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 imaging region by an active feedback mechanism².

The idea of locking-in to the particle is the base of the orbital tracking method. Instead of a raster scan a circular orbit is performed around the fluorescent particle. The intensity of the fluorescence along the orbit precisely localizes the particle position⁴.

The localized position of the particle can then be used to actuate the microscope scanners and re-center the orbit on the particle position. The galvanometer scanners of the microscope are driven by an analog voltage. The AC component of this voltage allows performing an orbit with the focused laser beam, i.e. a sine and a cosine wave applied to the X and Y scanning mirrors will allow performing a circular orbit. The DC offset of the signal facilitates changing the position of the orbit center. Once an orbit period is determined and the waveform for the AC signal is configured, the feedback system needs to update only the DC component of the signal.

A software able to read the fluorescent signal collected from the detectors is required to control the scanners and the detectors, to calculate the position of the particle and update the DC offset. For a successful feedback imaging a careful choice of the orbit parameters (size and timing) is required and these parameters have to be adjusted based upon the characteristics of the fluorescent particles that it is necessary to track.

The physical and mathematical foundations of the technique were described in the past^4,5 for both 2D and 3D applications. In this protocol we briefly describe the main hardware components of the setup, and in more detail the choice of the parameters and the sample preparation required for a typical experiment allowing us following the displacement of a lysosome at a high temporal resolution within a living cell.

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 nM of tubulin tracker green. Incubate cells for 1 hr at 37 °C. Optional step: Prior to incubation, perform an additional stain with a mitochondrial matrix staining dye (25 nM).
4. Wash the cells three times in HBSS to remove any unbound dye. Add fresh DMEM growth medium prior to imaging the cells.

2. Microscope Configurations

The microscope for the particle tracking described in the video protocol is assembled on the frame of a commercially available inverted microscope (Figure 4). However commercial modules for 3D orbital particle tracking are now available.

1. Use a Coherent Chameleon-Ultra II Ti:Sa femtosecond laser excitation light source, with a tunable output wavelength range between 690 nm-1,040 nm.
2. Ensure that the laser beam is aligned in the rear port of the microscope using IR-coated mirrors. The laser beam is attenuated using a rotating half-wave plate followed by a calcite linear polarizer. Attenuate the beam so that the average power at the sample is between 0.5 and 2 mW.
   NOTE: The laser beam is reflected by a pair of galvanometer-motor actuated mirrors that allow control of the position and trajectory of the focused beam in the sample plane. In a typical configuration the collimated laser beam exiting of the galvanometer mirrors is expanded (10X) passing through a beam expander, before entering the rear of the microscope objective after reflection on short-pass dichroic mirror.
3. Collect the fluorescence light from the sample by placing a high numerical aperture water objective (60X, NA 1.2) into the light path. Choose a fluorescence filter cube according to the desired emission wavelengths in either one or two channel configuration. Employ emission bandpass filters to further select the spectral range of the emitted fluorescence.
4. Place the sample onto a motorized stage, and adjust the fine motion of the microscope objective using an objective piezo-controller.
5. Direct the light from the filter cube into photomultiplier tubes where the signal is discriminated and sent to a digital I/O data acquisition card.
   NOTE: Computer generated waveforms are the analog output of the I/O card and are provided to the scanners control electronics. The photon counting input from the photomultipliers and the output signal to the scanners are measured and controlled via the I/O card by the Laboratory for Fluorescence Dynamics SimFCS software.

3. Imaging

1. Position the cells on the stage and focus using transmitted light illumination.
2. Switch to raster scan imaging to identify the cells that have incorporated the dye and to visualize the underlying microtubules. Identify the initial area where vesicle movement is present.
3. Once an isolated vesicle is identified, set the following parameters for orbital tracking:
   1. Select the radius of the orbit to define the size of the circular scan according to the size of the particle being tracked. For a point emitter, set the radius of the orbit equal to the waist of the Point Spread Function (PSF) of the excitation beam to maximize sensitivity and response.
   2. Set the axial distance to define the distance between the two orbits that are performed to localize the particle position along the axial direction. Set the axial distance to 1.5-3 times the PSF waist.
   3. Define the dwell time according to the brightness of the particle to set the time spent on each point of the orbit, which will also determine the photo-bleaching rate. Use a dwell time between 10-100 µsec.
   4. Set the number of points in each orbit to 64 or 128 to yield orbit periods in the order of 4-32 msec and provide a high temporal resolution for determining the particle position.
4. Change the DC offset signal sent to the mirrors in order to center the beam on the particle through the graphical user interface via a cursor selection in the raster-scanned image.
5. Begin tracking by switching the microscope mode from raster-imaging to orbital scanning.
6. Activate the feedback and data collection.

4. Trajectory Analysis

1. Use the software to display the fluorescence collected at each point along the orbit and at each time point in the form of an ‘intensity carpet’. The intensity collected along the carpet provides information on the interaction of the particle with other bright objects. Use the software to display both the trajectory information (i.e. the DC displacement over time of the x,y scanners and of the z-piezo) as well as the fluorescence intensity collected from the photomultiplier tube over time in the form of time series.
2. Use the time series representation and the ‘intensity carpet’ information to select only a region of interest in the trajectory.
3. Use the software to display a 2D projection of the selected portion of the particle trajectory. Select the appropriate controls to color-code the trajectory according to the particle fluorescence intensity.
4. Select the option to display the particle trajectory in 3D, and color-code it according to the fluorescence intensity. Rotate the trajectory in 3D using the controls to visualize the features of the lysosome motion along the microtubule.

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