ZFIN ID: ZDB-PUB-151202-2
Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy
Lauterbach, M.A., Ronzitti, E., Sternberg, J.R., Wyart, C., Emiliani, V.
Date: 2015
Source: PLoS One   10: e0143681 (Journal)
Registered Authors: Wyart, Claire
Keywords: Calcium signaling, Calcium imaging, Specimen sectioning, Fluorescence microscopy, Fluorescence imaging, Motor neurons, Zebrafish, Embryos
MeSH Terms:
  • Animals
  • Calcium/analysis
  • Calcium/metabolism*
  • Embryo, Nonmammalian
  • Microscopy, Fluorescence/methods*
  • Molecular Imaging/methods*
  • Motor Neurons/metabolism*
  • Spinal Cord/embryology
  • Spinal Cord/metabolism
  • Zebrafish/embryology
  • Zebrafish/metabolism
PubMed: 26625116 Full text @ PLoS One
Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.