Few-cycle all-fibre supercontinuum laser for ultrabroadband multimodal nonlinear microscopy

Abstract

Temporally coherent supercontinuum sources constitute an attractive alternative to bulk crystal-based sources of few-cycle light pulses. We present a monolithic fibre-optic configuration for generating transform-limited temporally coherent supercontinuum pulses with central wavelength at 1.06 mm and duration as short as 13.0 fs (3.7 optical cycles). The supercontinuum is generated by the action of self-phase modulation and optical wave breaking when pumping an all-normal dispersion photonic crystal fibre with pulses of hundreds of fs duration produced by all-fibre 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-fibre few-cycle pulsed sources make them powerful tools for applications benefitting from the ultrabroadband spectra and ultrashort 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 their successful performance in ultrabroadband multispectral and multimodal nonlinear microscopy.

Full text

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Few-cycle all-bre supercontinuum laser for
ultrabroadband multimodal nonlinear microscopy
Azahara Alamagro-Ruiz
FYLA LASER SL
Salvador Torres-Peiró
FYLA LASER SL https://orcid.org/0000-0003-3688-7232
Héctor Muñoz-Marco
FYLA LASER SL
Marina Cunquero
ICFO
Gustavo Castro-Olvera
ICFO
Romain Dauliat
University of Limoges, XLIM
Raphael Jamier
Xlim Research Institute
Oleksiy V. Shulika
Universidad de Guanajuato
Rosa Romero
Sphere Ultrafast Photonics, S.A
Paulo T. Guerreiro
Sphere Ultrafast Photonics, S.A
Miguel Miranda
Sphere Ultrafast Photonics, S.A
Helder Crespo
Sphere Ultrafast Photonics, S.A
Philippe Roy
University of Limoges, XLIM
Pablo Loza-Álvarez
ICFO
Pere Pérez-Millán (  ppmillan@fyla.com )
FYLA LASER SL https://orcid.org/0000-0002-5872-8278
Article

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Keywords: supercontinuum sources, lasers, ultrafast lasers, bre lasers, ultrabroadband multimodal
nonlinear microscopy
DOI: https://doi.org/10.21203/rs.3.rs-820062/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

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Abstract
Temporally coherent supercontinuum sources constitute an attractive alternative to bulk crystal-based
sources of few-cycle light pulses. We present a monolithic bre-optic conguration for generating
transform-limited temporally coherent supercontinuum pulses with central wavelength at 1.06 mm and
duration as short as 13.0 fs (3.7 optical cycles). The supercontinuum is generated by the action of self-
phase modulation and optical wave breaking when pumping an all-normal dispersion photonic crystal
bre with pulses of hundreds of fs duration produced by all-bre chirped pulsed amplication. Avoidance
of free-space propagation between stages confers unequalled robustness, eciency and cost-
effectiveness to this novel conguration. Collectively, the features of all-bre few-cycle pulsed sources
make them powerful tools for applications benetting from the ultrabroadband spectra and ultrashort
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 their successful performance in ultrabroadband
multispectral and multimodal nonlinear microscopy.
Introduction
Since seminal proposals that envisioned its advantages1,2, nonlinear optical (NLO) microscopy has
progressed rmly in the last decades3–5, to the point where it is today an indispensable facility in
microscopy services of large scientic institutions. Multiphoton excited uorescence (MPEF) and second
harmonic generation (SHG) are among the most relevant NLO microscopy techniques for biological
investigations. Common advantages to MPEF and SHG techniques, in contrast to classic single-photon
uorescence microscopy, are deep tissue penetration, 3D-sectioning imaging, out of focus scatter-free
imaging and small focal volume (thus high resolution in all three spatial axes). MPEF allows exogenous
and endogenous uorophore excitation and SHG enables observation of the spatial structure of non-
centrosymmetric materials and/or interfaces (e.g., changes on the index of refraction) in unlabelled
biological specimens6. Multimodal NLO microscopy usually combines MPEF and SHG for full
exploitation of the advantages of both techniques. 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
sample7,8. 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. Solid-state and optical bre
mode-locked lasers capable of pulse durations of around 100 fs are used regularly in NLO microscopy.
Less explored are few-cycle sources, which, by delivering pulses with durations in the range of 10 fs,
increase the eciency of the excited nonlinear effects by an order of magnitude, helping to reduce the
average power on the samples and maximise their viability. Besides, and inherently to the relationship
between time and frequency domains, 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 microscopy9,10. Currently, commercially available few-cycle sources are
provided only by a few bulk solid-state technologies, namely titanium-sapphire (Ti:Sa) oscillators and

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more recently optical parametric chirped pulsed amplication (OPCPA) systems. These sources are
complex, bulky, water-cooled, of cumbersome use and require frequent service and maintenance. They
have therefore a high cost of ownership, while their price is often inated due to lack of competing
technologies. This limitation in technology availability is what motivates our present work: the
development of fully optical bre-based few-cycle lasers as an alternative technology to bulk solid-state
lasers, to foster a universal adoption of multimodal and multispectral NLO microscopy, making it cost
effective, attractive and reliable for many scientic laboratories, industrial laboratories and hospitals
facilities.
In this work, we present rst a monolithic bre-optic conguration for generating temporally coherent
supercontinuum (SC) that provides transform-limited few-cycle pulses with durations as short as 13.0 fs
at a central wavelength of 1060 nm, the shortest pulse durations provided to date, to our knowledge, by
an all-bre source in the 1 mm spectral region (a spectral region of utmost relevance in biological
microscopy because it is the region within near UV to mid infrared where tissues present the lowest
absorption coecients, thus the highest achievable imaging depths11,12). Being all-bre, this type of
source is more simple, robust, ecient and cost-effective than previously reported few-cycle temporally
coherent SC sources13–16. Next, we report results on the performance of the few-cycle all-bre source in
multimodal and multispectral NLO microscopy: imaging of different biological specimens is obtained by
simultaneous TPEF of multiple uorophores whose single-photon peak absorption wavelengths are in the
band from 480 to 580 nm; TPEF and SHG are combined to obtain images of neurons (via TPEF) and of
muscle and pharynx (via SHG) of living
C. elegans
specimens; the penetration depth capability of the few-
cycle source is assessed by TPEF imaging of transparent specimens (zebrash embryos) and of
scattering tissues (Wistar rat retina); an image of the full retina, of ~ 170 µm depth, is obtained with
cellular resolution, showing better imaging resolution and depth than the image of the same sample
acquired with a commercial Ti:Sa laser at a central wavelength of 810 nm.
Results
Few-cycle all-bre temporally coherent supercontinuum.
Ultrafast bre lasers appear as an alternative to Ti:Sa oscillators and OPCPA systems, since they are
compact, air-cooled, turn-key, cost-effective and maintenance and alignment-free17,18. However, the
natural gain spectra of rare-earth elements (the active media in bre lasers) limit their emission
bandwidths and pulse durations to a few tens of nm and a few hundreds of femtoseconds, respectively.
Nevertheless, in the last years, research on supercontinuum (SC) generation has demonstrated successful
approaches to obtain very high-quality few-cycle pulses using ultrafast bre lasers as exciting sources of
particular nonlinear effects in photonic crystal bres (PCFs): pumping at a wavelength within the
attened top of the convex dispersion curves of all-normal dispersion (ANDi) PCFs (whose dispersion lies
completely in the normal dispersion region), spectral broadening appears due to the action of self-phase
modulation (SPM) and optical wave breaking (OWB)19. In this way, highly coherent SC emission can be
generated, preserving compressible pulses in the temporal domain. Different approaches to generate

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temporally coherent SC using ANDi PCFs have been proposed. In some congurations the seed stage (or
pump stage) of the coherent SC source is built with free-space optics technology: e.g., Ti:Sa lasers,
optical parametric oscillators (OPOs) or master oscillator power ampliers (MOPAs)13,14,20−24. In other
congurations the seed stage is a bre laser but light coupling to the ANDi PCF is performed with free-
space optics15,25−27. Chow
et. al.
28 proposed an all-bre conguration based on ANDi PCFs for the 1.55
µm band, but this work lacked a demonstration of transform limited or near-to-transform limited
compression of the generated SC, probably due to the long length of the ANDi bre used in the
experiment (64 m). To the best of our knowledge, none of the current state-of-the-art congurations that
demonstrate generation of transform-limited or near-to-transform-limited few-cycle temporally coherent
supercontinua using ANDi PCFs are an all-bre conguration (said all-bre conguration understood as a
monolithic bre-optic conguration where all its stages are bre based and coupled to each other by a
bre splice or a bre-based transition). In this section we describe an all-bre conguration for generating
temporally coherent supercontinuum that provides transform-limited few-cycle pulses with durations as
short as 13.0 fs. Recently we have reported rst evidence of the ability of this all-bre conguration to
deliver few-cycle transform-limited pulses29. In this section we describe in detail the monolithic
architecture of the system, the process of design, manufacture and optimization of ANDi PCFs according
to the diagnosis of different emission regimes depending on the bre geometry and we present results of
few-cycle emission of optimized time-domain quality, which are conrmed by independent pulse duration
measurement techniques: d-scan, and interferometric autocorrelation.
Temporally coherent spectral broadening in ANDi PCFs is achieved by SPM and OWB using short lengths
of ANDi PCFs (few to tens of cm) pumped by input pulses of high peak power (few to tens of kW) and
very short duration (few to hundreds of fs). As an example, Heidt
et al
.19 showed that a spectral
broadening of > 100 nm (with central wavelength at 1060 nm) can be obtained while maintaining perfect
coherence by pumping 1 m of ANDi PCF with input pulses of 5 kW peak power and 200 fs duration. In our
monolithic conguration, the ANDi PCF is a few tens of cm long and is excited by input pulses of > 15 kW
peak power and < 250 fs duration. Ti:Sa lasers, OPOs or MOPAs, with laser generation architectures
based on free-space congurations, deliver naturally pulses of this type. However, for an all-bre laser
architecture it is a challenge to offer pulses with these properties to be delivered to an ANDi PCF because
the light is completely conned in the cores of the optical bres, whose core diameters are typically below
10 mm. A peak power of 15 kW inside guiding bres of 10 mm core diameter yields intensities of > 15
GW/cm2 which, for propagation lengths of a few cm, are above the threshold for many undesired
nonlinear effects that distort the laser pulses propagating inside the bres and, particularly, destroy the
temporal coherence of the laser pulses. To overcome this problem, we use a chirped pulse amplication
(CPA)30 system with an all-bre conguration (see Fig.1), where the temporal compression stage is built
with a hollow core photonic bandgap bre (HC PBGF). Maintaining the temporal coherence throughout its
stages, such conguration delivers pulses of > 15 kW peak power and < 250 fs duration, to be used as
exciting pulses of the ANDi PCF. The end of the HC PBGF is fusion spliced to the ANDi PCF. To maintain
the integrity of the structure of both bres, the arc discharge parameters of the fusion procedure are set
for a weak splice, providing a coupling eciency of 0.4 or greater.

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Figure1. a), All-bre conguration of a temporally coherent supercontinuum source of few-cycle pulses.
GDD: group delay dispersion; GVD: group velocity dispersion; b), Qualitative representation of the spectral
and temporal properties of the pulsed optical signal as it evolves throughout the stages of the all-bre
source (stages 1 to 5) and at the output of the temporal compressor (stage 6) used to compress the pulse
down to its Fourier limited duration. Dli and Dti: spectral bandwidth and temporal duration of the pulses,
respectively, at the output of
i
-th stage; c), Properties of the pulsed signal at the output of each stage, for
an example of implementation of the all-bre conguration where the active bre is Yb-doped, thus with
laser emission in the 1 mm band. MFD: mode eld diameter, lc: central wavelength, PRR: pulse repetition
rate, DlFWHM: spectral bandwidth at full width at half maximum, DtFWHM: pulse duration at full width at
half maximum, Pavg: average power, Pp: pulse peak power, Ip: pulse peak intensity. Values are given for a
pump laser diode wavelength and average power of 976 nm and 3.75 W, respectively.
The rst stage (stage 1) corresponds to the seed of the system, a passively mode-locked all-bre
oscillator that delivers transform limited pulses of hundreds of femtoseconds duration and MHz range
pulse repetition rate (PRR) with a central emission wavelength lc. PRR and lc remain unchanged
throughout all stages of the system. Stage 2 is composed of a polarization maintaining (PM) single
mode optical bre and a PM fused bre combiner. The bres of this stage have a normal group velocity
dispersion (GVD > 0). The function of stage 2 is to stretch the pulses temporally so they can be amplied
in the next stage without generating nonlinear effects (the peak power remains below the threshold of
generation of nonlinear effects in the optical bre core). Also, it pre-compensates the anomalous
dispersion that the pulses will experience in stage 4. The fused bre combiner launches light from a laser
diode into the active bre of the next stage. Stage 3 is composed of a double clad PM rare-earth doped
active bre, with normal group velocity dispersion (GVD > 0). The function of stage 3 is to amplify the
pulses to the maximum possible peak power without biasing nonlinear effects that would distort the
temporal and spectral shape of the pulses. The amplication is produced progressively in the bre by
stimulated radiation in the active rare earth ions of the bre core, which are pumped to excited states by
the light coming from the laser diode of the previous stage through the rst clad of the bre (this clad is
passive, i.e., undoped). Stage 4 is composed of a hollow core photonic bandgap (HC PBG)
microstructured bre with anomalous group velocity dispersion (GVD < 0). The function of stage 4 is to
compress the pulses to achieve the peak power required at the input of the ANDi PCF to eciently
generate temporally coherent spectral broadening by SPM and OWB. Also, its length is chosen so that the
net group delay dispersion (GDD) suffered by the pulses from the oscillator output is slightly anomalous
(− 0.015 ps2 in the example of implementation of Fig.1). This way, the pulse still undergoes compression
in the rst segment of the ANDi PCF and SPM eciency is optimized by having the maximum peak
power achievable inside the ANDi PCF. Since the material of the HC PBG bre core is air, nonlinear effects
due to high peak power are avoided, hence the pulse does not experience additional spectral broadening.
Stage 5 is composed of a highly nonlinear ANDi PCF a few tens of cm long. GVD in this bre is normal
within a very broad spectral bandwidth (broader than 300 nm, centred at 1060 nm, in the implementation
example of Fig.1), with a quasi-at and symmetric shape (see Fig.2-d2). The function of this stage is to
spectrally broaden the spectrum of the pulsed signal by SPM under near-zero-dispersion conditions,

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which preserves the temporal coherence of the pulses so that they remain compressible down to pulse
durations corresponding to the Fourier limit of their spectrum. At the end of stage 5, the pulsed signal is
delivered to free-space and collimated. Stage 6 is composed of a free-space temporal compressor of
xed anomalous dispersion (GDD < 0) and variable normal dispersion (GDD > 0). It compresses the
temporally coherent pulses from the output of stage 5 down to close to their Fourier limited durations
(12.2 fs in the example of implementation of Fig.1). The variable compression is introduced by a pair of
glass wedges placed in the path of the optical signal. Dispersion is varied by changing their relative
position (insertion length), therefore changing the amount of glass material effectively traversed by the
optical signal.
The ANDi bre of Sect.5 was manufactured with F300 silica and exhibits a standard PCF solid core
design: the solid-core results from a missing hole that is surrounded by N rings of holes running along the
bre longitudinal axis. Holes are displayed in a periodic manner with a triangular lattice. The air holes
have a diameter d. The distance between hole centres, called pitch, is Λ. The microstructure of air holes is
surrounded by a jacket of uniform silica that confers the bre a typical diameter of 125 µm. Ecient
connement of the light inside the core is obtained for a value of N equal to 7. The required properties of
the GVD curve are calculated using the semi-empirical model proposed by Saitoh
et al.
31. Considering a
central wavelength of 1060 nm, a set of potentially valid dispersion curves is obtained combining the
values of Λ and d within the following ranges: Λ = [0.50 ; 0.64] µm ; d = [1.50 ; 1.70] µm. ANDi PCFs with
optimum values of Λ and d have been manufactured through an optimisation iterative process (see
section Methods). Figure2 summarizes the results of the optimisation process of the ANDi PCF
manufacture, from design to performance on temporally coherent spectral broadening. Figure2a shows a
scanning electron microscope (SEM) image of the cross section of the manufactured ANDi PCF that
presents the largest temporally coherent spectral broadening (bre 2). Figure2b shows a map of
calculated values of Λ and d that provide adequate dispersion curves for temporally coherent spectral
broadening. The red region denotes the condition of the dispersion maximum belonging to the exciting
laser bandwidth (1060 ± 15 nm). The blue region extends the condition to a broader bandwidth (1060 ± 30
nm). Section Methods offers an extended explanation of the elaboration of this map. Figures2c1-3 are
detailed SEM images of the core region of representative ANDi PCFs manufactured during the
optimisation process, with different values Λ and d (measured from the corresponding SEM images) and
numbered from 1 to 3. Figures2d1-3 show the calculated dispersion curves for bres 1 to 3, respectively.
Finally, Figs.2e1-3 show the spectra at the output of the ANDi PCF (stage 5 in Fig.1) obtained with bres
1 to 3, respectively, for the same exciting conditions (output signal of stage 4 in Fig.1) and same length
(20 cm), at 3 different driving currents of the laser diode that pumps the amplifying stage (stage 3 in
Fig.1). Fibre 1 presents a dispersion curve out of the target of ANDi condition, being the dispersion
parameter D ∈ [0; + 5 ps/nm km] within the exciting laser bandwidth. Consequently, an asymmetric
spectral broadening is observed, governed by temporally incoherent dynamics of rst stages of SC
construction in an anomalous GVD regime: modulation instability (MI), soliton ssion and Raman self-
frequency shift32,33. Contrarily, bre 2 presents an ANDi curve, being the dispersion parameter D ∈ [-5; -25
ps/nm km] within a full bandwidth of 300 nm, centred at a maximum dispersion wavelength of 1060 nm.

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SPM governs a symmetric temporally coherent broadening. The near-zero dispersion of the ANDi bre
limits pulse temporal stretching, thus driving high SPM eciency. The evolution of spectral broadening in
bre 2 is shown in Fig.2e2. The average pump power of 3.75 W is slightly above the average pump
power limit below which spectral broadening is due to SPM only. The shoulder peak at 930 nm in the
short-wavelength edge of the spectrum indicates the beginning of the appearance of OWB effects, which
are to be avoided to maintain “pure” temporally coherent spectral broadening by SPM19. Fibre 3 presents
an ANDi curve as well, being D ∈ [-25; -40 ps/nm km] within a full bandwidth of 300 nm, centred at a
maximum dispersion wavelength of 1045 nm. These values are more distant from zero dispersion than
those of bre 2. Consequently, SPM still broadens the spectrum while preserving temporal coherence, but
with less eciency (compared to bre 2), because the pulse suffers larger temporal stretching.
To conrm that our pulses at the output of the ANDi PCF are temporally coherent (thus compressible to
close to their Fourier transform limit), we use the free-space temporal compressor of stage 6 (Fig.1) and
compare results of two different methods of pulse characterization in the time domain: d-scan technique
and interferometric autocorrelation. The d-scan technique is based on performing a dispersion scan to
the pulses with a pulse compressor while measuring the spectrum of the resulting second-harmonic (SH)
signal. From this measurement, the phase and the amplitude properties of the electric eld of the pulses
are retrieved34. Figures3a1-4 show the properties of the pulse measured with the d-scan technique, for
the case of ANDi bre 2 and a pump diode average output power of 3.75 W. The compressor is designed
to provide a range of net GDD to the pulse from − 1000 fs2 (0 mm insertion length, Figs.3a1-2) to + 1500
fs2 (17 mm insertion length, Figs.3a1-2). Details on the design of this compressor and corresponding
application of the d-scan technique have been reported previously by us35. The compressor is adjusted to
achieve the best quality pulse (shortest FWHM pulse width and highest peak power ratio between main
peak and side-lobes). In these conditions, the pulses present very low GDD, third order dispersion (TOD)
and fourth order dispersion (FOD) values (+ 276 fs2, -5162 fs3 and − 1621 fs4, respectively) with 56 ± 4 %
of the energy in the main peak and with peak power ratio between the main peak and the side-lobes
greater than 8. The measured pulse width at FWHM of the compressed pulse is 13.0 fs (3.7 optical
cycles). This result demonstrates the high degree of temporal coherence of the pulsed signal, since it is
very close to the Fourier limited duration supported by its optical spectrum (12.2 fs). The fact that the
pulses are properly compressed by a compressor of negligible TOD proves that the pulses suffer very low
TOD (comparable to that of the compressor) while being spectrally broadened at the ANDi bre. This
effect results from the atness of the dispersion curve of the ANDi bre (Fig.2d2). Average and peak
power of the beam at the output of the compressor are Pavg = 160 mW and Pp =169.2 kW, respectively.
Focused on a sample area of e.g., 10 mm2, the photon irradiance of the beam during pulse propagation
on the sample would be 8.5 x 10 30 photons s − 1 cm − 2 (neglecting losses in the optics), a gure well
above typical multiphoton excitation thresholds. Figure3b shows an autocorrelation trace of the pulses
obtained at the output of stage 6 with an interferometric autocorrelator. This trace corresponds to the
pulsed signal compressed to its minimum duration by the same variable dispersion compressor
employed for the d-scan technique. From this trace, an estimation of the FWHM of the pulse intensity

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prole of 12.6 fs is obtained. Despite not providing unambiguously the real form of the pulse intensity
prole36,37, the autocorrelation method is widely known and trusted by microscopists. Hence, the fact that
it produces, independently, an estimated result of the pulse width comparable to the accurate result of the
d-scan method, is relevant for a straightforward use of the source in current NLO microscopy setups. To
the best of our knowledge, with the measurement of a temporal pulse width of 13.0 fs (3.7 optical cycles)
we report the shortest pulses obtained to date from an all-bre source in the 1 mm spectral region, being
> 3 times shorter than the shortest pulses delivered by previously reported all-ber sources in this region,
which, limited mainly by the gain spectrum of ytterbium and by bre dispersion and nonlinearity
management constraints, do not support durations below 42 fs38 − 41.
Broadband multispectral and multimodal nonlinear imaging
The output from stage 6 was coupled to an adapted inverted confocal microscope (Eclipse TE2000-U,
Nikon) (Fig.4a) modied for nonlinear imaging experiments. The variable dispersion compressor
described above was used to pre-compensate the dispersion of the optical elements in the path of the
microscope towards the sample, where the pulse duration was maintained below an estimated duration
of 16 fs (Fig.4c). The coupling system of the external illumination source included two galvanometric
mirrors (Cambridge Technology, UK) and a telescope arrangement. We used a dichroic mirror (FF825-
SDi01, Semrock) for sending the pulses to the illumination objective. The generated uorescent light
captured using this objective was collimated and sent through the same dichroic mirror, in a non-
descanned conguration, for detection using a photomultiplier tube (PMT) detector (H9305-04,
Hamamatsu). Three different microscope objectives, with different refractive index immersion media,
numerical apertures (NA) and magnications were used (see table with properties in Fig.4). Dispersion
pre-compensation was performed for every objective. The few-cycle all-bre source described above was
used as the illumination source for all image acquisitions, except for images in Figs.5F,H that were
generated with a pulsed Ti:Sa laser (MIRA 900-F, Coherent, 200 fs nominal pulse width) operating at a
central wavelength of 810 nm.
Possible leakage of fundamental laser light was ltered with a BG40 lter (FGB37-A, Thorlabs). The two-
photon excited uorescence (TPEF) signal was ltered with uorescence lter cubes (DAPI, FITC and
TRITC: Standard series, Nikon) and collected in the backward direction. We used a 25x NA1.10 water
immersion objective (Apo LWD, Nikon) for the second harmonic generation (SHG) signal collection in the
forward direction. The SHG signal was ltered with a bandpass lter (FF01-542/27 − 25, Semrock).
The dispersion pre-compensation performance of the variable compressor was evaluated by varying the
glass wedges insertion to adjust the duration of the pulse at the sample plane. For each image, we
measured the mean intensity of the generated TPEF signal as the function of glass insertion. The results
can be seen in Figs.4b1-3. The induced dispersion per glass insertion length was + 147 fs2 / mm. We see
that the pre-compensation system can eciently be used to maximise the uorescent signal from the
sample. A maximum TPEF intensity depending on the insertion of the glass was observed. This indicates
that the system is capable of pre-compensating the dispersion introduced by the different microscope

Page 10/27
objectives. The discrepancies found in the optimum insertion length when imaging different samples can
be attributed to changes in the refractive index of the samples.
Using the 25x objective under the optimised GVD settings of the compressor, we have successfully
imaged several samples. Importantly, good uorescence signal and depth were achieved using an
average power of the pulsed beam of ~ 4 mW, measured at the sample plane (the average power of the
beam at the output of the variable compressor was 160 mW; it was attenuated by a variable neutral
density lter and by intrinsic losses of the optical components through the microscope optical path). The
maximum penetration achieved corresponds to 220 µm (Figs.5A-D) in depth of the tail of a transgenic
line zebrash embryo (Caax-GFP) expressing GFP in all cell membranes. Zebrash embryos are
transparent, so they allow imaging at these large penetration depths. To test the penetration capabilities
of the laser within a scattering tissue two excised retinas were stained with the same cytoskeleton marker
(phalloidin), each conjugated with a different uorescent dye: AlexaFluor 405 and 647, to be excited with
a Ti:Sa Coherent MIRA 900-F laser (ΔλFWHM = 10 nm, λc = 810 nm) and with our few-cycle all-ber source
(ΔλFWHM = 150 nm, λc = 1060 nm), respectively. We proceeded to record the full retina (~ 170 µm) with
cellular resolution, acquiring z-stack images (Figs.5G-H). For both lasers, we used the same laser power
at the sample plane and similar step spacing for constructing the z-stacks. Then lateral re-slices of the z-
stack images were performed. Figures5E-F show the comparison of the lateral re-slice TPEF images
acquired with our few-cycle all-bre source and with a Coherent MIRA 900-F laser. In the image acquired
with our few-cycle all-bre source, all synaptic (bright regions) and nuclear (gap regions) layers that
characterize the tissue can be distinguished (Fig.5E). However, in the images acquired with the Coherent
MIRA 900-F laser it was only possible to distinguish four layers (Fig.5F). Larger imaging depth was
achieved using the few-cycle all-bre source, with which properly resolved images of the retina deeper
layers ONL and OS (Figs.5E,G) were obtained. It is interesting to mention that the rat retina is highly
autouorescent to light in the blue-green region of the spectrum. In addition, the external segment of the
photoreceptor cells where opsins (photopigments) are packaged, is highly absorbing to visible light.
Therefore, illumination sources in the IR spectrum combined with red uorescent dyes are ideal for depth
imaging to prevent the autouorescence generation/distortions in this tissue.
To test the capability of our few-cycle all-bre source to nonlinearly excite multiple markers we proceeded
to acquire TPEF images of multiple uorophores: GFP, SYTOX Green, Alexa Fluor 568, tagRFP and Alexa
Fluor 647. We also acquired SHG images of unlabelled tissues. Care was taken to use the corresponding
lters for acquiring the TPEF signals. Figure6A shows an image of a mouse intestine section stained
with SYTOX Green (FITC lter) labelling the nuclei shown in yellow, and Alexa Fluor 568 phalloidin (TRITC
lter) labelling the actin laments shown in blue. Figure6B shows the rhizome of
Convallaria majalis
stained with Fast Green and Safranin. Chloroplasts are shown in green (FITC lter) and cell walls are
shown in red (TRITC lter). In Fig.6C, it is also possible to see the autouorescence of a pollen grain. The
large emitted autouorescence spectrum was detected using two different uorescence lters, FITC lter
shown in green and TRITC lter shown in red. We have also been able to image
in vivo
samples with
cellular resolution. In particular, paralysed
C.elegans
specimens. In Fig.6D, we can see a
C.elegans

Page 11/27
OH15500 strain expressing tagRFP in all neurons. The SHG signal revealed all the different structures of
the pharynx of the animal: corpus, isthmus and posterior bulb. Moreover, we can see the muscle bres
and their contraction during swallowing over time. These structures are a high-priced reference while
imaging the neurons with TPEF. By using different emission lters, we have been able to split the
uorescence from the different uorophores to visualize multiple structures with a single illumination
source.
Discussion
We have presented a monolithic bre-optic source of transform-limited few-cycle pulses. The simplicity,
robustness and cost-effectiveness of its conguration are powerful factors in favour of this technology to
replace traditional solid-state sources of few-cycle pulses in various applications. The successful
performance of our source to provide high quality TPEF and SHG microscopy images anticipates its
utility in three-photon excited uorescence (3PEF) and third harmonic generation (THG) microscopy, as
well as in other NLO microscopy techniques, such as coherent anti-Stokes Raman scattering (CARS)42,
multiphoton uorescence lifetime imaging microscopy (MP-FLIM)43,44 and, consequently, in multimodal
and multispectral combinations between all mentioned techniques. The central wavelength of ~ 1 mm is
also favourable for microscopy compared to the ~ 800 nm central wavelengths of Ti:Sa lasers, as it
results in larger penetration depth in biological tissues and enables performing third-harmonic
microscopy without going into the deep-UV, as would be the case of a central wavelength of ~ 800 nm.
Utility of this type of source is anticipated also in applications of other disciplines that rely on the inherent
properties of few-cycle pulses, such as ultrafast spectroscopy, optical frequency comb generation and
frequency metrology45–47. We have presented pulse durations as short as 13.0 fs (3.7 cycles), with a
central wavelength of 1060 nm, but this is a non-fundamental limit that we expect to overcome with
enhanced bre manufacture precision to obtain atter and nearer to zero dispersion curves of the ANDi
PCFs. Based on the all-bre conguration reported here, our future work aims at increasing the output
average power to the few-watts level (by increasing the pulse repetition rate of the bre oscillator seed to
the GHz range48) and at obtaining few-cycle emission at central wavelengths of 1.5 mm and 2.0 mm,
particularly useful for localized nonlinear excitation of semiconductor materials transparent to these
wavelengths49.
Methods
All-normal dispersion photonic crystal bre (ANDi PCF).
The following text describes the technical properties of the nonlinear all normal dispersion (ANDi) optical
bres used in this work to generate temporally coherent supercontinuum emission.
Design of ANDi PCF

Page 12/27
Design of PCFs for this SC source is governed by the trade-off between technological limits and design
requirements. The SC shall be temporally coherent and be seeded by a femtosecond Yb-doped bre laser.
Thus, we need to use PCFs having all-normal dispersion (ANDi) within the laser line as well as within all
the spectral range where SC is expected to be generated. As the nominal value of the central wavelength
is 1060 nm, the natural choice of the material is silica. Therefore, as a starting point we have chosen the
geometry shown in Fig.7, which consists of a triangular array of the air holes in the silica glass matrix,
where the core is formed by omitting one hole in the centre of the structure. The main issue in the PCF
drawing process is controlling the shape and homogeneity of the diameters of the air holes. Not all
technologies allow fabricating PCFs with air holes of very small size (diameter) and, more importantly,
guarantee the stability of the structure along hundreds of meters and the reproducibility of such designs.
Therefore, the search of the designs interesting for us are implemented using the hole diameter as the
independent parameter. The minimum diameter of the holes is chosen to be dmin = 500 nm. The
maximum diameter is dmax = 4000 nm. The distance between the holes L is dened within the range d /
L = 0.2–0.8. This interval includes the values which guarantee the PCFs to be single mode (d / L ≤
0.43)50,51. The PCFs having d / L > 0.43 could be either single mode or multimode. The spectral
bandwidth of interest is 400 nm at a central wavelength l0 = 1060 nm. In order to have relatively
symmetric spectral broadening it is desirable to have a dispersion prole D(l) as symmetric as possible
relative to the excitation wavelength l052. Another requirement is to have a dispersion as small as
possible in absolute value to guarantee that self-phase modulation will be the dominating spectral
broadening mechanism.
To calculate the dispersion curves, we used the semi-empirical model proposed by Saitoh
et. al.
31. Within
this model, the theory of single-mode optical bres is applied to describe the dispersion properties of
index-guided PCFs. Being semi-empirical, this model allows iterating over a large volume of parameters to
lter out designs tted to technical requirements. Using this model, we scanned the (d, L) - space within
the limits mentioned above and applying the condition that the dispersion maximum occurs within the
laser bandwidth, Dmax ∈ D l, or to a slightly more extended range, Dmax ∈ D l ± 15 nm. The resulting
parameters’ map is shown in Fig.8. The red area corresponds to the stricter condition Dmax ∈ D l. The
variety of enumerated points corresponds to different pairs (d, L) whose dispersion curves are shown in
Fig.9. The yellow circle on the red area corresponds to the target design.
Fabrication Method
The bre has been fabricated using the standard stack and draw method, where 168, 1 mm outer
diameter capillaries have been stacked together around a 1 mm rod. The stack has been introduced and
chocked in an outer tube that will be part of the outer cladding of the nal bre. This preform, called
primary preform, has been drawn into several 1 m long microstructured capillaries (or canes) by applying
vacuum into interstitial holes while keeping the 168 inner holes of capillaries at atmospheric pressure.
One of these microstructured canes is then introduced into a jacket tube to reach the expected outer
diameter, pitch and hole diameters ranges during the nal drawing. Finally, the ne control of hole

Page 13/27
diameter and pitch is obtained by controlling a quadruple of parameters which are the furnace
temperature, the preform and drawing speeds, and the pressure applied to the holes. Despite the high
quality of the control devices and of the regulation of the electronic systems of the drawing tower,
stabilising and controlling the diameter of the small air holes (500–600 nm) with an expected accuracy
of 50 nm is challenging, especially achieving repeatability from a drawing to another. Indeed, many
factors inuence the repeatability and the ne control of the air hole diameter, among which, for example,
micro-geometrical uctuations along one tube or between external tubes (second drawing step) or
residual material stress and geometrical uctuations between microstructure capillaries (rst drawing
step). In order to obtain bre samples with stabilized and controlled dimensions, we have performed live
tests during the drawing. These consists in (i) periodically collecting 1 m long bre samples (every 500 m
with xed drawing parameters) and (ii) estimating the chromatic dispersion curve by launching kW-range
peak power pulses in the samples in order to observe the signature of nonlinear mechanisms. Indeed, an
anomalous dispersion will lead to asymmetric spectral broadening governed by MI, soliton ssion and
Raman self-frequency shift. This occurs when hole diameters are too large (e.g., larger than 630 nm, for a
pitch of 1650 nm). On the other hand, when the chromatic dispersion is all-normal but far from zero and
exhibits fast variations of group velocity over the spectral band of interest (e.g., holes diameter smaller
than 520 nm, for a pitch of 1580 nm), the shape of the spectrum is triangular and broadening eciency is
low. Furthermore, despite the 7-layer structure chosen to minimize connement losses, for such small d /
L ≤ 0.33, the fundamental mode experiences larger connement/bending losses that compromise the
achievement of good results. The pressure applied in the preform was consequently tuned in order to
reach the moment when we observed the development of a symmetric ecient broadening that is the
signature of the expected single contribution of SPM (e.g., for holes diameter of 596 nm and pitch of
1615 nm). The inertia of the process requires to waiting for several minutes (i.e., several hundred meters
of drawn ber) in order to obtain a sample with stabilized geometrical parameters although the applied
changes in pressure are as small as 0.1 kPa for a total applied pressure from 12 to 15 kPa depending on
the drawn preform and samples.
Instrumentation
Fibre splices in the example of implementation shown in Fig.1 were performed with a Fujikura FSM 100
P bre fusion splicer. Microscope images of PCFs sections shown in Fig.2 were obtained with a Zeiss
Ultra 55 scanning electron microscope. Image postprocessing and statistical analysis followed, to obtain
d
and Λ parameters for each sample shown in Figs.2c1-3. Spectra of Fig.2 were taken with 50 pm
spectral resolution using a Yokogawa AQ6373B optical spectrum analyser. D-scan measurements in
Fig.3 were taken with a Sphere Ultrafast Photonics d-micro system, which includes an optimised
compressor for microscopy. Autocorrelation traces in Figs.3 and 4 were obtained with 1 fs resolution
using a Femtochrome Research FR-103TPM interferometric autocorrelator.
Declarations
ACKNOWLEDGEMENTS

Page 14/27
The authors acknowledge support from the European EUREKA program, project E!11729 FEMTECPRO /
CDTI-INNO-20171026; the EU H2020-INNOSUP program, project FEMTOCOLORS (GA No 739697); the
program for Industrial Doctorates of the Spanish Ministry of Science and Innovation, grants DI-17-09578
and DI-15-07461; the sabbatical leave program 2017-2018 by the University of Guanajuato and grant CIIC
2018.316; the “Severo Ochoa” program for Centres of Excellence in R&D (CEX2019-000910-S) of the
Spanish Ministry of Economy and Competitiveness; the EU H2020 program (GA No 871277); Fundació
Privada Cellex, Fundació Mir-Puig, Generalitat de Catalunya through the CERCA program; Fundação para
a Ciência e a Tecnologia (FCT) (UIDB/04968/2020); Programa Operacional Temático Factores de
Competitividade (NORTE-01-0145-FEDER-022096); UT Austin Portugal Program, co-funded by
NORTE2020, PORTUGAL2020, FEDER and FCT (203269 - ExtreMed - NORTE-01-0247-FEDER-045932);
Laserlab-Europe EU-H2020 (871124).
The authors acknowledge L. Lynn and M. Krieg from the group of Neurophotonics and Mechanical
Systems Biology of ICFO, for providing the
C. elegans
worms.
AUTHOR CONTRIBUTIONS
A.A.R., S.T.P., H.M.M. and P.P.M. designed, developed and characterized the few-cycle all-bre sources.
O.V.S. and S.T.P. designed and performed simulations of the properties of the ANDi PCFs. R.D., R.J. and
P.R. manufactured the ANDi PCFs. R. R, P.T.G, M.M. and H.C designed and built the d-scan system. M.C,
G.C.O, S.T.P and P.L.A. designed and performed the experiments on nonlinear microscopy. All authors
analysed and discussed the experimental results. P.P.M coordinated the interdisciplinary collaboration
between groups. A.A.R and P.P.M wrote the manuscript. All authors revised the manuscript.
COMPETING INTERESTS
A.A.R., S.T.P., H.M.M. and P.P.M. declare employment with FYLA. P.P.M. declares personal nancial interest
(as co-founder and shareholder) with FYLA. S.T.P., H.M.M. and P.P.M. declare co-authorship of a patent.
R.R. declares personal nancial interest and employment with SPH. P.T.G. declares employment with
SPH. M.M. declares co-authorship of a patent and employment with SPH. H.C. declares co-authorship of
two patents and personal nancial interest (as co-founder and shareholder) with SPH. Patent 1:
Universidade do Porto; M. Miranda, H. Crespo, T. Fordell, C. Arnold, A. L’Huillier; WO2013054292A1;
US9,397,463 B2; Granted in the USA; involving the dispersion scan technique. Patent 2: SPH and
Universidad de Salamanca; B. Alonso, I. J. Sola, H. Crespo; WO2019003102A1; Published; involving the
selfcalibrating dispersion scan technique. Patent 3: FYLA; P. Pérez-Millán, S. Torres-Peiró, H. Muñoz-
Marco; EP3731352A1; US20200343681A1; JP2020181193A; Published; involving all-bre generation of
temporally coherent supercontinuum. SPH: Sphere Ultrafast Photonics, S.A. is a company that sells
devices for the temporal measurement and compression of ultrashort laser pulses. FYLA: FYLA LASER
SL is a company that sells ultrafast bre lasers.
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Figures

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Figure 1
a), All-bre conguration of a temporally coherent supercontinuum source of few-cycle pulses. GDD:
group delay dispersion; GVD: group velocity dispersion; b), Qualitative representation of the spectral and
temporal properties of the pulsed optical signal as it evolves throughout the stages of the all-bre source
(stages 1 to 5) and at the output of the temporal compressor (stage 6) used to compress the pulse down
to its Fourier limited duration. Δλi and Δτi: spectral bandwidth and temporal duration of the pulses,
respectively, at the output of i-th stage; c), Properties of the pulsed signal at the output of each stage, for
an example of implementation of the all-bre conguration where the active bre is Yb-doped, thus with
laser emission in the 1 μm band. MFD: mode eld diameter, λc: central wavelength, PRR: pulse repetition
rate, ΔλFWHM: spectral bandwidth at full width at half maximum, ΔτFWHM: pulse duration at full width
at half maximum, Pavg: average power, Pp: pulse peak power, Ip: pulse peak intensity. Values are given
for a pump laser diode wavelength and average power of 976 nm and 3.75 W, respectively.

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Figure 2
Summary of properties of three representative microstructured bres manufactured in this work,
numbered 1,2 and 3. a), SEM image of the cross section of an ANDi PCF (bre 2), with parameters d =
0.596 μm and Λ = 1.615 μm (detail of bre core region in Fig. 2c2); b), Positions of bres 1, 2 and 3,
according to their corresponding experimental values of d and Λ on the map of calculated values of d
and Λ that provide acceptable dispersion curves for temporally coherent spectral broadening (see
Methods section); c1-3), Detailed SEM images of the core region of bres 1,2 and 3; d1-3), Calculated
dispersion curves for bres 1 to 3, respectively; e1-3), Optical spectra at the output of the ANDi PCF (stage
5 in Fig. 1) obtained with bres 1 to 3, respectively, for same bre length (20 cm) and pump diode (stage
2 of Fig. 1) average output powers of 1.3 W (black line), 3.3 W (red line) and 3.75 W (blue line).

Page 22/27
Figure 3
Independent measurements of the properties of the pulsed signal after the stage of free-space
compression using two different techniques: SHG d-scan technique and interferometric autocorrelation.
a1-5), SHG d-scan retrieval results and resulting pulse in the spectral and time domain; a1), Measured,
calibrated d-scan trace; a2), Retrieved d-scan trace; a3), Red line: measured linear spectrum. Blue line:
retrieved spectral phase. Black line: 4th order polynomial t of the spectral phase; a4), Dashed green line:
temporal intensity prole of the transform limited pulse. Light green: temporal intensity prole of the
measured pulse; a5), Characterizing parameters of the pulse, obtained from the d-scan retrieval; b),
Interferometric autocorrelation trace of the pulse, compressed to its minimum achievable duration. A
pulse width of 12.6 fs is estimated assuming a deconvolution factor 0.71 for a gaussian shaped pulse.

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Figure 4
a), Setup diagram. The excitation laser (red) is focused with a microscope objective on the sample. The
TPEF signal (green) is collected in the backward direction. In the forward direction, the excitation laser
and the SHG signal (blue) are collected with a microscope objective. A dichroic mirror is used to separate
the laser light from the nonlinear signals. The TPEF signal is ltered from the SHG with a narrow
bandpass lter. The TPEF and SHG signal are detected using PMT detectors: the transmitted light from
the excitation laser is detected with a photodiode; b1-3), Mean intensity of the generated TPEF signal as
function of insertion length of the variable compressor glass wedges, for different objectives and sample

Page 24/27
specimens (Convallaria and pollen); c), Interferometric autocorrelation trace of the pulse, measured at the
sample plane, compressed to its minimum achievable duration, for the case of XL Plan N 25x objective.
An estimated duration of 15.6 fs is calculated from the autocorrelation trace (assuming a deconvolution
factor of 0.71 for a gaussian shaped pulse), which is only slightly longer than the duration at the output
of the source (12.6 fs, estimated with the same gaussian-shaped approximation, in Fig.3b). This
difference is mostly due to residual uncompensated higher order dispersion (TOD and above) of the
optics in the path of the beam, and not due to loss of spectral components.
Figure 5
A-D, TPEF images of the tail of a 2-days-old transgenic line zebrash embryo (Caax-GFP) expressing GFP
in all cell membranes. A-C, Intensity-normalised images corresponding to 26, 71, 150 µm depth. Scalebar:
40 µm. D, the lateral re-slice of a Z-stack composed of 300 images (0.71 µm step spacing). Scalebar: 20
µm. E-H, Comparison of TPEF imaging performance between our few-cycle all-bre temporally coherent
supercontinuum source (E, G) and a Coherent MIRA 900-F laser as illuminating sources (F, H), 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. E, Lateral re-slice of a Z-stack composed of 376 images (0.52
µm step spacing) acquired with the few-cycle all-bre source. F, Lateral re-slice of a Z-stack composed of
404 images (0.50 µm step spacing) acquired with the Coherent MIRA 900-F laser. Scalebar: 15 µm.

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Figure 6
A, MAX projection of TPEF images of a mouse intestine section. Nuclei (yellow) and actin laments (blue)
are shown. Scalebar: 10µm. B, MAX projection of a Z-stack of TPEF images of a rhizome of Convallaria
majalis. Chloroplasts (green) and cell walls (red) are shown. Scalebar: 5µm. C, MAX projection of a Z-
stack corresponding to 37 images (1.95 µm step spacing) of autouorescence TPEF images of a pollen
grain. FITC (green) and TRITC (red) ltered signals. Yellow corresponds to the overlap of

Page 26/27
autouorescence signal ltered with the FITC and TRITC lters. Scalebar: 10µm. Selected frames from
the same Z-stack. D, SUM projection of a Z-stack corresponding to 56 images (0.65 µm step spacing) of
SHG signal of the muscle and pharynx (grey) and TPEF from the neurons (red) of a living C. elegans
(OH15500 strain). Scalebar: 25 µm.
Figure 7
Cross section of the PCF to be drawn out, with diameter d and period (pitch) Λ.

Page 27/27
Figure 8
Acceptable values of geometrical parameters. Δλ is the exciting laser bandwidth, 30 nm, which is the
maximum bandwidth obtained in experimental measurements.
Figure 9
Corresponding dispersion curves of acceptable ANDi PCFs, according to the map of Fig. 8.