Low-cost Custom Fabrication and Mode-locked Operation of an All-normal-dispersion Femtosecond Fiber Laser for Multiphoton Microscopy
November 22nd, 2019
A method is presented to build a custom low-cost, mode-locked femtosecond fiber laser for potential applications in multiphoton microscopy, endoscopy, and photomedicine. This laser is built using commercially available parts and basic splicing techniques.
Femtosecond pulse lasers have broad applications in multiphoton miscroscopy. This protocol can be used to fabricate a femtosecond all-normal dispersion fiber laser that is compact, robust, and inexpensive. Compared with commercial solid-state ultrafast lasers, the laser produced in this technique costs much less because it consists of only commercially available parts.
Also, fiber lasers do not need water cooling, so the size of the system is smaller. Last but not least, the fiber components do not require alignment, which makes the system robust to vibration. Unlike commercially available systems, this laser does not have a cover to block unwanted beams.
Experienced personnel are needed to assemble and operate the laser. Some experiments appear to be unreproducable because it is very likely to miss some uncalled details when following written instructions. In video demonstration, the viewers won't miss anything.
Start by splicing single-mode fibers, or SMFs, in order to ensure proper performance of the splicing equipment before more valuable fiber optic materials are used. Strip approximately 30 millimeters of the fiber with a fiber-stripping tool. If working with fragile fibers, a razor blade can be used to carefully peel off the buffer.
Use a lint-free tissue with ethanol or isopropanol to clean the stripped fiber. A buzzing sound while wiping indicates that the fiber is sufficiently clean. Then, place the fiber holder on the fiber cleaver and make sure that the blade, fiber clamp of the cleaver, and the fiber holder are all clean.
Carefully load the fiber into the fiber holder leaving approximately 25 millimeters of stripped clean fiber at the free end of the cleaver to clamp. Gently close the fiber clamp on the cleaver. To avoid excess tension on the fiber, reopen and close the clamp.
Press the Cut button and the cleaver will automatically cut the fiber. Use tweezers with plastic-rounded tips to move the piece cut out from the fiber to a sharps disposal container and transfer the fiber holder to the fusion splicer. Repeat the procedure to cleave the second fiber.
The two fibers to be spliced together should have cleaved ends opposing each other by the fiber holders within the fiber splicer. Close the cover of the splicer and set parameters such as Core Diameter, Mode Field Diameter, and Cladding Diameter. Set the alignment method to Cladding, press the Set button, and the splicer will align automatically.
Press the Set button at every stop to confirm the quality of the alignment. The splice will be done automatically. Check the quality of the splice using the quality controls of the splicer as well as the camera view of the region.
A good splice has a uniform cladding boundary and uniform brightness along the fiber such that no splice juncture is visible. Then, open the splicer cover and one of the fiber holders. Optionally, a fiber sleeve may be added to protect the splice and the heater of the splicer can be used to mold the sleeve onto the fiber.
Splice the combiner output to the ytterbium-doped active fiber. Follow the previously described procedure to cleave the combiner output fiber. Due to the shape of its cladding, the active fiber should first be cleaved and spliced with a piece of single-mode fiber which will later be removed.
Cut the single-mode fiber about two centimeters from the splicing point with a wirecutter. Then, strip the entire length of the single-mode fiber and 0.5 centimeters of the active fiber which will leave the active fiber capped with two centimeters of bufferless single-mode fiber. Load the active fiber into the cleaver making sure that only the single-mode fiber is clamped by the fiber clamp.
From this point on, follow the previously described procedure for cleaving and splicing the fiber. Safety is the top priority. Remember to put every piece of fiber fragment in the sharp object box.
Also, laser safety goggles should be warn whenever the pump is operating. Turn on the oscilloscope and set the instrument to AC coupling mode with the trigger level set to 30 millivolts. Move the optical spectrum analyzer photodiode input fiber to monochromatic input and set the device to OSA mode.
Then, lock the phase of the laser by adjusting the wave plates. Rotate Quarter-Wave Plate 2 several degrees back and forth. The mode-locking spectrum consists of two stable peaks with a plateau between them.
Meanwhile, observe a stable pulse train on the oscilloscope. If the mode-locking spectrum is not observed, rotate Quarter-Wave Plate 1 several degrees in one direction and repeat the previous step. If the spectrum is still not observed, rotate the birefringent filter several degrees and repeat the process.
Mode-locked operation was verified upon completion of fiber laser fabrication. The pulse spectrum output from the laser oscillator was centered near 1070 nanometers with the characteristic cat ear shape which indicates mode-locking as predicted by numerical simulation. As a further diagnostic for mode-locking, the pulse duration and pulse repetition power spectra were measured using the autocorrelator and radio frequency spectrum analyzer respectively.
Pulse durations of 70 femtoseconds were measured. Pulse stability was tested by continuously monitoring the average output power and the pulse spectrum. When the laser setup was mounted on a floating optical table with vibration damping, the power drift was less than 3.5%over 24 hours without active cooling.
After verifying mode-locking, the imaging performance was tested using simple test targets and biological samples. Fluorescence was measured during adjustments of the pulse power which verified that the signal was quadratically dependent on the laser power delivered to the sample plane. Stained and unstained biological specimens were imaged using the custom-built fiber laser.
As an additional verification of two-photon excitation, collected hyperspectral images of multicolor fluorescent microspheres were compared with images taken by linear excitation with commercial diode lasers. Finally, the normalized spectra of green and red beads excited by the diode laser versus the custom FS fiber laser were compared. The free space component can be replaced by corresponding fiber parts which can further increase robustness and mobility.
The all-fiber system can be put on a cart for clinical scenarios. The free space component can be replaced by corresponding fiber parts which can further increase robustness and mobility. The all-fiber system can be put on a cart for clinical scenarios.
The impact of this technology is an open question. We anticipate that it will give researchers new access to femtosecond laser technology and enable them to develop new publications.
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Overview
This article presents a method for constructing a low-cost, mode-locked femtosecond fiber laser, which has potential applications in multiphoton microscopy, endoscopy, and photomedicine. The laser is built using commercially available parts and basic splicing techniques, making it an accessible option for researchers.
Key Study Components
Area of Science
- Neuroscience
- Optics
- Biomedical Engineering
Background
- Femtosecond pulse lasers are essential tools in multiphoton microscopy.
- They offer advantages over traditional solid-state ultrafast lasers, including cost-effectiveness and compactness.
- Fiber lasers eliminate the need for water cooling, reducing system size.
- These lasers are robust against vibration due to the lack of alignment requirements.
Purpose of Study
- To provide a cost-effective alternative to commercial ultrafast lasers.
- To facilitate the use of femtosecond lasers in various biomedical applications.
- To demonstrate the feasibility of building a laser using readily available components.
Methods Used
- Construction of a femtosecond all-normal dispersion fiber laser.
- Utilization of commercially available parts for assembly.
- Application of basic splicing techniques.
- Evaluation of the laser's performance in multiphoton microscopy.
Main Results
- The constructed laser is compact and robust.
- It significantly reduces costs compared to commercial alternatives.
- The system's design allows for easy integration into existing setups.
- Performance tests indicate suitability for multiphoton microscopy applications.
Conclusions
- This method provides a viable option for researchers needing femtosecond lasers.
- The low-cost design encourages broader access to advanced imaging techniques.
- Future work may explore further enhancements and applications in photomedicine.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables biopharma R&D teams to develop compact, low-cost femtosecond laser systems for multiphoton microscopy applications, reducing capital barriers to advanced imaging. By leveraging commercially available fiber components and standard splicing techniques, the approach supports rapid prototyping and deployment in preclinical imaging workflows. The system’s robustness and portability facilitate translational research and potential integration into point-of-care or intraoperative imaging platforms.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables high-resolution imaging of cellular and subcellular structures to validate target engagement and pathway modulation in disease models.
- Operational Value: Provides reproducible pulse characteristics essential for quantitative two-photon excitation and consistent fluorescence readouts.
Screening & Assay Development
- Scientific Value: Supports label-free and fluorescent imaging assays requiring precise temporal focusing for depth-resolved analysis of tissue samples.
- Operational Value: Delivers stable 70 fs pulses at 31 MHz repetition rate, ensuring consistent excitation efficiency across high-throughput screening plates.
Translational & Preclinical Research
- Scientific Value: Enables imaging of fresh tissue specimens and intravital models with minimal photodamage due to low average power requirements.
- Operational Value: The all-fiber design allows integration into handheld or endoscopic probes for minimally invasive preclinical imaging.
Pipeline & Workflow Integration
The laser system fits within the discovery continuum from target validation through preclinical imaging, where reliable pulse delivery is critical for mechanistic readouts and biomarker detection.
- Discovery Biology: Supports hypothesis testing via multiphoton imaging of protein localization, calcium flux, and metabolic activity in live cells.
- Screening: Enables assay readiness through consistent pulse energy and spectral stability, reducing variability in fluorescence-based readouts.
- Analytics: Provides quantitative outputs including pulse duration, spectrum, and power stability for correlating laser parameters with biological responses.
- Translational Research: Connects to preclinical continuity by enabling imaging of ex vivo tissues and small animal models with subcellular resolution.
- Enterprise Reuse: The modular, all-fiber architecture allows reuse across multiple imaging platforms and adaptation to different wavelength ranges via fiber components.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in imaging data through verified mode-locked operation and quadratic power dependence of two-photon signals.
- Operational Value: Standardization via commercially available components and documented splicing procedures reduces setup variability between labs.
- Strategic Value: Reduces late-stage biological risk by enabling early validation of drug effects in intact tissue systems using clinically relevant imaging modalities.
- Portfolio Impact: Supports risk-adjusted advancement by providing mechanistic imaging data to inform go/no-go decisions in lead optimization.
Implementation Considerations
- Expertise in fiber optic splicing and laser safety protocols is required for assembly and operation.
- Instrumentation needs include optical spectrum analyzers, autocorrelators, and RF spectrum analyzers for pulse characterization.
- Cross-team standardization requires training on mode-locking verification techniques to distinguish true from noise-like operation.
- Adaptation considerations include replacing free-space components with fiber-delivered versions to enhance robustness for clinical or industrial environments.
- Practical limitations include the need for careful handling of fiber fragments and alignment sensitivity during initial cavity setup.
