Super-resolution Imaging of Neuronal Dense-core Vesicles

Summary

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

Abstract

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.

Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

Introduction

A number of cellular processes depend on accurate and efficient vesicle-mediated trafficking of biomolecules to specific subcellular destinations. One prominent example is synaptic assembly, which is preceded by long-ranged, vesicle-mediated delivery of synaptic constituents from sites of biogenesis in the neuronal soma to potentially distal pre- and postsynaptic sites¹.

Fluorescence microscopy is a powerful and popular method of studying vesicle trafficking. Strengths of the technique include its sensitivity, specificity, and compatibility with live imaging². Unfortunately, until relatively recently, the technique has....

Protocol

1. Sample Preparation

1. Prepare DNA encoding a photoconvertible or photoactivatable chimera targeted to DCVs, using standard molecular biology techniques.
   Note: One possibility is DNA encoding a tissue plasminogen activator-Dendra2 chimera (tPA-Dendra2). Dendra2 is a photoconvertible protein that switches from green to red emission upon exposure to ultraviolet light¹².
2. Culture hippocampal neurons on high performance #1.5 cover slips for ~5-10 days, following standard protocols¹³.
3. Transfect developing hippocampal neurons using a cationic lipid reagent.
   Note: Viral-mediated infection is more effective for....

Results

Figure 1 shows one end product of imaging and processing. In this PALM image, lateral coordinates of localized fluorophores are shown using the centroid display mode, and the super-resolution image of the associated DCV is shown using the Gaussian display mode.

Figure 2A shows analogous widefield and PALM images of the soma and proximal processes of an eight days in vitro hippocampal neuron expressing tPA-Dendra2. Important features of the images incl.......

Discussion

PALM and related super-resolution fluorescence microscopy techniques have recently emerged as a valuable complement to better-established forms of optical microscopy and electron microscopy (EM)^22-24. Positive attributes of PALM include relatively simple sample preparation and minimal sample perturbation. In principle, PALM also can be used to study living cells. Probably the main negative attribute of PALM is time-consuming data acquisition, which significantly hampers studies of faster dynamic processes in l.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Zeiss PALM	Carl Zeiss, Inc.	Elyra PS.1	With Zeiss Efficient Navigation (ZEN) software and fluorescence filter Set 77 HE GFP/mRFP/Alexa633
Lipofectamine 2000 Transfection Reagent	Life Technologies	11668-019
Minimum Essential Medium	Life Technologies	11095-080
Phosphate Buffered Saline	Life Technologies	10010049
Paraformaldehyde	Electron Microscopy Sciences	19208
Sucrose	Sigma-Aldrich	S-8501
Growth Glass Coverslips 18 mm 1.5D	Fisher Scientific	NC0059095