Multiphoton microscopy imaging: diverse techniques for imaging neurons in vivo
Compared with the traditional single-photon wide-field fluorescence microscope, multiphoton microscopy (MPM) has the functions of optical sectioning and deep imaging. In 2019, Jerome Lecoq et al. discussed related MPM technology from three aspects: neuron imaging deep in the brain, massive neuron imaging, and high-speed neuron imaging.
In order to link neuron activity with complex behavior, it is usually necessary to image neurons in the deep cortex, which requires MPM to have the ability of deep imaging. Excitation and emission light will be highly scattered and absorbed by biological tissue, which is the main factor limiting the imaging depth of MPM. Although the scattering problem can be solved by increasing the laser intensity, it will bring other problems, such as burning the sample, defocusing and near-surface Fluorescent excitation. The best way to increase the depth of MPM imaging is to use longer wavelengths as excitation light.
In addition, for two-photon (2P) imaging, out-of-focus and near-surface fluorescence excitation are the two largest depth-limiting factors, while for three-photon (3P) imaging, these two problems are greatly reduced, but three-photon imaging due to fluorescence The absorption cross-section of the group is much smaller than that of 2P, so an order of magnitude higher pulse energy is required to obtain the same intensity fluorescence signal as that excited by 2P. Functional 3P microscopy is more demanding than structural 3P microscopy, which requires faster scanning in order to sample neuronal activity in time; higher pulse energy is required in order to collect sufficient signals within the dwell time of each pixel.
Complex behaviors often involve large brain networks with both local and long-range connections. To link neuron activity with behavior, it is necessary to monitor the activity of very large and widely distributed neurons at the same time. The neural network in the brain processes incoming stimuli within tens of milliseconds. To understand this fast neural network To study neuron dynamics, MPM is required to have the ability to rapidly image neurons. Fast MPM methods can be divided into single-beam scanning techniques and multi-beam scanning techniques.
Single-beam scanning technology enables high-speed traversal of neural tissue with a large field of view (FOV)
When using MPM to image neurons, random-access scanning—that is, the laser beam is quickly scanned at any selected point on the entire field of view—can scan only the neurons of interest, which not only avoids scanning any unlabeled Nerve fibers can also optimize the scanning time of the laser beam. Random-access scanning (Fig. 1) can be achieved with an acousto-optic deflector (AOD), which works by bonding a piezoelectric transducer with a radio-frequency signal to a suitable crystal. The resulting acoustic waves induce a periodic refractive index grating, Diffraction occurs when a laser beam passes through a grating. The intensity and frequency of the sound wave can be adjusted by the radio frequency electrical signal to change the intensity and direction of the diffracted light, so that one-dimensional horizontal arbitrary point scanning can be realized by using one AOD, and 3D can be realized by using a pair of AODs combined with other axial scanning technologies random access scanning. However, this technique is very sensitive to the motion of the sample and prone to motion artifacts. At present, fast raster scanning, that is, progressive scanning in FOV, is widely used because the algorithm can easily solve the motion artifacts.
AOD-based two-photon imaging of neocortical L2/3 neurons in vivo[2]
There are many ways to realize fast raster scanning, using a vibrating mirror for fast 2D scanning, combining a vibrating mirror and an adjustable electric lens for fast 3D scanning, but the adjustable electric lens cannot quickly focus in the axial direction due to the limitation of mechanical inertia Switching, which affects imaging speed, can now be replaced with a spatial light modulator (SLM).
Remote focusing is also a means to achieve 3D imaging, as shown in Figure 2. In the LSU module, the scanning galvanometer scans horizontally, and the ASU module includes the objective lens L1 and the mirror M, and the axial scanning is realized by adjusting the position of M. This technique can not only correct the optical aberration introduced by the main objective lens L2, but also enable fast axial scanning. To obtain more neuron imaging, the FOV can be enlarged by adjusting the objective lens design of the microscope, but the objective lens with large NA and large FOV is usually heavy and cannot move quickly for fast axial scanning, so large FOV systems rely on Telefocus, SLM and adjustable motorized lenses.
Schematic diagram of a remote focusing two-photon imaging system[3] Multi-beam scanning technology can simultaneously image different positions of neuronal tissue
This technique3 typically uses two independent paths for imaging two distant (>1-2 mm apart) imaging sites (Fig. 3C,D); for adjacent regions, it usually uses multiple beams of a single objective lens for imaging (Fig. 3E,F). The multi-beam scanning technique must pay special attention to the crosstalk problem between excitation beams, which can be solved by post-light source separation method or space-time multiplexing method. The post-hoc light source separation method refers to the use of algorithms to separate the beams to eliminate crosstalk; the time-space multiplexing method refers to the simultaneous use of multiple excitation beams, the pulses of each beam are delayed in time, so that the individual beams excited by different beams can be temporarily separated. fluorescent signal. More neurons can be imaged by introducing more beams, but multiple beams will increase the overlap of fluorescence decay time, which limits the ability to distinguish signal sources; and multiplexing has a negative impact on the working speed of electronic devices. High requirements; a large number of beams also requires higher laser power to maintain an approximate signal-to-noise ratio of a single beam, which can easily lead to tissue damage.
Large Area Imaging Technology
In recent years, the development of different MPM technologies has broadened the scope of our imaging of neural tissue, allowing us to image more neurons deep in the brain at a faster speed, which has greatly promoted neuroscience research and enabled us to Gain a clearer understanding of brain function.
