A Protocol for Real-time 3D Single Particle Tracking

Summary

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Abstract

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 µm²/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

Introduction

The emergence of advanced imaging techniques has opened a window to see ever more detailed structure of cellular phenomena, all the way down to the molecular level. Methods such as stochastic optical reconstruction microscopy (STORM)^1,2,3, photo-activated localization microscopy (PALM)^4,5,6,7, structured illumination microscopy (SIM)^8,9,10,

Protocol

1. Setup Layout and Alignment

1. Installation and collimation of the tracking excitation laser
   1. Affix the laser to the optical table using a home-built mount. The mount is a simple aluminum plate with mounting holes for the laser head and the optical table. The laser should be firmly attached to a metal mount for stability and heat dissipation. For this work, use a 488 nm solid state laser for illumination (Figure 1), though the wavelength can be selected to suit a particular fluorophore or experiment. A critical factor is that the laser wavelength fit the 2D-EOD working range, which is determined primarily by the wave....

Results

Fixed particle scanning (Figure 4) and freely diffusing 110 nm fluorescent particle tracking (Figure 5) were performed following the protocol above. The particle scanning was performed by moving the piezoelectric nanopositioner and bin photons while simultaneously calculating the particle's estimated position at each point in the scan. The scanning image shows a square of even intensity (Figure 4a) a.......

Discussion

Although many varieties of 3D single particle tracking methods have emerged in recent years, robust real-time tracking of high speed 3D diffusion at low photon count rates with a simple setup is still a challenge, which limits its application to important biological problems. The 3D-DyPLoT method described in this protocol addresses these challenges in a several ways. First, the excitation and detection pathways are simplified greatly compared to other implementations making alignment simple and robust. Secondly, the mov.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2D Electro-optic Deflector	ConOptics	M310A	2 required
Power supply for EOD	ConOptics	412	Converts FPGA ouput to high voltage for EOD
TAG  Lens	TAG Optics	TAG 2.0	Used to deflect laser along axial direction
XY piezoelectric nanopositioner	MadCity Labs	Nano-PDQ275HS	Used for moving the sample to lock the particle in the objective focal volume in
Z piezoelectric nanoposiitoner	MadCity Labs	Nano-OP65HS	Used to move the objective lens to follow the diffusing particle
Micropositioner	MadCity Labs	MicroDrive	Used to coarsely position sample and evaluate
Objective Lens	Zeiss	PlanApo	High numerical aperture required for best sensitivity. 100X, 1.49 NA, M27, Zeiss
sCMOS camera	PCO	pco.edge 4.2	Used to monitor the particle's position
APD	Excelitas	SPCM-ARQH-15	Lower dark counts beneficial
Field programmable gate array	National Instruments	NI-7852r
Software	National Instruments	LabVIEW
Tracking excitation laser	JDSU	FCD488-30
Lens	ThorLabs	AC254-150-A-ML	L1
Lens	ThorLabs	AC254-200-A-ML	L2
Pinhole	ThorLabs	P75S	PH
Glan-Thompson Polarizer	ThorLabs	GTH5-A	GT
Half-wave plate	ThorLabs	WPH05M-488	WP
Lens	ThorLabs	AC254-75-A-ML	L3
Lens	ThorLabs	AC254-250-A-ML	L4
Lens	ThorLabs	AC254-200-A-ML	L5
Lens	ThorLabs	AC254-200-A-ML	L6
Dichroic Mirror	Chroma	ZT405/488/561/640rpc	DC
Fluorescence Emission Filter	Chroma	D535/40m	F
10/90 beamsplitter	Chroma	21012	BS
PBS	Sigma	D8537
190 nm fluorescent nanoparticles	Bangs laboratories	FC02F/9942
110 nm fluorescent nanoparticles	Bangs laboratories	FC02F/10617
Coverslip	Fisher Scientific	12-545A
Powermeter	Thorlabs	PM100D
CMOS	Thorlabs	DCC1545M
Iris	Thorlabs	SM1D12D
Microscope	Mad City Labs	RM21