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 ...

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 border="0" cellpadding="0" cellspacing="0" width="525">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:197px;">Name</td>
			<td style="width:160px;">Company</td>
			<td style="width:168px;">Model</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysotracker DND 26</td>
			<td>Life technologies</td>
			<td>L-7526</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tubuline tracker Green</td>
			<td>Life technologies</td>
			<td>T34075</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mitotracker RED</td>
			<td>Life technologies</td>
			<td>M7512</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coherent Chamelon- ultra II TI</td>
			<td>Coherent</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcite polarize</td>
			<td>Melles Griot Glan&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Galvanometer-motor mirror</td>
			<td>Cambridge technologies</td>
			<td>M 6350</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dichroic mirror</td>
			<td>Chroma technologies</td>
			<td>700 DCSPXR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Motorized stage</td>
			<td>ASI</td>
			<td>MS2000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piezo&#160;</td>
			<td>PI</td>
			<td>P721-LLQ</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>H7422P-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data acquisition card</td>
			<td>IO tech</td>
			<td>PCI 1128-4000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging software</td>
			<td>LFD</td>
			<td>Global for images-SimFCS</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 traj...

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

Research

JoVE Journal

Bioengineering

10.1K 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

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

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 ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...