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2024

Microscopic "upstart", looking deeper: looking ahead to three-photon microscopic imaging technology

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Since the two-photon microscopy (2PM) was first reported in 1990, based on the nonlinear effect of femtosecond laser, the two-photon microscope has two obvious advantages compared with the microscope regular continuous light excitation (such as confocal microscope).AdvantagesThe two-photon excitation wavelength is longer, which can reduce the photobleaching and light damage to the sample to a certain extent; the optical slicing ability is brought based on the nonlinear effect, so that the imaging system does not need confocal small holes.¹

Compared with the visible light band, near-infrared light has higher penetration and lower attenuation in biological tissues, which makes the imaging depth deeper, making most of the two-photon microscopes excited by near-infrared wavelengths quickly become an important tool for the study of the structure and function of brain science. Analogous to other optical imaging technologies, such as optical coherence tomography (OCT) and photoacoustic imaging (PAM/PACT), other technologies have the same advantages in imaging depth, but it is usually difficult to achieve the submicron cell imaging resolution level of two-photon microscopy.

Further, a Cornell University research team found in 2013three-photon microscope(Three-photon microscppy, 3PM) Compared with two-photon, femtosecond laser can be used to a greater extent to excite and image deeper samples with higher nonlinear coefficients. Since then, three-photon microscopic imaging technology has gradually entered everyone's field of vision, highlighting the "power".

Fig.1 Quality comparison of two-photon and three-photon microscopic imaging at different imaging depths in mouse brain

As shown in Figure 1, in the examples of two-photon and three-photon microscopic imaging, with increasing depth (> 550 μm), the three-photon imaging resolution at deep tissue is significantly due to two-photon microscopy. Combining adaptive optics technology can further yield exciting results--Three-photon microscopy can even image neurons and blood vessels directly through the mouse skull,as shown in Figure 2.^2-5

Figure 2 Three-photon imaging of mouse neurons and vasculature through the intact skull using adaptive optics

It is well known that imaging depth is limited by the scattering and absorption of photons within biological tissue. As far as the excitation light is concerned, as the imaging position is deeper, fewer photons reach the focal plane. It seems that the loss of photons can be compensated by increasing the laser power, but too much light power on the surface of the biological sample may cause the risk of sample damage. Even when the excitation beam power is controlled below the tissue damage threshold, as the focal plane becomes deeper, the excitation beam produces varying degrees of background noise in the sample surface-to-focal plane "cone. When a certain depth is reached, the back noise intensity will completely annihilate the signal, eventually limiting the imaging depth. In two-photon imaging, once the target imaging depth exceeds the imaging signal-to-background ratio (SBR) limit, it is difficult to improve the image quality even if the excitation light power is increased. Three-photon imaging can effectively reduce the background and obtain a deeper range than two-photon imaging because of its higher nonlinear conditions.

Three-photon microscopic imaging seems to have such a significant advantage, but it still seems to face many technical problems in practical application.Allen Brain Institute (USA)"the current situation of three-photon imaging technology is similar to that of the early two-photon birth-that is, within a few years of the new imaging technology, people still need to spend a lot of energy to obtain high-quality and meaningful images in actual imaging," said the scholar." So,Compared with the more mature two-photon imaging technology, what are the difficulties in the development of three-photon imaging technology, and how to overcome them?

Selection of excitation wavelength

Fig. 3 Schematic curve of tissue attenuation (scattering absorption) from 800 to 2800 nm, mainly illustrating the biological imaging window at 1300 nm and 1700 nm

Compared with the two-photon process, three-photon excitation requires a longer incident wavelength. For example, the commonly used neural tracer GCaMPs green fluorescent protein has a two-photon excitation band of 920-940 nm, but the wavelength needs to be extended to ~ 1300 nm when excited by three photons. For another example, the red-shift probe requires a two-photon excitation wavelength of ~ 1100 nm, while the three-photon excitation is required at ~ 1700 nm.^4,5therefore, it can be seen that,Wavelength tunable lasers, compared to single-wavelength lasers, can meet the excitation wavelength tuning requirements of different fluorescent molecules, and occupy the mainstream in two-photon and three-photon imaging research. In practical applications, in terms of wavelength, there is still a large space for exploration for various stable tuned lasers, imaging windows, imaging probes and other issues.

Pulse width and compression

Femtosecond lasers used in two-photon imaging generate pulse widths of 100 to 150 fs. The higher nonlinearity on which the three-photon process relies depends to a large extent on the peak power of the pulse, so three-photon imaging requires a shorter pulse width (~ 50 fs). But in most cases,How to generate stable and shorter (~ 50 fs) femtosecond pulsed light is still one of the challenges of three-photon technology.. In addition, because the ultrashort pulse beam propagates in the material, there will be group delay dispersion (group delay dispersion, GDD), resulting in different spectral components are stretched in time, in the time domain reflected in the width of the pulse width, so that the pulse peak power is reduced, resulting in the reduction of multiphoton signal intensity and image signal-to-noise ratio.

In order to solve the above dispersion problem and generate the best nonlinear signal at the focal plane, pulse compression elements are often added to the optical system to pre-compensate the group delay dispersion (pre-chirp). However, a shorter pulse width means a larger spectral bandwidth, which makes the design and manufacture of pulse width compression components quite challenging. Therefore, in the course of the development of multi-photon imaging technology, many outstanding imaging researchers have focused on solving the femtosecond pulse management and constructing suitable pulse width compression components, so that the pulse width on the focal plane is the shortest.

The degree of pulse width compression is critical. When the pulse width compression is sufficient, the necessary conditions for three-photon signal acquisition are guaranteed. If the compression is insufficient and the pulse width is too large, even if the power is increased to the level of burning out the sample, the three-photon signal cannot be detected at all. From this, it can be seen that for three-photon imaging, the research of the laser itself is still promising and challenging.

Pulse energy, repetition rate and average power

From the realization of two photons to the realization of three photons, due to the difference in the excitation cross section of the two, the realization of three photons not only needs to shorten the pulse to about ~ 50 fs, but also needs to greatly increase the single pulse energy. However, commercially available microscope objectives and other optical components are rarely designed and optimized for high transmission and low dispersion for three-photon excitation wavelengths, which is likely to make the transmittance of the whole system only about 10%. Therefore, we should fully consider how to weigh the relationship between the three main parameters of peak power (pulse energy), repetition frequency and average power.

In most cases, the average power of the femtosecond laser ~ 100 mW can ensure that the biological sample is not damaged during spot scanning. The three-photon imaging femtosecond laser used should produce an average power of at least ~ 1W for the 10% optical system transmittance estimated above. This brings up interesting questions: is the average power of 1W output in the form of "1 MHz, 1μJ" or through "10 MHz,100 nJ? Currently, most three-photon imaging is performed at a repetition rate of 1 MHz, with a pixel dwell time of a few microseconds, and each pixel accumulates a signal generated by a single or multiple pulses, so a sufficient amount of signal can be generated at a frame rate of several frames per second. If the repetition frequency is increased to match the resonant frequency of the resonant mirror, the imaging frame rate can be further accelerated, but the resulting increase in average power may increase the risk of sample damage.

If the energy of a single pulse is increased blindly, it will produce more and more bright multi-photon signals under ideal circumstances, but the actual situation is that too high energy will lead to nonlinear damage of tissue. Similarly, although increasing the average power and repetition frequency can enhance the signal and achieve high-speed imaging, excessive power can also lead to excessive thermal effects. These factors make the three-photon imaging window in the wavelength range of the laser not wide, and the imaging window will vary with the sample and imaging depth, which complicates the parameter selection of the three-photon imaging laser. Studies have discussed the optimal repetition frequency as a function of tissue scattering and imaging depth, as shown in Figure 4. It can be seen from the green area in the figure that the optimal parameter range of incident light changes obviously with the increase of depth. For example, at a depth of 600 μm, the higher the repetition frequency, the better; while at a depth of 1mm, there is an optimal repetition frequency of 1 MHz.⁶

Figure 4 The best single pulse energy and repetition frequency for three-photon imaging in scattering tissues with effective attenuation lengths of 600 μm and 1mm, respectively.

Compared with the 80 MHz high repetition frequency of the traditional two-photon imaging femtosecond laser, three-photon imaging requires a laser with a lower repetition rate and higher single pulse energy, so a 1030 nm high-energy laser is often used to pump a tunable optical parametric amplifier (OPA) to tune it to a relatively suitable excitation wavelength. Although the wavelength, repetition frequency and power of most OPA are adjustable, they can only be operated with constant pump energy. Therefore, it is generally necessary to ensure sufficient energy margin in the deep tissue when imaging, while gentle energy control is required when imaging shallow planes.

It can be seen that laser energy, power, beam mode and long-term stability are also considered in the three-photon imaging technology. Especially when used for fast three-photon imaging, laser noise jitter has a prominent effect on image quality due to the short residence time of a single pixel.⁷In the era of digital optics, the rapid development of adaptive optics technology is also full of great potential and research value in enhancing the depth and contrast of multi-photon imaging.⁸

Future outlook

In retrospect, with the development of tunable lasers towards the direction of "miniaturization and ease of use", it has successfully promoted the development and application of two-photon microscopic imaging technology. Similarly, three-photon imaging lasers may also have similar development trends, such as: laser sources will become more reliable, more compact, easier to operate, and more flexible in parameter adjustment.

In the future, the development of three-photon microscopic imaging is inseparable from the in-depth study of new tunable light sources and imaging strategies. Excitingly, many teams at home and abroad are making progress, such as the MIT team's controlled bending of optical fibers,⁹The pulse spectrum is modified directly by self-phase modulation. For example, the Cornell team developed an adaptive excitation source (AES),^10,11The laser does not stick to a fixed repetition frequency operation, but with the help of complex and fine closed-loop feedback between the sample and the laser, the pulse is accurately generated when scanning to the top of the neuron, so that the average power can be guaranteed to be safe and controllable, and higher and faster instantaneous repetition frequency can be achieved. Although some of these new technological advances are still in the proof-of-principle stage, with the joint efforts of multidisciplinary researchers and engineers, it is very likely that the three-photon microscopy imaging paradigm will be widely used in future neuroimaging laboratories.

In recent years, China's scientific research team in the field of biomedical multiphoton microscopy technology progress, suchChina Laser MagazinePhotonics Research The team of Academician Cheng Heping/Professor Wang Aimin of Peking University reported in the third issue of this yeardual objective two-photon imaging（Click to view article),¹²Professor Kong Lingjie's team of Tsinghua University reported in the 6th issuedual-region two-photon imaging（Click to view article),¹³All show that multiphoton microscopy technology is steadily advancing towards the demand for three-dimensional, deep, high-resolution, multi-region neuroscience observations.

In recent years, my country's optoelectronic information field has also actively carried out advanced imaging technology innovation and application research, and gradually applied novel imaging methods to important scenes such as biology, astronomy, and remote sensing. Many sci-tech journals also focus on the frontier progress in the field of imaging in China, suchAdvanced Imaging(AI), which is jointly published by China Laser Magazine and Hangzhou Research Institute of Xidian University. The journal is co-edited by Professor Shao Xiaopeng, Xi'an Institute of Optics and Precision Mechanics, Chinese Academy of Sciences, and Professor Gigan, Sylvain of Sorbonne University, France. It is committed to publishing high-level basic and applied research progress in imaging and related fields, and promoting the rapid development of advanced imaging technology. Enhance academic exchanges and cooperation at home and abroad in this field, and promote the transformation of scientific research results.