Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)

Summary

We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.

Abstract

Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

Introduction

The visualization of complex cellular structures has long been integral to biological insights and discovery. Although fluorescence microscopy can image cells with high molecular specificity, its resolving power is limited by diffraction to ~ 200 nm in the image plane (x,y, or lateral dimension) and > 500 nm along the optical axis (z, or axial dimension)^1,2. Hence, the observation of ultrastructural features has historically been limited to electron microscopy (EM). Fortunately, the recent development of super resolution microscopy has circumvented this limit, enabling spatial resolution in the 10 - 100 nm range^1-6. In particular, super resol....

Protocol

1. Imaging Specimen Preparation

1. Since background fluorescence signals interfere with fluorescence from fluorophore labels, clean the coverglasses by first rinsing them in de-ionized water (ddH₂O) and then air-drying them using compressed air. Subsequently, perform plasma etching in a plasma cleaner for 15 sec, or longer if necessary.
2. To enable drift correction and iPALM calibration, use #1.5 round (22-mm diameter) pre-cleaned coverglasses embedded with fluorescent nanoparticles as fiducial marks, which serve as highly photostable fiducials for reliable calibration and drift correction. Due to the long acquisition time required to accum....

Results

Critical requirements for iPALM are the alignment, registration, and calibration of the optical systems. These are necessary to ensure proper interference within the 3-way beam splitter requisite for z-coordinate extraction. To enable continuous monitoring, constant point sources of fluorescence are necessary. This can be achieved using fluorescent Au or bi-metallic nanoparticles²³ whose photoluminescence arise from localized surface plasmon resonance (LSPR). They act as a stab.......

Discussion

The optical system of iPALM is based on a 4-π dual-opposed objective design, as shown in Figure 1A. The setup is constructed using both custom-machined and commercial opto-mechanical parts, as described earlier²³ and listed in Table 1. In addition to our setup, the Howard Hughes Medical Institute (HHMI) hosts a system that is accessible to the scientific community at the Advanced Imaging Center at the Janelia Research Campus. For the full mechanical drawi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
optical table	Newport, CA	RS4000	iPALM, installed on 4 Newport Stabilizer vibration isolators
vibration isolator for optical table	Newport, CA	S-2000
laser-642	Newport, CA	1185055	output power=100 mw
laser-561	Newport, CA	1168931	output power=200 mw
laser-488	Newport, CA	1137970	output power=200 mw
laser-405	Newport, CA	1142279	output power=100 mw
broadband dielectric mirrors	Thorlabs, NJ	BB1-E02	laser combiner
dichroic beamsplitter	Semrock, NY	LM01-427-25
acousto-optic tunable filter	AA Opto-Electronic, France	AOTFnC-VIS-TN
Linear polarizer	Newport, CA	05LP-VIS-B
baseplate	local workshop	customized
turning mirror (22.5°)	Reynard Corpporation, CA	customized	22.5° mirror
motorized optic mounts	New Focus, CA	8816
motorized XYZ translation stage	Thorlabs, NJ	MT3/M-Z6	sample holder
T-Cube DC servo motor controller	Thorlabs, NJ	TDC001
Piezo Phase Shifter	Physik Instrumente, Germany	S-303.CD
objective lens	Nikon, Japan	MRD01691	objective. Apo TIRF 60X/1.49 oil
translation stage	New Focus, CA	9062-COM-M
Pico Motor Actuator	New Focus, CA	8301
rotary Solenoid/Shutter	DACO Instruments, CT	5423-458
3-way beam splitter	Rocky Mountain Instruments, CO	customized	beamsplitter
Piezo Z/Tip/Tilt scanner	Physik Instrumente, Germany	S-316.10
motorized five-axis tilt aligner	New Focus, CA	8081
Picmotor ethernet controller	New Focus, CA	8752
Piezo controllers/amplifier/digital operation module	Physik Instrumente, Germany	E-509/E-503/E-517
band-pass filter	Semrock, NY	FF01-523/20	filters
band-pass filter	Semrock, NY	FF01-588/21
band-pass filter	Semrock, NY	FF01-607/30
band-pass filter	Semrock, NY	FF01-676/37
notch filter	Semrock, NY	NF01-405/488/561/635
motorized filter wheel with controllter	Thorlabs, NJ	FW103H
EMCCD	Andor, UK	DU-897U-CSO-#BV	3 sets
Desktop computers for controlling cameras and synchronization	Dell	Precision T3500	PC, 4 sets
coverslips with fiducial	Hestzig, VA	600-100AuF	sample preparation. fiducial marks with various density and spectra available
fibronectin	Millipore, MT	FC010
paraformaldehyde	Electron Microscopy Sciences, PA	15710	fixation. 16%
glutaraldehyde	Electron Microscopy Sciences, PA	16220	25%
triton X-100	Sigma aldrich, MO	T8787
HUVEC cells	Life Technologies, CA	C-015-10C
Medium 200	Life Technologies, CA	M-200-500
Large Vessel Endothelial Factors	Life Technologies, CA	A14608-01
Dulbecco's Phosphate Buffered Saline		14190367
Pennicillin/Streptomycin		15140122
Trypsin/EDTA	Life Technologies, CA	25200056
PIPES	Sigma aldrich, MO	P1851	PHEM
HEPES	1st base, Malaysia	BIO-1825
EGTA	Sigma aldrich, MO	E3889
MgCl2	Millipore, MT	5985
Alexa Fluor 647 Phalloidin	Invitrogen, CA	A22287	staining
sodium borohydride (NaBH4)	Sigma aldrich, MO	480886	quenching
glucose	1st base, Malaysia	BIO-1101	imaging buffer
glucose oxidase	Sigma aldrich, MO	G2133
catalase	Sigma aldrich, MO	C9322
cysteamine	Sigma aldrich, MO	30070
Epoxy	Thorlabs, NJ	G14250
vaseline	Sigma aldrich, MO	16415	sample sealing
lanolin	Sigma aldrich, MO	L7387
parafin wax	Sigma aldrich, MO	327204
Immersion oil	Electron Microscopy Sciences, PA	16915-04	imaging. Cargille Type HF