser for two-photon microscopy:
- Material Specification: A 9 mm-long, Type IIa SCD crystal was utilized. The crystal featured broadband Anti-Reflection (AR) coatings, maintaining 1-3% reflectivity across the 796-1200 nm range.
- Pump Source Configuration: A femtosecond Ti:Sapphire laser generating 200 fs pulses at 80 MHz was used, tuned specifically between 840 nm and 900 nm to drive the second-Stokes emission.
- Polarization Alignment: The pump beam was precisely polarized parallel to the [111] axis of the diamond crystal using a half-wave plate to maximize the Raman gain efficiency.
- Cavity Design: The DRL was configured as a four-mirror ring cavity (M1, M2 concave, M3, M4 plane) with a radius of curvature (ROC) of 200 mm for the concave mirrors.
- Output Coupling Optimization: Mirror M4 served as the output coupler, designed with 30% transmission (T=30%) specifically for the second-Stokes wavelengths (1040-1300 nm) while maintaining high reflectivity (HR) for the first Stokes.
- Wavelength Tuning: Continuous tuning of the second-Stokes output (1080 nm to 1200 nm) was achieved by synchronously tuning the Ti:Sapphire pump laser and adjusting the DRL cavity length via translation of mirror M4.
- Imaging Setup: The DRL output was filtered using a 1000 nm long pass filter (LP) to isolate the Stokes emission from the residual pump light, and then coupled into a multi-photon microscope using high numerical aperture (NA 1.3 or NA 1.4) oil immersion objectives.
6CCVD Solutions & Capabilities
Section titled â6CCVD Solutions & CapabilitiesâThe successful implementation of the Diamond Raman Laser hinges entirely on the availability of high-quality, precisely engineered Single Crystal Diamond (SCD). 6CCVD is an expert supplier of MPCVD diamond materials and customization services necessary to replicate, optimize, and extend this research.
| Research Requirement | 6CCVD Solution & Value Proposition |
|---|---|
| High-Purity Raman Gain Medium | Optical Grade SCD (Single Crystal Diamond): We supply high-purity, low-birefringence Type IIa SCD wafers and plates, essential for high-efficiency nonlinear optics and maximizing the 1332 cm-1 Raman shift. |
| Custom Dimensions & Thickness | Precision Sizing: The paper required a 9 mm-long crystal. 6CCVD offers custom SCD substrates up to 10 mm thick and plates up to 500 ”m thick, cut and polished to exact specifications required for laser cavities. |
| Large Area Polycrystalline Diamond | PCD Wafers up to 125 mm: For scaling DRL technology or integrating diamond heat spreaders in high-power systems, 6CCVD provides large-area Polycrystalline Diamond (PCD) wafers up to 125 mm in diameter. |
| Surface Quality for Low Loss | Ultra-Low Roughness Polishing: Our SCD material is polished to Ra < 1 nm, minimizing scattering losses critical for high-Q laser cavities and maximizing conversion efficiency in Raman applications. |
| Integrated Coatings & Metalization | Custom Metalization Services: We offer in-house metalization (Au, Pt, Ti, W, Cu) and dielectric coating consultation. We can deliver SCD crystals pre-coated to meet specific reflectivity requirements across the NIR/SWIR spectrum (e.g., custom AR coatings for 796-1200 nm). |
| Engineering Support for Optimization | Expert Consultation: 6CCVDâs in-house PhD team can assist researchers in optimizing diamond specifications (purity, orientation, and thickness) to maximize Raman shift efficiency and pulse compression potential for similar nonlinear microscopy and laser projects. |
For custom specifications or material consultation, visit 6ccvd.com or contact our engineering team directly.
View Original Abstract
Abstract We demonstrate that the second-Stokes output from a diamond Raman laser, pumped by a femtosecond Ti:Sapphire laser, can be used to efficiently excite red-emitting dyes by two-photon excitation at 1,080 nm and beyond. We image HeLa cells expressing red fluorescent protein, as well as dyes such as Texas Red and Mitotracker Red. We demonstrate the potential for simultaneous two-color, two-photon imaging with this laser by using the residual pump beam for excitation of a green-emitting dye. We demonstrate this for the combination of Alexa Fluor 488 and Alexa Fluor 568. Because the Raman laser extends the wavelength range of the Ti:Sapphire laser, resulting in a laser system tunable to 680-1,200 nm, it can be used for two-photon excitation of a large variety and combination of dyes.