Proceedings of the
9th International Conference of Asian Society for Precision Engineering and Nanotechnology (ASPEN2022)
15 – 18 November 2022, Singapore
doi:10.3850/978-981-18-6021-8_OR-15-0165
A new light-field microscope system for high- resolution 3D bio-imaging
1Graduate School of Engineering, Shizuoka University, Johoku 3-5-1, Naka-ku, Hamamatsu 432-8561, Japan
2Research Institute of Electronics, Shizuoka University, Johoku 3-5-1, Naka-ku, Hamamatsu 432-8561, Japan
3Graduate School of Integrated Sciences for Life, Hiroshima University,1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8528, Japan
ABSTRACT
With the recent development of systems biology, which views living organisms as "systems" consisting of small interacting elements such as molecules, cells, and even individuals, three-dimensional observation of biological phenomena defined on the millisecond scale is required. Therefore, a three-dimensional observation technique with both high spatial and temporal resolution to observe the behavior of each element in three-dimensional space is indispensable. Scanning microscopes such as confocal microscopes and light-sheet microscopes, which are generally used for three-dimensional observation, have a temporal resolution of about 30 fps at the fastest, resulting in a time difference between the start and end planes of scanning when performing spatial observation. Therefore, we focused on light-field microscopes that can acquire images in a single shot without scanning. However, light-field microscopes have the disadvantage of low spatial resolution. In addition, in order to clarify the correlation between neural firing patterns and behavior in methods such as optogenetics, where activity patterns are controlled by optical manipulation, feedback control is required to measure the activity of the neural network and perform optical manipulation of specific neurons according to the activity state of the network. In the case that such a control is required, the activity of the neural network must be measured. A three-dimensional observation technique with real-time performance is required when such control is needed. In light of the above, a light field microscope with high spatial and temporal resolution and real-time performance is desired. We have been developed a new light-field microscope for real-time observation of nematode cells. C. elegans has a transparent body, which allows observation of all cells from outside the body. C. elegans has been shown to have neural mechanisms similar to those of mammals, and the elucidation of these mechanisms has potential applications in the medical field. Conventional light-field microscopy has sufficient temporal resolution, but spatial resolution is insufficient for observation of C. elegans. There are two conventional light-field systems. One is called One F system, the other is called BA system. The One F system, which has an image sensor positioned at the focal plane of a microlens array (MLA) has a deep depth of field but a low xy resolution. The BA system can be constructed by placing the MLA and image sensor at the focal plane of the lens (MLA), and has the feature of high xy resolution but shallow depth of field. We have developed a new system, the One F-BA composite system that combines these two systems. When the sample is in the focal plane of the objective lens, the system behaves like the One F system. When the sample is out of the focal plane, the system behaves like the BA system, providing high xy resolution and a deep depth of field. With the proposed method, xy resolution of 2-5[µm] and depth of field of 70[µm] were achieved, enabling real-time observation of nematode cells.
Keywords: Light-filed microscopy, 3D measurement, Bio-imaging, C.elegans
1Graduate School of Engineering, Shizuoka University, Johoku 3-5-1, Naka-ku, Hamamatsu 432-8561, Japan
2Research Institute of Electronics, Shizuoka University, Johoku 3-5-1, Naka-ku, Hamamatsu 432-8561, Japan
3Graduate School of Integrated Sciences for Life, Hiroshima University,1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8528, Japan
ABSTRACT
With the recent development of systems biology, which views living organisms as "systems" consisting of small interacting elements such as molecules, cells, and even individuals, three-dimensional observation of biological phenomena defined on the millisecond scale is required. Therefore, a three-dimensional observation technique with both high spatial and temporal resolution to observe the behavior of each element in three-dimensional space is indispensable. Scanning microscopes such as confocal microscopes and light-sheet microscopes, which are generally used for three-dimensional observation, have a temporal resolution of about 30 fps at the fastest, resulting in a time difference between the start and end planes of scanning when performing spatial observation. Therefore, we focused on light-field microscopes that can acquire images in a single shot without scanning. However, light-field microscopes have the disadvantage of low spatial resolution. In addition, in order to clarify the correlation between neural firing patterns and behavior in methods such as optogenetics, where activity patterns are controlled by optical manipulation, feedback control is required to measure the activity of the neural network and perform optical manipulation of specific neurons according to the activity state of the network. In the case that such a control is required, the activity of the neural network must be measured. A three-dimensional observation technique with real-time performance is required when such control is needed. In light of the above, a light field microscope with high spatial and temporal resolution and real-time performance is desired. We have been developed a new light-field microscope for real-time observation of nematode cells. C. elegans has a transparent body, which allows observation of all cells from outside the body. C. elegans has been shown to have neural mechanisms similar to those of mammals, and the elucidation of these mechanisms has potential applications in the medical field. Conventional light-field microscopy has sufficient temporal resolution, but spatial resolution is insufficient for observation of C. elegans. There are two conventional light-field systems. One is called One F system, the other is called BA system. The One F system, which has an image sensor positioned at the focal plane of a microlens array (MLA) has a deep depth of field but a low xy resolution. The BA system can be constructed by placing the MLA and image sensor at the focal plane of the lens (MLA), and has the feature of high xy resolution but shallow depth of field. We have developed a new system, the One F-BA composite system that combines these two systems. When the sample is in the focal plane of the objective lens, the system behaves like the One F system. When the sample is out of the focal plane, the system behaves like the BA system, providing high xy resolution and a deep depth of field. With the proposed method, xy resolution of 2-5[µm] and depth of field of 70[µm] were achieved, enabling real-time observation of nematode cells.
Keywords: Light-filed microscopy, 3D measurement, Bio-imaging, C.elegans
