FYLA laser for nonlinear optical microscopy

Temporally coherent broadband source, as this FYLA laser Cyclone, constitute an attractive alternative to Titanium–sapphire Lasers. We present a monolithic fiber optic configuration for generating transform-limited temporally coherent pulses and duration as short as 13.0 fs (3.7 optical cycles).
The supercontinuum light is generated by the action of self-phase modulation and optical wave breaking when pumping an all-normal dispersion photonic crystal fiber with pulses of hundreds of fs duration produced by all-fiber chirped pulsed amplification. Avoidance of free-space propagation between stages confers unequalled robustness, efficiency, and cost-effectiveness to this novel configuration. Collectively, the features of all-fiber few-cycle pulsed sources make them powerful tools for applications benefitting from the ultra-broadband spectra and ultra-short pulse durations. Here we exploit these features and the deep penetration of light in biological tissues at the spectral region of 1 mm, to demonstrate the successful performance of Cyclone a FYLA Laser in ultra-broadband multispectral and multimodal no-linear microscopy.


Why is Cyclone the perfect laser for nonlinear microscopy?

Cyclone, the FYLA ultra-broadband femtosecond fiber laser, appears as an alternative to Ti:Sa oscillators and OPCPA systems used for the most relevant Nonlinear optics (NLO) microscopy techniques, multiphoton excited fluorescence (MPEF) and second harmonic generation (SHG).
FYLA femtosecond laser is compact, air-cooled, turn-key, cost-effective and maintenance and alignment-free.
The two technical specifications of this FYLA all-fiber broadband source that makes it perfect for nonlinear microscopy techniques are:

  • The FYLA few-cycle source, delivers pulses with durations less than 20 fs that increase the efficiency of the excited nonlinear effects by an order of magnitude. Besides, the spectral composition of few-cycle pulses is extremely broad (bandwidths typically > 200 nm for sources operating in the near-IR), further enabling multispectral (simultaneous, if required) NLO microscopy.
  • Cyclone delivers femtosecond pulses. This is extremely important as the best trade-off between high photon irradiance and harmless average power levels is offered by lasers delivering pulses with durations in the femtosecond range. Multimodal NLO microscopy usually combines MPEF and SHG for full exploitation of the advantages of both techniques and these advantages rely on the excitation of the samples by laser pulses that provide very high photon irradiances (typically > 10 27 photons s − 1 cm− 2 ), to increase the probability of the rare event of simultaneous absorption of more than one photon by the sample.

The results of using Cyclone FYLA laser for nonlinear microscopy techniques

To analyse the efficiency of the FYLA laser we asked Marina Cunquero, from ICFO Institute of Photonic Sciences, in charge of the microscope tests.

Penetration depth assessment
Using the 25x objective under the optimised GVD settings, we have successfully imaged several samples. Importantly, fluorescence signal and depth were achieved using ~4mW of laser power (measured at the sample plane). The maximum penetration achieved corresponds to 220 µm (Figure 3) in depth of the tail of a transgenic line zebrafish embryo (Caax-GFP) expressing GFP in all cell membranes. Zebrafish embryos are transparent, so they allow imaging at this large penetration depths.


Figure 3: TPEF images of the tail of a 2-days-old transgenic line zebrafish embryo (Caax-GFP) expressing GFP in all cell membranes. (A-C) Intensity-normalised images corresponding to 26, 71, 150 µm depth. (D) the complete resliced image of a Z-stack composed of 300 images (0.71 µm step spacing). Scalebar: (A-C) 40 µm; (D) 20 µm.

To test the penetration capabilities of the laser within a scattering tissue, we proceed to image the full retina of a rat (~170 µm) with cellular resolution. Figure 4 shows the comparison of the resliced TPEF images acquired with Cyclone FYLA laser (Δλ=200nm, centred at 1060nm) and Coherent MIRA 900 laser (Δλ=10nm, centred at 810nm). Both excised rat retinas were stained with either Alexa Fluor 647-phalloidin and Alexa Fluor 405-phalloidin, being the first one to be excited with the FYLA system and while the second one with the Coherent MIRA 900.


Figure 4: Comparison of SCH FYLA, now called Cyclone, and Coherent MIRA 900 laser for TPEF imaging of an excised rat retina (retinal ganglion cells side up) stained with Alexa Fluor 647-phalloidin and Alexa Fluor 405-phalloidin, respectively. (A) Reslice of 376 images (0.52 µm step spacing) acquired with FYLA laser. (B) Reslice of 404 images (0.50 µm step spacing) acquired with Coherent MIRA 900 laser. Scalebar: 15 µm.

In both cases, the stains were used to visualize the actin of the cytoskeleton of the retinal neurons. We used same laser powers and similar step spacing for constructing the z-stacks. Images were treated in the same way for posterior comparison.
In the image acquired with FYLA system we clearly distinguish the synaptic (bright regions) and nuclear (gap regions) layers that characterize the tissue. It is interesting to mention that the rat retina is highly autofluorescent when illuminated with light in the blue-green spectrum.
In addition, the external segment of the photoreceptor cells where opsins (photopigments) are packaged, is highly absorbent to visible light. Therefore, illumination sources in the IR spectrum combined with red fluorescent dyes are ideal for depth imaging to prevent the autofluorescence generation/distorsions in this tissue. In particular, the FYLA laser resulted in a highly efficient system to image these type of samples.
The simplicity, robustness, and cost-effectiveness of FYLA Laser configuration are powerful factors in favour of this technology to replace traditional solid-state sources of few-cycle pulses in various applications.
For more information, please check the complete research Few-cycle all-fibre supercontinuum laser for ultrabroadband multimodal nonlinear microscopy.