# Multiphoton imaging via spectro-temporally tailored femtosecond pulses for dynamically reconfigurable excitation and multimodal analysis of biological samples

Erwan Ferrandon, Jérémy Saucourt, Bruno P Chaves, Alexis Bougaud, Manal

Arbati, Vincent Couderc, Stéphanie Durand-Panteix, Claire Carrion, Fabert

Marc, Benjamin Wetzel

► To cite this version:

Erwan Ferrandon, Jérémy Saucourt, Bruno P Chaves, Alexis Bougaud, Manal Arbati, et al. Multiphoton imaging via spectro-temporally tailored femtosecond pulses for dynamically reconfigurable excitation and multimodal analysis of biological samples. 19ème colloque de la Société française des Microscopies, SFμ, Jun 2025, Toulouse, France. hal-05238589

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Submitted on 29 Sep 2025

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# Multiphoton imaging via spectro-temporally tailored femtosecond pulses for dynamically reconfigurable excitation and multimodal analysis of biological samples

Erwan FERRANDON<fnref n="1" />, Jérémy SAUCOURT<fnref n="1" />, Bruno P. CHAVES<fnref n="1" />, Alexis BOUGAUD<fnref n="1" />, Manal ARBATI<fnref n="1" />, Vincent COUDERC<fnref n="1" />, Stéphanie DURAND-PANTEIX<fnref n="2" />, Claire CARRION<fnref n="2" />, Marc FABERT<fnref n="1" />, Benjamin WETZEL<fnref n="1" />

## Session : Avancées instrumentales / Imagerie Corrélative et Multimodale

Multiphoton microscopy has enabled significant progress in the observation of biological samples inherent to the specificities of their pulsed laser sources. The resulting nonlinearity makes it possible to reveal biological structures without fixation or labeling, such as the generation of second harmonics (SHG) on collagen fibers or the third harmonic (THG) on lipids[1-2-3]. Multimodal imaging enables bundling different imaging modalities on a single microscope, by e.g. adding vibrational information as Coherent Anti-Stokes Raman Scattering (CARS) with SHG, THG and multiphoton fluorescence via suitable excitation and detection[4]. However, multimodal imaging performances strongly depend on the characteristic of the illumination sources, for which femtosecond lasers feature limited tunability and scalability: illuminations are often inadequate (i.e. pulse duration, spectral selectivity, bandwidth coverage), yielding limited performances and hinder acquisition's important data when observing samples where the endogenous response can be induced at specific excitation wavelength(s).

Here, we experimentally implement femtosecond pulses with reconfigurable spectro-temporal properties towards flexible multimodal microscopy, via dynamically adjustable multiphoton excitations. Specifically, dual femtosecond lasers with synchronized emissions at 1040 and 1550nm are leveraged for reconfigurable pump-probe excitations. Using spectral pulse shaping techniques, on-chip temporal processing[5-6] prior to fiber propagation and nonlinear spectral broadening, the excitation at 1550nm can be adjusted before sample illumination. By means of spectro-temporal reconfigurability[7] we can conjointly scan and excite several fluorescence responses and vibrational bands on biological samples in order to extract the most complete endogenous information possible[8-9]. Spectral reshaping further enables multiplexing imaging of stained biological samples.

Probing the excitation parameters, multichannel dataset with variable spectral responses can be acquired (Fig.1A-B), enabling image processing via Principal Component Analysis (PCA)[10] and introducing advanced machine-learning strategies to gain insight into sample characteristics (Fig.1C). This approach opens opportunities for image segmentation, analysis and unsupervised learning, especially in the field of medical analysis and the detection of pathologies.

<footnote n="1">¹ XLIM Research Institute, CNRS UMR 7252, Université de Limoges, 87060 Limoges, France</footnote>

<footnote n="2">² CNRS, INSERM, CHU Limoges, BISCEm, UAR 2015, US 42, Univ. Limoges, F-87000, Limoges, France</footnote>
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**Figure 1:** Acquisition of a renal blood vessel section embedded in paraffin, observed simultaneously with synchronized 1040nm and 1550nm femtosecond lasers. In this example, the 1550nm laser excitation can be dynamically adjusted in the temporal and spectral domain by a reconfigurable on-chip component, to tune the strength of multiphoton absorption around 1550nm and/or adjust the temporal overlap and resonance selection of CARS signals. (A) Image acquisition with randomly selected chip parameters; (B) Image acquisition of the same area with optimal chip parameters for enhanced peak power and temporal overlap between two laser excitation, providing complementary response on different detection channels; (1) Detection channel 410-465nm: Three-photon emission from 1040nm excitation wavelength; (2) Detection channel 495-540nm: SHG from 1040nm; (3) Detection channel 575-640nm: Two-photon emission from 1040nm; (4) Detection channel 750-800nm - Near infrared two-photon emission from hybrid excitation and CARS response (3100cm⁻¹). (C) Example of image reconstruction via PCA. Two hundred images with variable illumination conditions are analyzed to reconstruct a six channels images with synthetic colors (from C-I, the first principal component, to C-VI, the sixth). Here, the six channels used for color-coding each provide at least from 35% to 2% of the PCA content (i.e. 60% in total information on 6 channels). In this case, one can observe 200 hundred images clearly by PCA processing.
Références :

[1] P. Friedl, K. Wolf, UH. von Andrian, G. Harms, Biological second and third harmonic generation microscopy. (2007) Curr Protoc Cell Biol Chapter 4: Unit 4 15.

[2] P-C. Cheng, CK. Sun, Nonlinear (harmonic generation) optical microscopy. In: Pawley JB, ed. Handbook of biological confocal microscopy. (2006) 3rd ed. New York, NY, USA: Springer Science+Business Media LLC. 703–721.

[3] PJ. Campagnola, Second-Harmonic Generation Imaging microscopy of structural proteins in arrays in tissues. (2008) In: Masters BR, So PTC, eds. Handbook of biomedical nonlinear optical microscopy. New York, NY: Oxford University Press. pp 377–411.

[4] JX. Cheng and X. Sunney Xie. Coherent Anti-Stokes Raman Scattering Microscopy: Instrumentation, Theory, and Applications. The Journal of Physical Chemistry B 2004 108 (3), 827-840

[5] B. Wetzel, M. Kues, P. Roztocki, C. Reimer, P.L. Godin, M. Rowley, B.E. Little, S.T. Chu, E.A. Viktorov, D.J. Moss, A. Pasquazi, M. Peccianti, R. Morandotti, Customizing supercontinuum generation via on-chip adaptive temporal pulse-splitting Nature Communications, Vol. 9, pp. 4884 (2018)

[6] S. Sciara, H. Yu, M. Chemnitz, N. Montaut, B. Crockett, B. Fischer, R. Helsten, B. Wetzel, T. A. Goebel, R. Kramer, B. Little, S. Chu, S. Nolte, Z. Wang, J. Azana, W. Munro, D. Moss, R. Morandotti, Quantum key distribution implemented with d-level time-bin entangled photons, Nature Communications, Vol. 16, pp. 171 (2025)

[7] V. T. Hoang, Y. Boussafa, L. Sader, S. Février, V. Couderc, B. Wetzel, Optimizing supercontinuum spectro-temporal properties by leveraging machine learning towards multi-photon microscopy, Frontiers in Photonics, Vol. 3, pp. 940902 (2022)

[8] X. Gao, X. Huang, Z. Chen, et al. Supercontinuum-tailoring multicolor imaging reveals spatiotemporal dynamics of heterogeneous tumor evolution. Nat Commun 15, 9313 (2024).

[9] L. Wei, Z. Chen, L. Shi, et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (2017).

[10] J. T. Vanderplas, Python data science handbook: essential tools for working with data, Second edition. Sebastopol, CA: O’Reilly Media, 2022.

Adresse mail : erwan.ferrandon@unilim.fr / benjamin.wetzel@xlim.fr

This work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme under GA 950618 (STREAMLINE). B.W. acknowledges the support of the French ANR through the LabEx Σ-LIM (ANR-10-LABX-0074), the OPTIMAL project (ANR-20-CE30-0004) and the Région Nouvelle Aquitaine (SPINAL project). This research benefited from the support of the Platinum platform, with funding the European Union and the Nouvelle Aquitaine council under the PILIM program.