Broadband Femtosecond Laser 950-1150nm | SIMTRUM Photonics

Temporally coherent broadband source, as this laser, 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 Laser in ultra-broadband multispectral and multimodal no-linear microscopy.

1.1 Why is Cyclone the perfect laser for nonlinear microscopy?

Cyclone, the 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). Femtosecond laser is compact, air-cooled, turn-key, cost-effective and maintenance and alignment-free.

The two technical specifications of this all-fiber broadband source that makes it perfect for nonlinear microscopy techniques are:

  - The 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.

1.2 The results of using Cyclone laser for nonlinear microscopy techniques

To analyse the efficiency of the 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 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 system and while the second one with the Coherent MIRA 900.

Figure 4: Comparison of SCH, 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 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 the 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 laser resulted in a highly efficient system to image these type of samples.

The simplicity, robustness, and cost-effectiveness of Laser configuration are powerful factors in favour of this technology to replace traditional solid-state sources of few-cycle pulses in various applications.

2. Multiphoton Microscopy I: Increasing the Photon Flux with 15 Femtosecond Pulses

Massively increasing the photon flux with shorter excitation pulses (15 fs)

Two-Photon Excitation Fluorescence (TPEF) microscopy (also known as two-photon microscopy) is the method of choice for deep three-dimensional imaging of living tissues. Deep imaging is intrinsic to TPEF microscopy since it uses longer excitation wavelengths (near-IR) that scatter less than the shorter visible wavelengths traditionally used in confocal microscopy. This reduces background illumination coming from the scattered light and increases the contrast ratio at higher depths. As an example, in-vivo brain images at depths of 1mm can be achieved with TPEF microscopy.

Two-photon excitation occurs when two independent photons are absorbed simultaneously by a media. This requires two photons of the right energy to coincide in time and space on such media; an unlikely event which needs an extremely large excitation photon flux. The larger the photon flux, the higher the probability that two-photons will be absorbed simultaneously. In TPEF microscopy, higher photon flux leads to higher efficiency and hence improved image quality and resolution.

Traditionally in TPEF microscopy, the delivery of the large photon flux required for two-photon excitation has been implemented with broadly tunable solid-state femtosecond lasers with pulses of the order of 100 fs and practical repetition rates of approximately 80MHz. These can deliver very high peak powers with large photon flux levels, as needed for two-photon microscopy. However, the average power provided by these lasers (in the range of 1 – 4 Watts) can cause thermal damage as a result of the photo-thermal interaction with the media due to linear absorption of the fundamental excitation wavelength. This effect is particularly important in in-vivo imaging where temperatures in excess of 40 ºC lead to irreversible damage. As a result, the average power provided by traditional solid-state lasers has to be attenuated to be practically used in TPEF microscopy. This has a direct impact on the peak power which is also reduced accordingly.

An alternative to increase the peak power, maintaining the average power low and hence avoiding thermal damage is to shorten the pulse duration. This reduces the time interval in which the photons land on the media, enhancing the probability to be absorbed simultaneously.

Recently, for the first time, state-of-the-art supercontinuum all-fiber laser technology has enabled a commercial laser with pulses as short as 15 fs: Cyclone.

Compared to the traditional 100 fs lasers, Cyclone´s 15 fs pulses lead to an extraordinary 7-fold of photon flux, for equal average power levels.

3. Multiphoton Microscopy II: Brighter Images with Shorter Excitation Pulses

In previous weeks, we showed how an NIR laser such as Cyclone with a pulse duration of 15fs, offers over a 7-fold of photon flux when compared with a standard 100fs laser, for similar repetition rates and average power.

But what is the real impact on the image brightness of such enormous improvement of the number of photons available per time and area?

Theoretically, in two-photon microscopy the image brightness is directly related to the excitation efficiency which is quadratically dependent on the photon-flux and the fluorophore´s second order nonlinear excitation cross-section (GM).

As an example, we can calculate the excitation efficiency of the fluorescent protein mRFP when illuminated at 1050nm by a 15fs laser (such as the cyclone, compared to a 100fs laser. With a wider 200nm bandwidth, the 15fs laser excites mRFP across 900 to 1200nm, a much broader spectral region compared to the 11nm of the 100fs laser.

Blue Curve: Peak power of Cyclone 15fs all-fiber fiber laser centered at 1050nm

Orange Curve: Peak power of 100fs fiber laser centered at 1050nm

Grey Curve: Second-order nonlinear excitation cross Section (GM) of mRFP

Considering the peak power and the excitation cross-section at each wavelength, the excitation efficiency of mRFP can be calculated. The result is that a 50% improvement in efficiency is obtained with a 15fs laser such as the cyclone compared to the traditional 100fs lasers.

Blue Curve: Peak Power of Cyclone 15fs all-fiber fiber laser

Orange Curve: Peak Power of 100fs laser

Grey Curve: Second-Order Nonlinear Excitation Cross Section (GM) of mRFP

multiphoton microscopy

2P fluorescence microscopy images of a mouse intestine section stained with SYTOX Green labelling the nuclei (yellow) and Alexa Fluor 568 phalloidin labelling the actin filaments (blue).

4. Multiphoton Microscopy III: A Novel Concept in Multiphoton Microscopy

Imagine simultaneously imaging a wide range of fluorophores without having to think about selecting the optimum excitation wavelength of your laser. In two-photon microscopy, this is often a complex and sometimes an impossible task when using traditional 100fs excitation lasers.

Transform-limited 100fs solid-state lasers emitting in the IR have a spectrum in the range of 10 – 20nm and as a result, they can only simultaneously excite fluorophores whose excitation spectra falls within this 10-20nm spectrum.

To simultaneously excite a larger variety of fluorophores with a single laser, lasers with broader bandwidths and shorter pulse durations are required.

For example, a laser with 15 fs pulses, such as Cyclone laser, centered at 1050nm, delivers 15fs pulses and a bandwidth of 200nm. All green and red fluorophores within this bandwidth (which extends across 900 to 1200nm) can be excited simultaneously by such laser.

This makes simultaneous imaging of multiple fluorophores a viable, practical and simple alternative for two-photon microscopy.

Two-Photon Fluorescence Microscopy Image of a Mouse Intestine. Section stained with

Sytox Green: labelling the nuclei (magenta). FITC Filter

Alexa Fluor 568 Phaloidin: labelling the actin filaments (green). TRITC Filter

Both fluorescent markers were simultaneously excited with Cyclone and fluorescence was filtered with Nikon´s fluorescence cubes.

Image taken at ICFO-SLN the Super-Resolution Light Microscopy at ICFO-Institute of Photonics Sciences, Barcelona, Spain.

Acknowedgements to Sphere Photonics for the D-SCAN pre-compressor

5. New Horizons in Two-Photon Microscopy

Cyclone is a new class of femtosecond fiber laser for two-photon microscopy. It is a new proposal, it is differential. Powered at the core by proprietary technology, it enables the simultaneous excitation of the largest variety of fluorescent probes. It provides images with higher brightness. It introduces a new era of simplicity and cost.

Cyclone provides an extremely wide spectral bandwidth which expands in the NIR across the 900-1200nm spectrum. This overlaps with the two-photon excitation spectra of most green and red-shifted fluorescent labels, including eGFP, mRFP and DsRED. This remarkably exceeds the range of fluorescent labels that can be simultaneously excited with conventional femtosecond lasers, including broadly tunable lasers and single-line femtosecond fiber lasers.

Cyclone offers a highly flexible and versatile solution or two-photon excitation fluorescence microscopy, enhancing the features that can be imaged simultaneously on a sample, of particular importance for in-vivo and ex-vivo microscopy.

An image of a pollen grain using the pollen autofluorescence excited by the Cyclone laser shows the great image quality and simultaneous excitation at different spectral channels.

Not only the spectrum is broader but Cyclone also delivers shorter pulses. Combined with a dedicated state-of-the-art dispersion pre-compensator, pulses of the order of 15-20fs can be delivered on the microscope sample plane. This leads to an extraordinary peak power and an unparalleled photon flux at the sample plane, reaching more than 7-fold the photon flux of conventional femtosecond lasers with pulses in the range of 100-200fs.

The larger photon flux associated to the superior Cyclone peak power leads to an increased number of photons that reach the sample per area and time. This enhances the two-photon excitation efficiency up to a 49-fold when compared with conventional fixed-wavelength or broadly tunable lasers with pulse durations in the range of 100-200 fs. When used with fluorescent label DsRED, over a 50% efficiency is achieved.

The red-shifted NIR wavelengths of Cyclone laser combined with the increased excitation efficiency leads to better image brightness and deeper penetration. A 200 microns deep sample of a zebra fish is simply imaged by the Cyclone laser.

Cyclone broad spectral bandwidth not only enables the excitation of a large range of indicators but it also permits the multicolor excitation across the 900-1200 nm range with a single scan, making simultaneous excitation of different probes possible. This eliminates microscope alignment issues associated to broadly tunable lasers.

Images of a mouse intestine and a convallaria illustrate the great image quality that can be achieved when illuminating these samples with the broad bandwidth of the Cyclone laser.

Cyclone delivers a bandwidth of 200 nm, across the 900-1200nm spectral range, with pulses of 15 fs and a repetition rate of 75MHz, enhancing two-photon excitation and enabling the individual or simultaneous excitation of bountiful indicators.