3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

Summary

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

Abstract

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope¹. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns². The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam^3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell^6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 µm/sec, typically displaying a directed motion along the microtubule network⁸.

Introduction

A large number of approaches have been developed to date to track fluorescent particles in three dimensions using a microscope. Most approaches rely on the use of fast cameras, ideally suited to track in two dimensions, typically combined with customized modifications of the emission optics of the microscope to achieve tracking in the axial direction. Laser scanning microscopes (either confocal or two photons) conventionally can track a fluorescent particle by performing a time sequence of z-stacks, although this process is typically time consuming, and yields reasonable time resolution (10 Hz) only if the particle being tracked is kept in the center of a small raster....

Protocol

1. Sample Preparation

1. Maintain CHOK1 cells in tissue culture flasks using DMEM supplemented with 10% fetal bovine serum and 100 I.U/ml of penicillin 50 µg/ml of streptomycin. Incubate the cells in a 5% CO₂ humidified incubator at 37 °C.
2. Harvest and then plate CHOK1 cells on a 14 mm diameter micro-well with a surface thickness of 0.16 mm. Seed cells for optimal density for imaging, around 60-70% confluency.
3. Incubate cells overnight at 37 °C, 5% CO₂. Wash the cells three times in HBSS (Hank Buffered Saline Solution), and incubate cells in a solution containing 50 nM of Lysotracker DND26 green and 150 n....

Results

According to this protocol fast 3D single particle tracking can be performed inside living cells using a modified two-photon microscope to track the displacement of fluorescently labeled lysosomes. The experiment performed consists of tracking an isolated lysosome moving inside the cell after the endosome maturation process⁹. The lysosomes were stained using a fluorescent green dye and excited at 930 nm exploiting 2-photon excitation. Our data show that it is possible to obtain x,y,z displacement traj.......

Discussion

Despite the tremendous progresses of fluorescence microscopy techniques and instrumentations over the last years, achieving trajectories of fluorescent particles in three dimensions with a high temporal resolution has remained a challenge in the field. If high temporal resolution has been achieved tracking particles in two dimensions, extension to the axial direction typically brings a drastic reduction in the frequency response of the system¹⁰.

In this video-protocol we focused on .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lysotracker DND 26	Life Technologies	L-7526
Tubuline tracker Green	Life Technologies	T34075
Mitotracker RED	Life Technologies	M7512
Coherent Chamelon- ultra II TI	Coherent
Glan Taylor Calcite Polarizer	Melles Griot	03PTA001
Galvanometer-motor mirror	Cambridge Technologies	M 6350
Dichroic mirror	Chroma Technologies	700 DCSPXR
Motorized stage	ASI	MS2000
Piezo	PI	P721-LLQ
Photomultiplier tube	Hamamatsu	H7422P-40
Data acquisition card	IO tech	PCI 1128-4000
Imaging software	LFD	Global for images-SimFCS