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Updated: May 10, 2026
Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo
Published on: June 26, 2013
Let there be light: Targeted fluorescent labels illuminate cryo-FIB/ET of synapses
Spencer J Rothfuss1, Qiangjun Zhou2
1Brain Institute, Vanderbilt University, Nashville, TN 37240-7933, USA; Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37240-7933, USA; Chemical and Physical Biology Program, Vanderbilt University, Nashville, TN 37240-7933, USA.
Focused ion beam milling, guided by fluorescent tags, precisely targets and images chemical synapses. This method effectively removes interfering material, revealing detailed synaptic ultrastructure for advanced microscopy.
Area of Science:
- Neuroscience
- Cell Biology
- Microscopy
Background:
- Focused ion beam (FIB) milling is a technique used to expose subsurface cellular structures.
- High-resolution imaging of neuronal networks requires access to specific subcellular regions like chemical synapses.
- Existing methods may struggle with precise targeting and removal of extraneous material.
Purpose of the Study:
- To develop and validate a method for targeting FIB milling to specific cellular structures, namely chemical synapses.
- To improve the resolution and clarity of synaptic ultrastructure imaging using electron microscopy.
- To demonstrate the utility of fluorescent tags in guiding FIB milling and subsequent imaging.
Main Methods:
- Utilized fluorescent tags to label chemical synapses within neuronal networks.
- Employed focused ion beam (FIB) milling, guided by fluorescence, to precisely ablate overlying cellular material.
- Performed high-resolution electron microscopy on the exposed synaptic regions.
Main Results:
- Successfully targeted FIB milling to chemical synapses using fluorescent markers.
- Demonstrated effective removal of confounding cellular material surrounding the synapses.
- Achieved high-resolution imaging of synaptic ultrastructure, showcasing intricate details.
Conclusions:
- Fluorescent tag-guided FIB milling is a powerful approach for high-resolution imaging of chemical synapses.
- This technique overcomes limitations in accessing and visualizing synaptic ultrastructure.
- Enables detailed investigation of neuronal connectivity and function at the ultrastructural level.
