Two-photon microscopy (TPM) has been widely used in biological imaging owing to its sub-micron lateral resolution, intrinsic optical sectioning, and deep penetration abilities. TPM enables the observation of cellular and sub-cellular dynamics in deep live tissues within highly complex and heterogeneous environments such as the mammalian brain, thereby providing critical in situ and in vivo information.
However, because of the nonuniformity of the refractive index of biological tissues, the laser is distorted and scattered during propagation. Consequently, the focus point becomes a diffuse spot, which leads to a decreased imaging depth and poor resolution of TPM.
Adaptive optics (AO) technology was first applied to TPM in 2000, where a genetic algorithm was used to calculate the wavefront distortion and a deformable mirror (DM) was used to correct the aberration introduced by biological samples. Since then, various AO schemes have been developed for a wide range of high-resolution microscopes to advance the development of biological exploration.
In this study, the sources and characteristics of aberrations in TPM are examined, and different detection and correction methods in AO are summarized. The different applications of AO in TPM in recent years are comprehensively reviewed.
For AO technology, wavefront detection methods are generally divided into direct wavefront detection, which uses a wavefront sensor (WS) to detect wavefronts, and indirect wavefront detection, which estimates the aberrated wavefront using iterative algorithms.
In 2010, the pupil-segmentation AO method was proposed by Ji et al. This method involves the division of the pupil into several sub apertures and the use of spatial light modulator (SLM) to modulate the wavefront phase, and the imaging resolution of a fixed mouse cortex slice was restored to the near-diffraction-limited (Fig.2). In 2012, Tang et al. proposed an iterative multiphoton adaptive compensation technique that exploits the nonlinearity of multiphoton signals to determine and compensate for distortions and focus light inside deep tissues. The technique was tested using a variety of highly heterogeneous biological samples, and an imaging resolution of approximately 100 nm was obtained (Fig.3). In 2014, Wang et al. adopted a digital micromirror device (DMD) to rapidly modulate the intensity or phase of light rays of multiple pupil segments in parallel to determine the wavefront aberration (Fig.4). Subsequently, Park et al. developed a multi-pupil adaptive optics (MPAO) method in 2017 that allows the simultaneous correction of a wavefront over a field of view of 450 µm×450 µm, thereby expanding the correction area to nine times larger than those of conventional methods (Fig.7). Recently, Rodríguez et al. developed a compact adaptive optics module and incorporated it into both TPM and three-photon microscopy to correct tissue-induced aberrations. They also demonstrated that their technology allows the in vivo high-resolution imaging of both neuronal structures and somatosensory-evoked calcium responses in the spinal cord of mice at great depths (Fig.5).
Generally, the indirect wavefront detection system is relatively simple and easy to implement, but it is also time-consuming and has high computational expense. In the typical TPM, the excited fluorescence is limited to a small area near the focus. Such fluorescence points act as a natural guide star for the wavefront sensor to allow the application of direct wavefront detection in TPM. From 2010 to 2013, Cha et al. and Tao et al. used Shark-Hartmann wavefront sensor (SHWS) to detect the emission light and DM to correct the excitation light by injecting foreign fluorescent substances into the sample as a guide star for the in vitro imaging of mice brain tissue (Fig.10). In 2014, Wang et al. proposed descanning technology to accumulate all the transmitted optical signals for wavefront detection and improve the quality and efficiency of direct wavefront detection. The SLM was used to correct the excitation light and in vivo structural imaging was performed on the brain neurons of mice (Fig.11). In 2019, Liu et al. used near-infrared fluorescent dye as a guide star for direct wavefront detection, but replaced the SLM with DM. Structural imaging was carried out on the microvessels and neurons of mice, and an imaging depth of up to 1100 µm was obtained (Fig.12).
Compared with indirect wavefront detection, direct wavefront detection is faster and more accurate. However, the optical system for direct wavefront detection is complex, which reduces the imaging signal-to-noise ratio. To effectively use the two detection methods, the real distorted wavefront should be obtained in the optical detection path without a wavefront sensor. In 2017, Papadopoulos et al. proposed the focus scanning holographic aberration probing (F-SHARP) method, which directly measures the point spread function (PSF) of the distorted wavefront using interference technology and corrects the wavefront using the phase conjugation of the PSF (Fig.14). In 2022, Qu et al. combined a conjugate AO with the F-SHARP system and used a phase-locked amplifier to simplify the measurement steps of PSFs, which improves the measurement speed and correction accuracy (Fig.15).
Currently, there is an increasing demand for high-resolution structural and functional neuroimaging systems owing to the rapid development of brain science. However, the aberrations caused by tissues are complex, irregular, and rapidly changing and fast aberration detection and accurate correction systems are required. Therefore, it is necessary to use high-performance adaptive elements such as a high sensitivity wavefront sensor and correction elements with a high refresh rate and large compensation range. In addition, fast and accurate compensation algorithms can also improve the AO performance. In summary, the effective combination of various detection, correction and control techniques is the focus of in vivo microscopic imaging, which provides valuable information for scientific research.
