image: a Schematic diagram of coaxial imaging. b Schematic diagram of mixed detection in off-axis imaging. c Schematic diagram of separated detection in off-axis imaging. Images beside each system diagram represent the point spread function along the xz direction
Credit: Zhang, J., Qiao, W., Jin, R. et al.
In recent advancements in life sciences, optical microscopy has played a crucial role in acquiring high-quality three-dimensional structural and functional information. However, the quality of 3D images is often compromised due to the intense scattering effect in biological tissues, compounded by several issues such as limited spatiotemporal resolution, low signal-to-noise ratio, inadequate depth of penetration, and high phototoxicity. Although various optical sectioning techniques (confocal, two-photon, structured illumination, and light sheet microscopy) have been developed to address these challenges, each method adheres to distinct imaging principles for specific applications. As a result, the effective selection of suitable optical sectioning techniques across diverse imaging scenarios has become crucial yet challenging.
In a new paper published in Light: Science & Application, a team led by Professors Qingming Luo and Jing Yuan from Huazhong University of Science and Technology/Hainan University, China, in collaboration with Professor Shiqi Chen's team from the Chinese University of Hong Kong, have found that we can divide existing optical sectioning methods into coaxial and off-axis imaging by whether the illumination and detection optical axes coincide (Fig. 1). The xz cross-section view of the system's point spread function reveals that off-axis imaging distinguishes in-focus from out-of-focus information during raw data acquisition. This feature typically results in superior sectioning capabilities, making it ideal for imaging thick samples.
Based on coaxial and off-axis imaging characteristics, recommendations for optical sectioning methods in different applications have been provided according to the geometry of biological samples and specific needs (Figure 3). The coaxial system is comparable to the off-axis system in imaging thin samples with low background signals or thick samples with details to be resolved far bigger than the resolution limit, where the coaxial system is preferred due to its popularity and accessibility. However, the off-axis system is indispensable in imaging thick samples with detailed features close to the resolution limit.
The diversity of biological samples and specific experimental requirements present various challenges for imaging technology. Frequently changing imaging systems to adapt to different experiments is cumbersome and time-consuming. Therefore, there is potential for developing a single system that can implement multiple optical sectioning techniques. Off-axis detection separates the system's point spread function for recording, making integrating multiple techniques possible (Figure 4). In the future, further exploration of the recombination of the point spread function could enable more optical technology applications, providing a powerful and flexible new tool for biological research.
Journal
Light Science & Applications
Article Title
Optical sectioning methods in three-dimensional bioimaging