Y-Laser and Fatonics delivered the AURORA-3P three-photon source for bioimaging

2025.12.30

Three-photon microscopy is an advanced nonlinear optical imaging technique that uses ultra-short pulsed infrared lasers as the light source. Due to the strong penetration ability of infrared light, three-photon imaging can penetrate deep into tissues without the significant scattering or absorption that occurs with visible light at the surface. This gives the technique a significant advantage in high-resolution imaging of deeper tissues. Compared to traditional single-photon and two-photon microscopy, three-photon imaging provides greater imaging depth and clearer cellular or tissue structure information, making it highly suitable for applications in neuroscience and biomedical research.

Currently, the exploration of the brain mainly relies on optical microscopy, but the scattering of light by neural tissue prevents the light beam from penetrating deeply into the brain.
The insufficient imaging depth significantly hinders the quality of brain neural imaging.Therefore, achieving deep brain imaging has become an important research focus in brain neural imaging.
The emergence of multiphoton microscopy has alleviated, to some extent, the challenge of insufficient imaging depth. Multiphoton imaging is based on the multiphoton excitation effect. Fluorescent molecules have discrete energy levels, with the lowest energy level called the ground state, where most molecules reside, and the higher energy levels called the excited states. When illuminated with light of a specific wavelength, electrons in the ground state can absorb multiple photons and transition to the excited state. After remaining in the excited state for a certain period, the electrons relax and transition back to the ground state, emitting a photon with a corresponding wavelength. If the incident light wavelength is longer, it may cause the ground state molecules to absorb multiple photons simultaneously, leading to multiphoton excitation.

Three-photon vs Two-photon

		In the past 30 years, the emergence of two-photon microscopy (2PM) has greatly accelerated brain imaging research, offering extremely high spatial resolution and the ability to image deeper layers of the brain. However, the imaging depth of 2PM is limited to tens to hundreds of microns. To perform fluorescence imaging in deeper biological tissues at the millimeter scale, other imaging techniques need to be developed.

		Single dendritic spine-level resolution under a 640 µm field of view. Thy1-YFPH transgenic mouse (left) and wild-type mouse brain cortex injected with AAV-hSyn-GCaMP6s virus (right). Probe model: FHIRM-U. Imaging depth: 0-60 µm projection (left), 200-260 µm projection (right). Excitation wavelength: 920 nm. Imaging of freely moving mouse. Right image: Miniature three-photon microscope records structural and functional dynamics in the mouse brain cortex (L1-L6) and hippocampus CA1.

The concept of three-photon microscopy (3PM) was proposed in 1996, and it is expected that this technology could further extend the depth of fluorescence imaging. It utilizes the three-photon excitation effect, where fluorescent molecules absorb three long-wavelength photons and release a fluorescence photon through radiative transitions. However, due to the extremely high excitation pulse power required for 3PM, and the need for the development of long-wavelength lasers that are not yet mature, it has not received widespread attention. Recently, however, it has been validated that the optimal excitation wavelength for deep brain imaging is around 1300 nm and 1700 nm, which is too long for the two-photon excitation (2PE) of most existing fluorescent probes. Additionally, higher-order nonlinear effects provide a higher nonlinear threshold compared to 2PE, which is essential for high-contrast imaging in scattering media.

CC: Corpus callosum. Green represents GCaMP6s-labeled neuronal calcium signals, while magenta represents third-harmonic signals from the dura mater, microvasculature, and white matter interface.

On-site delivery：

In response to the growing demand in the neuroscience imaging market, Y-Laser Femtosecond and V-Quick Photonics collaborated to develop and successfully deliver a femtosecond laser for three-photon imaging, designed for mass production, to Beijing Supervision Science Biotechnology Co., Ltd. This product competes with models such as Light Conversion's CRONUS-3P, Class5 Photonics' WD-1300, and Coherent's MONACO-1300 series.

Specification：

1）Lower repetition rates and higher pulse energies are available upon request.

2）Continuous dispersion control; −3000 fs² to +3000 fs² compensation for microscopy.
3）Pump laser: 50W / 50µJ / 1030nm, supports higher power customization. Please contact us for details.
4）Measured after compression at 1/e²
5）Optional expansion for 650-900nm output available. Please contact us for details.

Measured parameters：


Typical spectrum (1250-1800nm)		Three-photon system + compressor output power comparison (drive laser :1MHz/40uJ/301fs@HELIOS-40W）


Typical pulse width（1300nm@55.4fs）		Typical pulse width（1700nm@60.5fs）


Output spot @1300nm (compressor outlet 500mm）		GDD Rang

AURORA-3P series 24H output power stability RMS = 0.4289% @ 1300nm

In vivo imaging for biological imaging applications：

Note: The three-photon imaging system is provided by Beijing TRANSCEND VIVOSCOPE Co., Ltd.