Multi-Z confocal microscopy

Fast, volumetric imaging over large scales has been a long-standing goal in biological microscopy. Scanning techniques such as fluorescence confocal microscopy can acquire 2D images at high resolution and high speed, but extending the acquisition to multiple planes at different depths requires an axial scanning mechanism that drastically reduces the acquisition speed. To address this challenge, we report an augmented variant of confocal microscopy where the key innovation consists to use a series of reflecting pinholes axially distributed in the detection plane, each one probing a different depth within the sample. As no axial scanning mechanism is involved, our technique provides simultaneous multiplane imaging over fields of view larger than a millimeter at video-rate. A high resolution version of multi-z microscopy is achieved with the use of a diffractive optical element in the excitation path. We have also demonstrated a version of Multi-Z confocal microscopy that operates in a line-scan mode using reflecting slits, and can produce multiplane imaging at 400 Hz frame rates. We have also demonstrated a version of Multi-Z confocal microscopy that operates in point-scan mode using a polygon scanner, and can produce multiplane imaging at near kilohertz frame rates, allowing in-vivo voltage imaging in mice.

Video-rate 4-plane calcium imaging of GCaMP6f-labeled neurons in mouse hippocampus in vivo.
Augmented 8-plane imaging of fixed mouse brain vasculature.

 

 

 

 

 

 

Zebrafish heart, 30 fps
Zebrafish tail, 20 micron interplane separation

 

 

 

 

 

 

 

  • B. Zhao, M. Koyama, J. Mertz, “High resolution multi-z confocal microscopy with a diffractive optical element”, Biomed. Opt. Exp. 14, 3057-3071 (2023). link
  • T. D. Weber, M. V. Moya, K. Kilic, J. Mertz, M. N. Economo, “High-speed multiplane confocal microscopy for voltage imaging in densely labeled neuronal populations”, Nat. Neurosci. https://doi.org/10.1038/s41593-023-01408-2 (2023). link
  • J.-M. Tsang, H. J. Gritton, S. L. Das, T. D. Weber, C. S. Chen, X. Han, J. Mertz, “Fast, multiplane line-scan confocal microscopy using axially distributed slits”, Biomed. Opt. Express 12, 1339-1350 (2021). link
  • A. Badon, S. Bensussen, H. J. Gritton, M. R. Awal, C. V. Gabel, X. Han, J. Mertz, “Video-rate large-scale imaging with Multi-Z confocal microscopy”, Optica 6, 389-395 (2019). link