Nonlinear microscopy is a powerful technique for imaging inside living organisms with a submicrometer resolution at millimeter depths. In conjunction with genetically-encoded calcium indicators and opsins, multiphoton fluorescence (MPEF) microscopy has revolutionized neuroimaging and is becoming a standard tool in neuroscience, while label-free methods such as second- and third-harmonic generation (SHG and THG), and coherent anti-Stokes and stimulated Raman scattering (CARS and SRS) have been developed into ultrasensitive structural and chemical imaging techniques.

The nonlinear optical processes of nonlinear microscopy require high light intensities, which can be reached at low average power when ultrashort light pulses are tightly focused. This feature is exploited to provide optical sectioning and to improve imaging contrast deep inside scattering tissues. Multiphoton excitation occurs when two or more photons simultaneously pass in the vicinity of a molecule, and their combined energy is used for excitation leading to fluorescence. The simultaneous arrival of several photons can also result in a harmonic generation – radiation at double or triple of the excitation laser frequency. Harmonic generation is an intrinsic label-free contrast determined by and used to characterize the molecular order and homogeneity of the sample.

Higher-order, three- and four-photon-excited (3PEF and 4PEF), fluorescence microscopy deserves special attention as it enables imaging at depths that cannot be achieved with conventional microscopy techniques, especially in strongly scattering samples such as the brain. Most importantly, modern laser sources can operate at the required repetition rates and deliver the necessary pulse energy for real-time functional brain imaging at biologically-relevant depths.

LIGHT CONVERSION product portfolio with recently released nonlinear microscopy-dedicated femtosecond laser sources, CRONUS-2P and CRONUS-3P, covers applications in functional neuroimaging, optogenetics, and deep imaging using medium-repetition-rate three-photon excitation and fast high‑repetition-rate two-photon imaging, as well as widefield and holographic excitation using high-power laser sources. See the CRONUS series comparison table below, while the complete list of laser sources for nonlinear microscopy and examples of the state-of-the-art applications are available in our latest brochure.

  • High pulse energy for deep imaging
  • 1250 – 1800 nm tuning range for 3P imaging
  • Down to 50 fs pulse duration for high peak power
  • Automated wavelength and GDD control for optimal signal
  • Market-leading pulse-to-pulse energy stability
  • Watt-level output at high repetition rate for fast imaging
  • Two tunable and one fixed output for simultaneous multibeam excitation
  • Automated GDD control for shortest pulses at the sample
  • Industrial-grade design for high power and beam stability
  • Combination of best collinear and non-collinear OPA features
  • Ultrashort pulses in NIR (650 – 900 nm and 1200 – 2500 nm)
  • Single-shot – 2 MHz repetition rate
  • < 100 fs pulse duration
  • Adjustable spectral bandwidth
  • Optional long pulse mode for gap-free tunability
  • Two simultaneous independently tunable outputs
  • 210 – 16000 nm tuning range
  • Single-shot – 2 MHz repetition rate
  • Up to 60 W, 0.5 mJ pump
  • Compact and cost-effective
  • CEP-stable option
  • 11, 40, or 76 MHz repetition rate
  • Down to 50 fs pulse duration
  • High-power models, up to 20 W
  • High-energy energy models, up to 0.6 µJ
  • Industrial-grade design for high output stability
  • CEP stabilization or repetition rate locking
  • Tunable pulse duration, 100 fs – 20 ps
  • Maximum pulse energy of up to 4 mJ
  • Down to < 100 fs right at the output
  • Pulse-on-demand and BiBurst for pulse control
  • Up to 5th harmonic or tunable extensions
  • CEP stabilization or repetition rate locking
  • Thermally-stabilized and sealed design
  • Tunable pulse duration, 190 fs – 20 ps
  • Maximum output of 120 W and 2 mJ
  • Single-shot – 2 MHz repetition rate
  • Pulse-on-demand and BiBurst for pulse control
  • Up to 5th harmonic or tunable extensions
  • Air-cooled model
  • Compact industrial-grade design

Effect of tissue fixation on the optical properties of structural components assessed by non-linear microscopy imaging

M. A. Markus, D. P. Ferrari, F. Alves, and F. Ramos‑Gomes, Biomedical Optics Express 8 (14), 3988 (2023).

We need to talk about laser pulse energy stability

L. Kontenis, M. Urbšas, J. Berzinš, and K. Neimontas, in Multiphoton Microscopy in the Biomedical Sciences XXIII, A. Periasamy, P. T. So et al., eds. (SPIE, 2023).

Deep tissue multi-photon imaging using adaptive optics with direct focus sensing and shaping

Z. Qin, Z. She, C. Chen, W. Wu, J. K. Y. Lau, N. Y. Ip, and J. Y. Qu, Nature Biotechnology (2022).

Ketogenic diet uncovers differential metabolic plasticity of brain cells

T. Düking, L. Spieth, S. A. Berghoff, L. Piepkorn, A. M. Schmidke, M. Mitkovski, N. Kannaiyan, L. Hosang, P. Scholz, A. H. Shaib et al., Science Advances 37 (8) (2022).

Machine learning-enabled cancer diagnostics with widefield polarimetric second-harmonic generation microscopy

K. Mirsanaye, L. U. Castaño, Y. Kamaliddin, A. Golaraei, R. Augulis, L. Kontenis, S. J. Done, E. Žurauskas, V. Stambolic, B. C. Wilson et al., Scientific Reports 1 (12) (2022).

Biodegradable Harmonophores for Targeted High-Resolution In Vivo Tumor Imaging

A. Y. Sonay, K. Kalyviotis, S. Yaganoglu, A. Unsal, M. Konantz, C. Teulon, I. Lieberwirth, S. Sieber, S. Jiang, S. Behzadi et al., ACS Nano 3 (15), 4144-4154 (2021).

Direct focus sensing and shaping for high-resolution multi-photon imaging in deep tissue

Z. Qin, Z. She, C. Chen, W. Wu, J. K. Y. Lau, N. Y. Ip, and J. Y. Qu, (2021).

Exploring two-photon optogenetics beyond 1100~nm for specific and effective all-optical physiology

T. Fu, I. Arnoux, J. Döring, H. Backhaus, H. Watari, I. Stasevicius, W. Fan, and A. Stroh, iScience 3 (24), 102184 (2021).

Fast optical recording of neuronal activity by three-dimensional custom-access serial holography

W. Akemann, S. Wolf, V. Villette, B. Mathieu, A. Tangara, J. Fodor, C. Ventalon, J. Léger, S. Dieudonné, and L. Bourdieu, Nature Methods 1 (19), 100-110 (2021).

In-vivo tracking of harmonic nanoparticles: a study based on a TIGER widefield microscope [Invited]

L. Vittadello, C. Kijatkin, J. Klenen, D. Dzikonski, K. Kömpe, C. Meyer, A. Paululat, and M. Imlau, Optical Materials Express 7 (11), 1953 (2021).

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