The incidence and mortality rates of digestive tract cancers are rising quickly globally, greatly endangering human life and health. Most digestive tract tumors come from precancerous lesions, and the development of early cancer detection and diagnosis technology is crucial to improving people’s health. To date, histopathological examination is still the "gold standard" for the clinical diagnosis of cancer, but this method has limitations, such as time-consuming and in vitro detection. Additionally, while biopsy sampling can examine the pathological characteristics of the suspected lesion area at the cellular scale, it cannot achieve full coverage of the suspected lesion area, so there is a certain risk of missed detection and false detection. Therefore, there is an urgent need to develop real-time, in vivo, in situ histological diagnostic techniques at the cellular scale to achieve early diagnosis of GI (gastrointestinal) cancers.

Two-photon endomicroscopy is a new type of endomicroscopic imaging technology based on the principle of two-photon excitation, with the technical advantages of optical-sectioning capability, deep penetration, low phototoxicity, and label-free imaging. This technique can realize structural imaging and functional imaging, which has great potential for applications in life science and clinical medicine.

Piezoelectric ceramic scanning two-photon endomicroscopy is the current preferred solution for two-photon endomicroscopy imaging technology. In recent years, this technique has achieved technological breakthroughs and new applications. This paper summarizes piezoelectric ceramic scanning two-photon endomicroscopic imaging technology and the research progress and introduces its application in the field of biomedical imaging.

Section 2 introduces three typical two-photon endomicroscopy systems: fiber bundle proximal scanning scheme, MEMS distal scanning scheme, and piezoelectric ceramic-driven fiber distal scanning scheme (Fig. 1). Subsequently, the system structure and breakthroughs in core device technology of piezoelectric ceramic scanning two-photon endomicroscopy in recent years are summarized (Fig. 2). It mainly includes low-dispersion low-loss transmission double-cladding fiber, high-imaging resolution miniature objective, and high resonant frequency piezoelectric ceramic fiber scanner.

On this basis, we introduce in Section 3 the recent research progress of the representative piezoelectric ceramic scanning two-photon endomicroscopy in this field. In the abroad research progress, the works from the following research groups are summarized, including Chris Xu’s group from Cornell University (Fig. 3), Frédéric Louradour’s group from Université de Limoges (Fig. 4), Xingde Li’s group from Johns Hopkins University (Fig. 5), Ki-Hun Jeong’s group from the KAIST (Fig. 6), and a joint team of Bernhard Messerschmidt’s and Juergen Popp’s groups from the GRINTECH and the Leibniz Institute of Photonic Technology, respectively (Fig. 7). In the domestic research progress, the work from the following research groups is summarized, including Ling Fu’s group from the Huazhong University of Science and Technology (Fig. 8), and a joint team of Lishuang Feng’s and Aimin Wang’s groups from the Beihang University and the Peking University, respectively (Fig. 9). It can be concluded that the capability of this technology for in situ, real-time, noninvasive, and high-resolution structural and functional imaging of biological tissues and organs has been fully verified. A part of the research units continues to focus on the research of a two-photon endomicroscopy integrated probe. The capability of the piezoelectric ceramic scanning two-photon endomicroscopy technology can be improved further by optimizing the core device and introducing new principles and methods; parts of the research units have conducted the development of a miniaturized endomicroscopy system to meet the clinical biosafety and compatibility requirements and develop its application in the biomedical imaging field.

In Section 4, we summarize two-photon endomicroscopy applications in structural and functional imaging of tissues and brain imaging of freely-moving animals. The following research groups’ work, including Xingde Li’s group from the Johns Hopkins University [Fig. 10 (a)-(i) and Fig. 12], a joint team of Liwei Liu and Junle Qu’s group from Shenzhen University [Fig. 10 (j)-(r)], and Heping Cheng’s group from the Peking University (Fig. 11), is summarized.

As a subcellular-scale optical biopsy technology, two-photon endomicroscopy can achieve real-time structural and functional imaging of biological tissues in situ, which has important scientific research value and broad clinical application prospects. The following recommendations are considered for the future development of two-photon endomicroscopy: 1) further breakthroughs in core device performance to improve the imaging capability and throughput of piezoelectric ceramic scanning two-photon endomicroscopy; 2) research on two-photon endomicroscopy technology based on MEMS scanning mirrors; 3) research on disposable endomicroscopy technology; 4) exploration of two-photon imaging technology-based multimodal imaging technology. It is foreseeable that piezoelectric ceramic scanning two-photon endoscopic imaging technology, as one of the important research directions of two-photon imaging technology, is expected to open a new paradigm of optical biopsy imaging applications for life science research and clinical medicine applications.