Multiline orthogonal scanning temporal focusing (mosTF) microscopy for scattering reduction in in vivo brain imaging

Yi Xue; Josiah R Boivin; Dushan N Wadduwage; Jong Kang Park; Elly Nedivi; Peter T C So

doi:10.1038/s41598-024-57208-6

Multiline orthogonal scanning temporal focusing (mosTF) microscopy for scattering reduction in in vivo brain imaging

Sci Rep. 2024 May 13;14(1):10954. doi: 10.1038/s41598-024-57208-6.

Authors

Yi Xue^{1

2

3}, Josiah R Boivin⁴, Dushan N Wadduwage^{2

5

6}, Jong Kang Park⁵, Elly Nedivi^{4

7

8}, Peter T C So^{9

10

11}

Affiliations

¹ Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
² Laser Biomedical Research Center, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
³ Department of Biomedical Engineering, University of California, Davis, CA, 95616, USA.
⁴ Picower Institute, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
⁵ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
⁶ Center for Advanced Imaging, Faculty of Arts and Sciences, Harvard University, Cambridge, MA, 02138, USA.
⁷ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
⁸ Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
⁹ Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. ptso@mit.edu.
¹⁰ Laser Biomedical Research Center, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. ptso@mit.edu.
¹¹ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. ptso@mit.edu.

Abstract

Temporal focusing two-photon microscopy has been utilized for high-resolution imaging of neuronal and synaptic structures across volumes spanning hundreds of microns in vivo. However, a limitation of temporal focusing is the rapid degradation of the signal-to-background ratio and resolution with increasing imaging depth. This degradation is due to scattered emission photons being widely distributed, resulting in a strong background. To overcome this challenge, we have developed multiline orthogonal scanning temporal focusing (mosTF) microscopy. mosTF captures a sequence of images at each scan location of the excitation line. A reconstruction algorithm then reassigns scattered photons back to their correct scan positions. We demonstrate the effectiveness of mosTF by acquiring neuronal images of mice in vivo. Our results show remarkable improvements in in vivo brain imaging with mosTF, while maintaining its speed advantage.

MeSH terms

Algorithms
Animals
Brain* / diagnostic imaging
Brain* / metabolism
Image Processing, Computer-Assisted / methods
Mice
Microscopy, Fluorescence, Multiphoton / methods
Neurons / metabolism

Abstract

MeSH terms

Grants and funding