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Nonlinear Microscopy
FUNCTIONAL 3P NEUROIMAGING
Recording of real-time single-neuron activity in the deep brain
layers of awake animals is crucial for understanding behavior
as well as brain connectivity and function. These applications
have been advanced by neuron imaging and stimulation
using high-power, high-pulse-energy, medium‑repetition‑ratelasers tunable in the SWIR range, which spans the biologicaltransparency windows at 1.3 µm and 1.7 µm. For two‑and three-photon-excited fluorescence (2PEF, 3PEF) andharmonic‑generation (SHG, THG) imaging in deep tissues,
dispersion-controlled femtosecond pulses from ORPHEUS line
OPAs and microscopy-dedicated CRONUS-2P and CRONUS-3P
lasers are truly a state-of-the-art choice.

3P microscopy with adaptive optics for focus sensing and shaping to compensate for both aberrations and scattering. ORPHEUS-F
excitation at 1300 nm enabled imaging up to 1.1 mm below the pia within the intact brain.
Courtesy of Jianan Y. Qu group, the Hong Kong University of Science and Technology. Source: Zh. Qin et al., Deep tissue multi-photon imaging using adaptiveoptics with direct focus sensing and shaping, Nature Biotechnology 40 (2022).

LABEL-FREE IN VIVO WIDEFIELD SHG IMAGING
Nonlinear excitation requires very high peak intensities
and thus has been traditionally limited to laser-scanningmicroscopy using tightly focused beams. For some in vivoand high-throughput applications, however, laser scanning
is too slow. With improved femtosecond laser technology
delivering ever-increasing average power, it is now possible

to excite nonlinear signals over a large area using widefield
nonlinear microscopy. Since optimal excitation conditions
are application dependent, the tunable repetition rate andpulse energy of the PHAROS and CARBIDE lasers as welltheir industrial reliability and low-noise performance are key
parameters when building widefield setups.

Label-free in vivo widefield SHG imaging of fruit fly larva using PHAROS femtosecond laser.
Courtesy of Virgis Barzda group, University of Toronto.

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Despite the advances in 3-photon excitation sources providing
longer wavelengths and higher pulse energies, certain
imaging challenges are still better addressed by tunablehigh-repetition-rate oscillator-based lasers. This is especiallytrue when imaging speed is the primary factor. For theseapplications, the CRONUS-2P laser offers the ultimate solution

with its optically synchronized three-outputs, two of which
are independently tunable. A three-beam source enables avariety of multiphoton excitation pathways, many of which areinaccessible using traditional single- and two-beam solutions.
Furthermore, independent tunability of the two beams
enables new coherent Raman scattering modalities.

2P optogenetic stimulation of individual neurons using CRONUS-2P.
Courtesy of Albert Stroh group, University Medical Center Mainz and Leibniz Institute for Resilience Research. Source: T. Fu et al., Exploring two-photon
optogenetics beyond 1100 nm for specific and effective all-optical physiology, iScience 24 (2021).

RASTER-SCANNING 2P/3P MICROSCOPY
For applications requiring a fixed-wavelength femtosecond
laser, such as multiphoton-driven fluorescence (MPEF), excited
at 1 µm, and harmonic-generation (SHG, THG) microscopy, theFLINT oscillator is a high-performance solid-state source in aproven, industrial-grade package and a compact footprint. In

particular, the FLINT oscillator provides stable 24/7 operation
with excellent noise performance, characterized by a RIN
that is <140 dBc/Hz above 200 kHz and shot-noise-limited at-160 dBc/Hz above 1 MHz.

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2P OPTOGENETICS

SHG and THG images of H&E-stained colon using FLINT femtosecond oscillator.
Courtesy of Virgis Barzda group, Vilnius University.

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Cancer diagnosis and surgical treatment that rely on imaging
require specificity and high throughput. Polarization‑resolved
second-harmonic generation (P-SHG) microscopy shows
potential in visualizing structural changes in collagennetworks and the extracellular matrix accompanying
tumor development. Furthermore, P-SHG is label-free andcompatible with live tissue imaging at depth. However,traditional raster-scanning is too slow for clinical applications,and the structural sensitivity of P-SHG is often hard toCourtesy of Virginijus Barzda group, University of Toronto, and Brian C. Wilson group, Princess Margaret Cancer Centre. Source: Mirsanaye et al., Unsupervised
determination of lung tumor margin with widefield polarimetric second-harmonic generation microscopy, Scientific Reports 12 (2022).

SHG, THG, AND 2P IMAGING
Fixation methods such as formalin are commonly used for thepreservation of tissue with the aim of keeping their structure
as close as possible to the native condition. However, fixatives
chemically interact with tissue molecules and may thus
modify their structure. Taking advantage of the second- and2PEF

forward scattered SHG

third‑harmonic generation (SHG and THG) emission capabilities
of such components, nonlinear two-photon (2P) microscopy
was used to evaluate the effect that preservation methods,
such as chemical fixatives, have on the nonlinear capabilities ofprotein components within mouse tissues.
forward scattered THG

mergedFreshback scattered SHG

2 days 4 °C – 4%PFA

78interpret. Nonlinear widefield microscopy utilizes amplified
femtosecond lasers to increase imaging throughput and thefield of view, while machine learning (ML) enables data-driven
analysis that can, for example, automate tumor margin
delineation and scoring. PHAROS and CARBIDE lasers, inconjunction with ML‑augmented widefield microscopy, havethe potential to bring the benefits of nonlinear microscopy to
the scale required for biomedical and clinical applications.

Large-area widefield polarization-resolved SHG microscopy of human lung tissue tumor margin using the PHAROS laser. Imageparameters such as SHG intensity, R-ratio, and degree of circular polarization, as well as SHG circular and linear dichroism, are usedin unsupervised machine learning to determine the tumor boundary.

4%PFA

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WIDEFELD POLARIMETRIC SHG MICROSCOPY

SHG signals fromcollagen, 2PEF and THG
signals from elastin
in vibratome sectionsof mouse kidney after
different treatments,using CRONUS-2P
femtosecond laser
source.
Courtesy of Frauke Alves andFernanda Ramos-Gomes,
Max-Planck Institute forMultidisciplinary Sciences,
Germany.
DOI: 10.1364/BOE.488453

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Adult zebrafish heart ventricle section from a scar formation
study. The brightfield image is stained with Masson’s trichrome(MT), connective tissue is blue, muscle is red/brown.

SHG and THG images reveal collagen and muscle structure atthe periphery of bulbus arteriosus, while MT-stained elastin isvisualized in the center in THG.

Adult zebrafish heart ventricle section, FLINT femtosecond oscillator used for imaging.
Samples courtesy of Justas Lazutka at the Vilnius University Life Sciences Center. Nonlinear imaging courtesy of the Barzda group at the Vilnius University
Department of Physics.

HOLOGRAPHIC 2P OPTOGENETICS
Traditional full-brain neurostimulation using CW light lacks
specificity, whereas point-by-point laser-scanning, despite
being highly specific, can be slow and is not simultaneous.
Holographic multiphoton neurostimulation, on the otherhand, is capable of random-access-style volumetric neuronactivation and is therefore used for advanced behavioral

neuroscience studies. Holographic stimulation is exceptionallydemanding on the laser source, requiring very high average
power, combined with complex on-demand pulse train
control – features that are well supported by the CARBIDE andPHAROS laser families.

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COMBINED SHG AND THG IMAGING

Holographic 2P optogenetic stimulation of mouse olfactory bulb neurons using laser system with PHAROS femtosecond laser.
Courtesy of Shy Shoham and Dmitry Rinberg groups, New York University. Source: J. V. Gill et al., Precise holographic manipulation of olfactory circuits reveals
coding features determining perceptual detection, Neuron 108 (2020).

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