Three photon imaging of multi-color deep layers with a single wavelength 1300 nm light source

Three photon (3P) excitation of fluorescent proteins and probes has aroused great interest, especially in the field of neuroscience applications. As confirmed by Chris Xu and other researchers, an important reason is that the 1300 nm and 1700 nm wavelength windows used for three photon excitation bring deeper penetration.

Three photon excitation can also provide a higher signal-to-noise ratio than two-photon excitation, and there is almost no defocused fluorescence. This allows deeper imaging in the living mouse brain by penetrating the cerebral cortex>1 mm thick.
It is also important that the 1300 nm wavelength meets the three photon excitation energy requirements of green fluorescent proteins and probes (such as dextran), while 1700 nm can be used to stimulate longer wavelength targets, such as tdTomato.

Some researchers hope to stimulate multiple probes at the same time to conduct more complex research on mammalian brain. This information rich data can accelerate the process of researchers to understand the relationship between neural connections and activities and important functions.

The short wave and long wave probes can be imaged simultaneously by using two excitation wavelengths. However, there are some limitations. First, it is complex (and more expensive) to generate two wavelengths of light and combine them in a microscope. Then is the power load problem; Using two laser sources to excite living tissue means using twice the laser power on the sample. In addition, until recently, there was no simple ("integrated") light source for 1300 nm or 1700 nm, so researchers usually use a laser of about 1040 nm to pump tunable OPA to generate 1300 nm or 1700 nm.

However, the combination of the two developments makes the three photon excitation of short wavelength and long wavelength probes easier, which can be accepted by a wider range of users.

1300 nm excited long wave probe

Several recent three photon imaging studies have shown that 1300 nm can be used to excite green and red fluorescent probes. The filters are then used to detect light from both probes in both cameras. For example, Timo van Kerkoerle and his doctoral student Marie Guillemant used Monaco and Opera － F to generate 1300 nm laser, demonstrating that dextran and tdTomato labeled interneurons were simultaneously stimulated in the mouse prefrontal cortex by three photon excitation (as shown in the figure). The z-stack image here shows that the redshift probe excitation signal is significant even at a depth of about 1 mm.

Data provided by Dr. Timo van Kerkoerle and Marie Guillemant, Neuropin, CEA Saclay.

Fig. Three photon imaging of dextran (green) and tdTomato (red) labeled interneurons in mouse prefrontal cortex, with a depth of about 1 mm.

Many commonly used red fluorescent molecules used to be excited to the lowest energy state at 1700 nm before imaging. This work shows that these commonly used red fluorescent molecules can also be excited to higher energy electronic states at 1300 nm. This new excitation mechanism allows dual green and red three photon fluorescence imaging in the mouse brain using only a 1300 nm laser.

We believe that these findings of Timo van Kerkoerle and Chris Xu will obviously affect the future development direction of imaging and three photon excitation of functional fluorescent probes.

Integrated 1300 nm pulse light source

The recently launched Coherent Monaco 1300 integrated laser provides a 1300 nm femtosecond light source for three photon excitation. Since the brightness of the three photon image is proportional to the third power of the laser peak power, this laser is very suitable for three photon imaging because of its short pulse width and high peak power.

Easy to use automatic light source with pulse width less than 50 fs.

Up to 2.5 W power output, 1, 2 or 4 MHz repetition frequency can be selected, and fast image acquisition is supported.
High quality (M2 ＜ 1.3), improving the imaging quantity, imaging efficiency and image resolution of the microscope in three-dimensional space.

The integrated structure also includes two common functional options to simplify three photon imaging and improve image brightness.

Full power control (TPC) function with dynamic power attenuation/gating

Compact pulse compressor (CPC) providing dispersion precompensation to obtain ideal pulse width at the sample

The bright future of triphoton imaging

The use of a single laser source to stimulate multiple probes is expected to promote the development of neuroscience. This technology is based on the deep mapping of multiple cell types across the cortex to achieve rapid and informative imaging of brain capacity. The development of three photon excitation technology and related mechanisms will undoubtedly help guide researchers who are deciding whether to choose tunable or single wavelength three photon excitation light sources, and highlight the value of solutions (such as Coherent Monaco 1300).

Source: Coherent Gaoyi Laser