Research Article: Automated Remote Focusing, Drift Correction, and Photostimulation to Evaluate Structural Plasticity in Dendritic Spines

Date Published: January 23, 2017

Publisher: Public Library of Science

Author(s): Michael S. Smirnov, Paul R. Evans, Tavita R. Garrett, Long Yan, Ryohei Yasuda, Anna Dunaevsky.


Long-term structural plasticity of dendritic spines plays a key role in synaptic plasticity, the cellular basis for learning and memory. The biochemical step is mediated by a complex network of signaling proteins in spines. Two-photon imaging techniques combined with two-photon glutamate uncaging allows researchers to induce and quantify structural plasticity in single dendritic spines. However, this method is laborious and slow, making it unsuitable for high throughput screening of factors necessary for structural plasticity. Here we introduce a MATLAB-based module built for Scanimage to automatically track, image, and stimulate multiple dendritic spines. We implemented an electrically tunable lens in combination with a drift correction algorithm to rapidly and continuously track targeted spines and correct sample movements. With a straightforward user interface to design custom multi-position experiments, we were able to adequately image and produce targeted plasticity in multiple dendritic spines using glutamate uncaging. Our methods are inexpensive, open source, and provides up to a five-fold increase in throughput for quantifying structural plasticity of dendritic spines.

Partial Text

Structural changes in dendritic spines, tiny postsynaptic protrusions on the dendritic surface of neurons, are considered to be the basis of synaptic plasticity, learning, and memory [1–5]. Among several forms of spine structural plasticity, structural long-term potentiation (sLTP) of single dendritic spines has been extensively examined as a structural correlate of functional LTP (fLTP), an electrophysiological model of learning and memory [3, 5, 6]. Applying two-photon glutamate uncaging at a single dendritic spine induces a rapid and long lasting spine enlargement at the stimulated spine, but not the surrounding spines [3]. The signaling cascades necessary for sLTP have been studied by combining sLTP imaging with pharmacological and genetic manipulation. Both sLTP and fLTP depend on NMDA-type glutamate receptors (NDMAR), Ca2+/Calmodulin-dependent kinase II (CaMKII) and several small GTPase proteins including Ras, Cdc42, Rac1 and RhoA [4, 7–11]. However, due to the low-throughput nature of the measurement, the study of sLTP has been limited to only a few proteins, and among more than 1000 proteins expressed in spines [12–14], it is largely unknown which ones are necessary for spine structural plasticity.

In order to achieve rapid and reliable focusing required for auto-focusing system, we employed ETL to our two-photon microscope by placing it at a conjugate plane of the back-focal plane of the objective (Fig 1A). A pre-ETL lens resizes the beam to fit the full aperture of the ETL, while two more lenses serve to resize the beam to fit the galvanometers. In this setup, regardless of ETL shape, the beam size is constant at the galvanometers and the back aperture plane of the objective. This setup minimizes the loss of beam intensity and spherical aberration.

We have designed an easily implementable module for Scanimage to allow for multi-position scanning and photoactivation of dendritic spines to study postsynaptic plasticity. Furthermore, we described an implementation of an ETL in the excitation path which minimizes the signal loss and distortion. The ETL served both to increase imaging speed and remove sample drift caused by rapid stage or objective movements when changing focus. Our optical implementation of the ETL as a remote focusing element is combined with a straightforward user interface which is able to align and control the ETL current, and as a result, the axial focus. Furthermore, our software interface allows users to tilt the imaging plane in 3D by rapidly modulating the axial focus position in phase with the Y-scanning galvanometer. We expect this feature to be particularly useful in neuroscience, where long, straight neuronal projections can be scanned along their own axis, resulting in a significant increase in scanning efficiency. We found that an ETL-based system is relatively low price and easily implemented in any two-photon microscope. While the focusing speed of an ETL (15 ms) is sufficient for multi-position imaging, tilted-plane imaging needs to be performed fairly slowly, at around 1Hz, to allow the ETL sufficient refocusing time along the imaging plane.

We successfully designed and implemented an automated system capable of reliably measuring sLTP in multiple dendritic spines. The implementation of an ETL in the excitation path, combined with galvanoic mirror scanning, allowed us to quickly switch between imaging positions with minimum perturbation to the sample. The customizable autofocus and drift correction system allows our software to track and stimulate individual dendritic spines over extended imaging sessions. By dramatically increasing the throughput of spine imaging and stimulation experiments, our system will accelerate studies to understand molecular basis of spine structural plasticity. In addition, the flexible implementation of software would allow researchers to use it for many imaging/photostimulation experiments.




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