This project was supported by grants NIH NIGMS 8P41 GM103540-28 and P50-GM076516

1. Sample Preparation
<ol>
	<li>Maintain CHOK1 cells in tissue culture flasks using DMEM supplemented with 10% fetal bovine serum and 100 I.U/ml of penicillin 50 &#181;g/ml of streptomycin. Incubate the cells in a 5% CO2 humidified incubator at 37 &#176;C.</li>
	<li>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.</li>
	<li>Incubate cells overnight at 37 &#176;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&#160;hr at 37 &#176;C. Optional step: Prior to incubation, perform an additional stain with a mitochondrial matrix staining dye (25 nM).</li>
	<li>Wash the cells three times in HBSS to remove any unbound dye. Add fresh DMEM growth medium prior to imaging the cells.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the sample onto a motorized stage, and adjust the fine motion of the microscope objective using an objective piezo-controller.</li>
	<li>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.</li>
</ol>
3. Imaging
<ol>
	<li>Position the cells on the stage and focus using transmitted light illumination.</li>
	<li>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.</li>
	<li>Once an isolated vesicle is identified, set the following parameters for orbital tracking:
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#181;sec.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Begin tracking by switching the microscope mode from raster-imaging to orbital scanning.</li>
	<li>Activate the feedback and data collection.</li>
</ol>
4. Trajectory Analysis
<ol>
	<li>Use the software to display the fluorescence collected at each point along the orbit and at each time point in the form of an &#8216;intensity carpet&#8217;. 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.</li>
	<li>Use the time series representation and the &#8216;intensity carpet&#8217; information to select only a region of interest in the trajectory.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

<ol>
	<li>So, P. T., Dong, C. Y., Masters, B. R., Berland, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+photon+excitation+fluorescence+microscopy.">Two photon excitation fluorescence microscopy.</a> Annu Rev Biomed Eng. 2, 399-429 (2000).</li><li>Dupont, A., Lamb, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+three+dimensional+single+particle+tracking.">Nanoscale three dimensional single particle tracking.</a> Nanoscale. 3, 4532-4541 (2011).</li><li>Estrada, L. C., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+nanometer+images+of+biological+fibers+by+directed+motion+of+gold+nanoparticles.">3D nanometer images of biological fibers by directed motion of gold nanoparticles.</a> Nano Lett. 11, 4656-4660 (2011).</li><li>Kis-Petikova, K., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distance+measurement+by+circular+scanning+of+the+excitation+beam+in+the+two+photon+microscope.">Distance measurement by circular scanning of the excitation beam in the two photon microscope.</a> Microscopy Research and Technique. 63, 34-49 (2004).</li><li>Levi, V., Ruan, Q., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3+D+particle+tracking+in+a+two+photon+microscope+application+to+the+study+of+molecular+dynamics+in+cells.">3 D particle tracking in a two photon microscope application to the study of molecular dynamics in cells.</a> Biophys J. 88, 2919-2928 (2005).</li><li>Lund, F. W., Wustner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+single+particle+tracking+and+temporal+image+correlation+spectroscopy+for+quantitative+analysis+of+endosome+motility.">A comparison of single particle tracking and temporal image correlation spectroscopy for quantitative analysis of endosome motility.</a> Journal of Microscopy. 252, 169-188 (2013).</li><li>Nath, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spreading+of+Neurodegenerative+Pathology+via+Neuron+to+Neuron+Transmission+of+beta+Amyloid.">Spreading of Neurodegenerative Pathology via Neuron to Neuron Transmission of beta Amyloid.</a> Journal of Neuroscience. 32, 8767-8777 (2012).</li><li>Balint, S., Vilanova, I. V., Alvarez, A. S., Lakadamyali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+live+cell+and+superresolution+microscopy+reveals+cargo+transport+dynamics+at+microtubule+intersections.">Correlative live cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 3375-3380 (2013).</li><li>Huotari, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosome+maturation.">Endosome maturation.</a> Embo Journal. 30, 3481-3500 (2011).</li><li>Ragan, T., So, P. T., Kwon, H. S., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+particle+tracking+on+the+two+photon+microscope.">3D particle tracking on the two photon microscope.</a> Multiphoton Microscopy in the Biomedical Sciences. 2, 247-258 (2001).</li><li>Konig, K., So, P. T. C., Mantulin, W. W., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+response+to+near-infrared+femtosecond+laser+pulses+in+two+photon+microscopes.">Cellular response to near-infrared femtosecond laser pulses in two photon microscopes.</a> Optics Letters. 22, 135-136 (1997).</li><li>Caviston, J. P., Holzbaur, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+motors+at+the+intersection+of+trafficking+and+transport.">Microtubule motors at the intersection of trafficking and transport.</a> Trends Cell Biol. 16, 530-537 (2006).</li><li>Cardarelli, F., Lanzano, L., Gratton, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+directed+molecular+motion+in+the+nuclear+pore+complex+of+live+cells.">Capturing directed molecular motion in the nuclear pore complex of live cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 9863-9868 (2012).</li></ol>

The authors have nothing to disclose.

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 the demonstrative application of tracking an individual particle in three dimensions within a living cell with a high temporal resolution (32 msec in this experiment) using a two-photon microscope. The setup required for performing such an experiment is typically available to laboratories performing laser scanning microscopy. Both instrument add-ons for 3D orbital tracking as well as the control software are available on the market, for commercial laser scanning microscopes such as those manufactured by Olympus. The use of a pulsed high power infrared laser source, where available, is advantageous in the design of the experimental setup since it does not require de-scanning of the emitted fluorescence. Furthermore it was demonstrated in the past that infrared irradiation is more benign towards the cell, under specific conditions11, given the reduction of out of focus fluorescence and photo-bleaching.
Although fast tracking of a fluorescent particle in two dimensions can be performed using a sensitive camera, the absence of any three-dimensional information can be often limiting in interpreting the biophysical meaning of the trajectory information. For instance, vesicles moving along microtubule frequently run up against microtubules intersection regions8,12. In this situation, the use of a 3D tracking technique overcomes the limit of using 2D projections of a 3D structure.
Due to the time response of our objective nanopositioner piezo device, the period of the movement in the z-axis is limited to about 30 msec, while in the x,y directions it can go down to a few msec. The localization precision of the method scales as the signal to noise ratio 4,5. The only apparent limitation of the method, i.e. the loss of a global view of the environment of the particle, is compensated by the possibility of reconstructing a temporal intensity carpet13, tracing the history of the interactions of the fluorescent particle with neighboring fluorescent sources, displaying either the same or a different spectral emission.
We show that the motion of lysosomes, vesicles that undergo directed motion along microtubules, powered by either kinesins or dyneins, can be followed within a living cell after staining using a commercially available dye. Recently, 2D tracking of lysosomes was correlated to the super-resolution imaging of the microtubule network, in the effort of detecting the routes that the vesicles take at microtubule-microtubule intersections8. 3D trajectories of the lysosomes display a more complex behavior than unidirectional motion. Although an average directed motion can be observed, bending, twisting and possible rotations along the tubules can be observed from these trajectories.
We have identified two critical steps in performing these experiments: first, it is necessary to achieve a labeling density of the lysosomes that is at the same time high enough to obtain brightly stained particles, and that at the same time provides a density of particles low enough to allow tracking of individual particles. In the case of two crossing particles, the microscope may switch from one particle to another one while tracking. The second critical step concerns the choice of the pixel dwell time, allowing a reasonable balance between high signal to noise ratio and tracking speed. We have observed that tracking speed in the order of 32 msec per tracking cycle are typically sufficient to follow lysosomes undergoing fast directed motion at speeds below 1 &#181;m/sec.
In summary, orbital tracking allows to measure in three dimensions and with a temporal resolution of &#60;40 msec the intracellular motion of fluorescently labeled vesicles. The 3D orbital tracking has the potential to be employed in the future for the study of different highly dynamic cellular processes such us the study of the chromatin dynamics real-time transcription and diffusion of plasmonic nanoparticles in the intracellular medium.

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 past4,5 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.

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

3d orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles

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 beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. 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 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

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 trajectories with a temporal resolution of 32 msec (Figure&#160;1a). The carpet analysis displays the intensity recorded along each orbit over time (Figure&#160;1b). During the tracking the integrated intensity of the emitted fluorescence can be recorded (Figure&#160;2a). The reconstructed 3D trajectory displays a directed motion, as can be expected from a vesicle moving on the microtubule network. Additionally, it is possible to correlate the 3D trajectory to a raster scan image of the surrounding region (Figure&#160;2b). This confirms the movement of the particle along the direction of a microtubule bundle.
The cells were additionally stained with a red marker for mitochondria to record eventual interaction of the vesicle with these organelles during their movement. It is possible to correlate the 3D trajectory of the particle inside the cell (Figure&#160;3a) to the intensity carpet recorded from the mitotracker fluorescence emission (Figure&#160;3b). This allows us to record the proximity interactions of the vesicle with the mitochondrial network as a function of their position within the cell. The emission peak recorded in the &#8220;mitochondria channel&#8221; does not give functional information about the way the organelles interact. It can provide only information on the time of their interaction and the relative spatial position of the fluorescent object with respect to the particle being tracked. This relative position is extracted from the angle within the orbit where the emission peak is recorded. A schematic representation of the microscope we used to perform this experiment can be observed in Figure 4.
<img alt="Figure 1" src="/files/ftp_upload/51794/51794fig1highres.jpg" /> 
Figure 1.&#160;Displacement vs time trajectories and intensity carpet. a) x, y and z lysosome displacement vs. time traces. b) The intensity trajectory (i.e. the intensity collected along each circular orbit vertical axis is time) provides information on the fluorescence surroundings of the particle. Bright spots outside of the boxed correspond to interactions of the particle with other bright objects, and are associated to jumps in the trajectory as the orbits re-centers on the brightest fluorescence source. The boxed region corresponds to the portion of the trajectory in between two jumps.
<img alt="Figure 2" src="/files/ftp_upload/51794/51794fig2highres.jpg" /> 
Figure 2.&#160;Following the 3D trajectory of a lysosome. a) 3D reconstruction of a portion of the trajectory of a lysosome collected using particle tracking. The trajectory is color coded for the fluorescence intensity collected from the particle (red, high; blue, low photon counts). Axes scales are in nm. Note that x,y,z axes have different ranges. b) 2D projection of the 3D trajectory, displaying an overlay of the trajectory on top of the microtubule imaged in a raster scan immediately after the trajectory was collected. c) Mitochondria stained with mitotracker.
<img alt="Figure 3" src="/files/ftp_upload/51794/51794fig3highres.jpg" /> 
Figure 3.&#160;Extending the orbital tracking to more than one channel (excitation at 930 nm). a) 3D trajectory of a lysosome within the cell. b) Intensity carpet in the green channel (the fluorescence collected in this channel is used for tracking). c) Intensity carpet in the red channel, where mitochondria were stained with a red fluorescent matrix targeted dye. The regions where the trajectory gets in contact with the mitochondria (arrowheads) appear as intensity bumps in the carpet, as the beam orbiting around the lysosome crosses into the mitochondrial matrix.
<img alt="Figure 4" src="/files/ftp_upload/51794/51794fig4highres.jpg" /> 
Figure 4.&#160;Schematics of the experimental setup: modified two-photon microscope. Schematic representation of the laser set-up: a 2-Photon Ti:Sa laser with a range 690-1,040 nm was used, a polarizer and an half waveplate are used to control the excitation power. All the other items are listed in the figure using the indicated abbreviations. We used a 60X objective with a NA of 1.2.

Watch this Scientific Journal Video about 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles at JoVE.com

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

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.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

3d-orbital-tracking-modified-two-photon-microscope-an-application-to

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.2K Views.  University of California, Irvine. 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. 

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

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Neuroscience

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Developmental Biology

A Protocol for Real-time 3D Single Particle Tracking

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 &#181;m2/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.

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.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Biology

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...

Visualization of Vesicle Motilities in Neurons Using Fluorescence Imaging

Source: Tsuruta, F., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55488">Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes.</a> J. Vis. Exp. (2017).
This video demonstrates how neuronal vesicles are labeled with specific fluorescent proteins to visualize their movement. First, a plasmid coding for a fluorescent protein is incubated with a transfection agent and then introduced into a culture of neurons. Subsequently, images of the neurons are acquired and analyzed to track the vesicles&#39; movement.

Wizualizacja ruchliwości pęcherzyków w neuronach za pomocą obrazowania fluorescencyjnego

1. Imaging Vesicle Motility

	Prepare the plasmids that will label each type of vesicle. For instance, use EGFP-Rab5 for early endosomes, EGFP-Rab7 for ...

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

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A Protocol for Real-time 3D Single Particle Tracking

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

4D Microscopy of Yeast

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Wizualizacja ruchliwości pęcherzyków w neuronach za pomocą obrazowania fluorescencyjnego