Light Conversion Expands CRONUS Femtosecond Laser Line to Advance Nonlinear Microscopy

Nonlinear microscopy is a powerful technique for imaging inside living organisms with submicrometer resolution at up to millimeter depths. In combination with genetically encoded fluorescent indicators and opsins, multiphoton microscopy has revolutionized intravital and neuroimaging and is becoming a standard tool in bioscience.

To further increase support for these demanding applications, Light Conversion is expanding its CRONUS microscopy-dedicated line of femtosecond lasers with two additional CRONUS-2P configurations. The new models offer greater flexibility in selecting excitation sources optimized for diverse experimental requirements – allowing users to focus on their research rather than the light source behind it.

MULTIPHOTON MICROSCOPY ENABLES DEEP-TISSUE IMAGING

One of the main challenges of imaging complex biological samples is optical scattering. As imaging depth increases, the signal-to-noise ratio quickly deteriorates. Serendipitously, most biological tissue is more transmissive at longer wavelengths in the near-infrared range needed for multiphoton excitation of commonly used fluorophores. 

Rather than relying on a single photon of a given wavelength to excite a fluorescent molecule, two-photon imaging uses two photons of half the energy – and twice the wavelength – to simultaneously interact with the fluorescent molecule and promote it to the excited state. Because fluorescence is generated only at the focal point, the technique provides inherent optical sectioning, allowing high-contrast images to be acquired several hundred micrometers deep inside tissue.

ADDRESSING THE CHALLENGES OF MULTIPHOTON IMAGING

Simultaneous photon–molecule interactions require extremely high photon densities, which can only be achieved using ultrashort laser pulses. However, pulse energies sufficient for nonlinear excitation may also trigger higher order photodamage mechanisms that can impair biological tissue. Additionally, near-infrared light is absorbed by water present in biological tissues, increasing the risk of sample heating. For this reason, carefully managing the power and energy delivered to the sample is essential – particularly when imaging sensitive specimens such as developing tissues.

To enable simultaneous observation of different tissue structures within a sample, researchers frequently employ multiple fluorescent labels or indicators with minimal spectral overlap. This approach, however, requires careful optimization of the imaging setup to selectively address different fluorophores.

An effective solution for meeting these requirements is the use of multiple independently tunable excitation wavelengths. This allows researchers to optimize excitation conditions for each fluorophore while minimizing the energy load on the sample. As a result, imaging contrast can be improved while maintaining efficient optical sectioning deep inside biological tissue.

The CRONUS-2P platform provides these capabilities by delivering multiple watt-level synchronized outputs, short pulse duration, and integrated group delay dispersion (GDD) control – making it well suited for demanding nonlinear microscopy applications.

This architecture enables simultaneous excitation of multiple fluorescent probes, calcium indicators, or optogenetic actuators at their absorption maxima. It also allows researchers to spectrally shift second-harmonic generation (SHG) and third-harmonic generation (THG) signals to simplify detection or achieve resonant enhancement.

NEW CRONUS-2P MODELS EXPAND IMAGING FLEXIBILITY

To broaden accessibility and better match specific microscopy workflows, Light Conversion now offers the CRONUS-2P femtosecond laser platform in three configurations, allowing researchers to select the system best suited to their experimental requirements.

The baseline CRONUS-2P configuration provides one tunable output covering 700–1100 nm together with a fixed output at 1025 nm, both accessible simultaneously within a compact 633 × 230 mm footprint – less than half the size of many comparable light sources.

The CRONUS-2P-XR model extends the tunable wavelength range to 680–1300 nm while maintaining the fixed 1025 nm output, increasing compatibility with a broader range of fluorophores and nonlinear imaging modalities.

For applications requiring maximum flexibility, the CRONUS-2P-DUAL configuration provides two independently tunable outputs covering 680–960 nm and 940–1300 nm, which can operate either separately or simultaneously. A third output fixed at 1025 nm is accessible in parallel, and configurations with identical tunable outputs are also available.

The synchronized tunable outputs further enable advanced coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) experiments, supporting applications such as dual-band imaging, access to a broader range of vibrational resonances, resonant enhancement, and constant-difference dual-beam tuning.

CRONUS LASERS FOR ADVANCED MICROSCOPY

The CRONUS-2P platform is part of Light Conversion’s broader CRONUS series of microscopy-dedicated femtosecond laser sources, which also includes the CRONUS-3P for advanced nonlinear microscopy.

Together, these lasers support applications in functional neuroimaging, optogenetics, and deep-tissue imaging using medium-repetition-rate three-photon excitation, as well as two-photon imaging. The CRONUS series also enables widefield and holographic excitation techniques requiring high-power femtosecond sources.

With the expanded CRONUS-2P lineup, researchers can select the configuration that best matches their experimental requirements while maintaining the imaging flexibility provided by synchronized multi-channel excitation.