Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Summary

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers within single cells.

Abstract

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

Introduction

While cellular structures exist on a wide range of spatial scales, fluorescence imaging of cellular organization on length scales smaller than ~250 nm is restricted in conventional microscopy due to the physical constraint of the diffraction limit. This limit was overcome with the advent of fluorescence photoactivation localization microscopy (FPALM¹) and similar techniques^2,3, which can localize large numbers of individual molecules with precision of ~10 nm, to generate images with resolution of a few tens of nanometers. FPALM is based on using optical control to activate and inactivate subsets of molecules (for a full description of FPALM,....

Protocol

Please note: A diagrammatic representation of optical components referenced in this protocol can be found in Figure 1.

1. Cell Sample Preparation

1. Plate cells at an optimized density (for NIH-3T3 cells, this is roughly 2-5 x 10⁴ cells/cm²) in wells of an 8-well chamber. Cells should be plated in complete media appropriate to the cell type, although media should be made without antibiotics and without phenol red, which contributes to background fluorescence. Note that conditions for cell experimentation, such as the optimal range of passage numbers, may differ for individual cell lines.<....

Results

Influenza hemagglutinin (HA) forms clusters on the order of tens of nanometers to micrometers, and these clusters variably colocalize with actin (Figure 5). These spatial distributions corroborate coarser scale imaging of these two proteins²⁸, and the dependence of the HA spatial distributions on actin¹⁹. Multicolor FPALM images can be further used to describe the density, area and perimeter of these clusters, and the degree of colocalization between the two species at both the nano.......

Discussion

Localization-based super-resolution imaging provides many powerful capabilities for biological imaging. The route from individual optical components placed on the table to a functional super-resolution microscope capable of simultaneously imaging multiple fluorescent species in a biological sample presents a number of challenges. Some aspects of the alignment are more critical than others; we endeavor below to provide guidance to prospective users dealing with the most difficult aspects of the route.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LabTek II chambers	Nunc
Fluorescent beads	Invitrogen	F-8801	Beads for calibration
Tetraspeck beads	Invitrogen	T-7279	Four color beads for calibration
Objective immersion oil	Zeiss	518F	Immersion oil for high NA objective (dependent on choice of objective)
HPLC water	Fisher Scientific	W5-4
Media	ATCC	30-2003	Or Cellgro 10-090
Antibiotics	GIBCO	15070-063
serum	Thermo Scientific	SH30087.03
Lipofectamine	Invitrogen	52887
Optimem I	GIBCO	11058-021
Trypsin	MPBiomedicals	1689149
paraformaldehyde	Fisher Scientific	AA433689M	CAUTION: Toxic