We would like to thank Oxford Nanoimaging for their constructive feedback and guidance. This work was funded by the 5UM1CA121947-10 to R.P.M. and the 1R01DA040394 to D.P.D.

1 Propagation and maintenance of cell lines
<ol>
	<li>Acquire human osteosarcoma cells (U2OS) and place the cells in the growth medium supplemented with 10% exosome-free Fetal Bovine Serum and 1x Penicillin/Streptomycin solution. 
	NOTE: Exosome-free Fetal Bovine Serum was generated following the protocol presented in McNamara et. al.26.</li>
	<li>Maintain the U2OS cells in a copper-coated incubator at 37 &#176;C in 5% CO2 and passage cells in T175 flasks26,27. The cells must be maintained in mid-logarithmic growth phase to prevent subpopulations from arising, or from the accumulation of apoptotic debris during the stationary phase.</li>
</ol>
2 Exosome isolation and purification
<ol>
	<li>Grow the U2OS cell lines to full confluency in 10 separate T175 flasks with 50 mL of media per flask. Remove the cell supernatant and successively passage it through a 0.45 &#181;m and 0.22 &#181;m vacuum filtration apparatus.</li>
	<li>Subject the supernatant to crossflow filtration (also known as tangential flow filtration) on a filtration system equipped with the 750 kDa hollow-fiber cartridge in order to remove smaller proteins or metabolites28.
	<ol>
		<li>Passage the supernatant through the tangential flow filtration operator at a constant forward pressure of 30 psi, while maintaining a retention pressure of &#60;20 psi to produce a &#916; pressure of 10 or more psi. Place a magnetic stir bar in the retentate tank and set to 150 rpm26.</li>
		<li>Maintain the feed rate at 40 mL/min or higher. Collect the permeate in a reservoir and dispose of it.</li>
	</ol>
	</li>
	<li>Concentrate the supernatant to 30 mL, and then equilibrate with four volumes of 1x PBS. Collect the crossflow filtered and equilibrated solution in a 50 mL conical tube.</li>
	<li>Precipitate the EVs at 4 &#176;C overnight in a final concentration of 40 mg/mL polyethylene glycol with a molecular weight of 8,000 Da. Centrifuge the EVs at 1,200 x g at 4 &#176;C for 1 h and resuspend in 500 &#181;L of 1x PBS.</li>
	<li>Incubate the EVs with 50 &#181;g/mL of photoswitchable red membrane intercalating dye and 50 &#181;g/mL of RNase A in 1x PBS at 4 &#176;C for 1 h.</li>
	<li>Remove excess dye through a column on a protein purification system attached to a fraction collector. Identify EV fractions through UV absorbance and pool them into a microcentrifuge tube26.</li>
	<li>Affinity-select a total of 200 &#181;L of EVs using anti-CD81 magnetic beads equilibrated in 1x PBS.
	<ol>
		<li>Dilute 100 &#181;L of the anti-CD81 magnetic beads in 1x PBS to a total volume of 1 mL and allow them to bind at 4 &#176;C for 2 h in a 2 mL microcentrifuge tube with continuous rocking.</li>
	</ol>
	</li>
	<li>Wash the anti-CD81 beads and EV three times with 1x PBS. Elute CD81+ EV from the solution with 100 mM Glycine at pH 2.0 for 30 min at 37 &#176;C.</li>
	<li>Pipette the purified CD81+ EV into an equal volume of 100 mM Tris-HCl at pH 7.5 in 1x PBS. 
	&#8203;NOTE: An aliquot of purified CD81+ EV solution may be reserved for Nanoparticle Tracking Analysis or electron microscopy in order to confirm the purity of the solution and presence of EVs26.</li>
</ol>
3 Fixation and preparation
<ol>
	<li>Place affinity-purified EVs onto glass-bottom &#181;-slide 8-well plates in a total volume of 200 &#181;L (can dilute EV sample in 100 mM Tris-HCl at pH 7.5 in 1x PBS) and allow to adhere to the surface overnight at 4 &#176;C.</li>
	<li>Without removing the existing solution from the 8-well plate, fix EVs onto the plates by adding 200 &#181;L of 4% paraformaldehyde in 1x PBS to the EV-containing solution in each well and allow to incubate for 30 min at room temperature21.</li>
	<li>Carefully remove paraformaldehyde and excess solution with a micropipette in order to not disturb the EVs. Wash the EV with 1x PBS to remove excess paraformaldehyde. Perform the wash procedure three times. Remove excess 1x PBS.</li>
	<li>Prepare 250 &#181;L of dSTORM B-cubed buffer solution per sample by creating a solution of 5 mM protocatechuate dioxygenase (Part A) diluted in imaging buffer (Part B) per manufacturer&#39;s protocol. 
	NOTE: The concentration of the enzyme in the buffer may be doubled to 10 mM if photobleaching occurs.
	<ol>
		<li>Add 250 &#181;L of the prepared buffer to each well per manufacturer&#39;s protocol for 20 min at room temperature before imaging to scavenge oxidizing molecules. 
		&#8203;NOTE: The EV can then be either viewed immediately or stored at 4 &#176;C for up to a week. Replace the buffer following storage before every visualization.</li>
	</ol>
	</li>
</ol>
4 Direct stochastic optical reconstruction microscopy calibration
<ol>
	<li>Prepare the beads required for the calibration of the super-resolution microscope by diluting 100 nm microspheres to a concentration of 0.5% in molecular biology grade water and pipetting 200 &#181;L into each well of a glass-bottom &#181;-slide 8-well plate.
	<ol>
		<li>Allow the beads to settle in the wells for 1 h at room temperature.</li>
	</ol>
	</li>
	<li>Without removing the existing solution, add 200 &#181;L of 4% paraformaldehyde in PBS to each well to the calibration bead solution and allow to incubate for 30 min at room temperature.</li>
	<li>Carefully remove the paraformaldehyde with a micropipette to not disturb the beads and wash the beads three times with 1x PBS. Prepare the buffer according to step 3.4.</li>
	<li>Remove 1x PBS and add 250 &#181;L of the prepared buffer to each well. Allow the buffer to sit for 20 min before visualization.</li>
	<li>Before placing anything on the stage, connect to the 3-D microscope using the Connect the Microscope button. Add 100x oil to the objective and place the center of the well on top of the objective. In the Acquire setting, turn on the 473 and 640 nm excitation lasers and click on View.
	<ol>
		<li>Without the 3-D lens activated, view the beads under the photon saturation setting by clicking on Photon Counts in the Image Display options. Set the initial laser powers to 8.4 mW for the 473 nm laser and to 11.6 mW for the 640 nm laser.</li>
		<li>Decrease the focus of the laser to around -300 nm or the focal plane of the calibration beads to produce a clear resolution of the individual beads. Once the Z-plane is focused, further adjust the laser power levels to account for variation in each field of view.</li>
	</ol>
	</li>
	<li>Under the instrument functions, complete 3-D mapping calibration and channel mapping calibration to obtain the errors on the X-, Y-, and Z-axis. Set the max number of FOVs to 20, the target number of points to 4,000, the max distance between channels to 5.0 pixels, and the exclusion radius between channels to 10.0 pixels during channel mapping calibration.</li>
	<li>Ensure that the calibration produces a point coverage of &#62;90% and mapping quality that is good. Save the given calibration data for future image acquisitions.</li>
</ol>
5 Visualization of EV in three dimensions
<ol>
	<li>Add 100x oil onto the objective and place the prepared EVs into the microscope. Without the 3-D lens activated, turn on the 640 nm excitation laser and initially raise it to between 1.2 mW and 12.5 mW depending on the intensity of the signal and field of view to excite the red membrane intercalating dye stained EVs.
	<ol>
		<li>Under the Image Display options, switch the viewing method from photon saturation to percentiles to better visualize the EVs. Adjust laser power to minimize noise, while maximizing signal. Maintain all the other parameters.</li>
		<li>Adjust the focus of the Z-plane by clicking the up or down icon on the Z-axis. 
		NOTE: The Z-plane should be focused between -200 and -350 nm, but will vary depending on the field of view.</li>
	</ol>
	</li>
	<li>Set the exposure time to 20 ms, the frame capture to 10,000 frames, and the initial laser power to between 1.2 mW and 12.5 mW, or the laser power determined in step 5.1, depending on the intensity of the signal and field of view.
	<ol>
		<li>Activate the 3-D lens using the icon and start the acquisition by clicking on the Acquire button.</li>
	</ol>
	</li>
	<li>Throughout image acquisition, raise the laser power by 3 increments of 10 every 1000 frames, or enough to maintain a high signal-to-noise ratio. Do not adjust the Z-plane during acquisition. 
	&#8203;NOTE: The laser may be raised to a maximum of 90 mW laser power.</li>
</ol>
6 Post-acquisition modification and EV tracing
<ol>
	<li>After the image acquisition, toggle over to the Analyze viewing window. Perform drift correction on the unfiltered image, and then activate filters. Adjust photon count, localization precision, sigmas, and frame index according to Table 1.</li>
	<li>Overlay an XYZ plane view tool along the X-axis of individual EVs from the field of view and export .CSV files of photoswitching events.</li>
	<li>Bisect individual EVs on the XY-axis in an X by Y field of view using a line histogram tool, which bins photoswitching events into set distance groups.</li>
	<li>Take images of single EVs and save them as .tiff files.</li>
	<li>Create 3-D videos of individual EVs using a 3-D visualization tool and color according to placement along the Z-axis.</li>
</ol>

M.G.C. has no conflicts of interest to declare. R.P.M and D.P.D. receive material support from Oxford Nanoimaging (ONI) Inc. and Cytiva Inc. (formerly GE Healthcare). R.P.M. and D.P.D. declare competing interests for the possible commercialization of some of the information presented. These are managed by the University of North Carolina. The funding sources were not involved in the interpretations or writing of this manuscript.

EVs have become a popular area of study due to their important role in many intracellular processes and cell-to-cell signaling1,30. However, their visualization proves to be difficult as their small size falls below the diffraction limit of light microscopy. Direct stochastic optical reconstruction microscopy (dSTORM) is a direct method of visualization that bypasses the diffraction limit by capturing photoswitching events of individual fluorophores over time and...

Extracellular vesicles (EVs) are membrane-bound vesicles released by all cell types. They contain lipids, proteins, metabolites, and nucleic acids and transfer these materials locally between cells and distally between tissues and organs. There are three primary subtypes of EVs: apoptotic bodies, microvesicles, and exosomes1,2. Here, we focus our discussion on exosomes and their associated proteins.
Exosomes are secreted vesicles originating from the inward budding of early endosomes into the multivesicular body (MVB). The MVB then fuses with the plasma membrane, releasing the exosomes into the extracellular space to travel to other cells3,4. Exosomes exist on a spectrum of sizes ranging from 40 to 150 nm and are enriched with endosomal transmembrane proteins known as tetraspanins (CD9, CD63, CD81), membrane-bound endosomal sorting complex required for the transport (ESCRT), and lipid raft-associated proteins1,2, 5,6,7.
Characterizing the biochemical makeup of exosomes has become a popular field for researchers to better understand their functional nature. Many methods exist for visualizing and characterizing exosomes, including nanoscale flow cytometry, nanoparticle tracking analysis (NTA), scanning and transmission electron microscopy (TEM), surface plasmon resonance, resistive pulse sensing, and traditional light microscopy, each of which contains intrinsic pros and cons8,9. TEM and cryo-EM can achieve nanometer-based resolution, but often require dehydrating and freeze-fracture steps, thereby shrinking or lysing EVs10,11. NTA relies on light scattering, allowing for the characterization of hundreds of EVs at a time, but is an indirect measurement of particle size and cannot easily distinguish between EVs, viruses, and protein aggregates12,13,14,15,16. Nanoscale flow cytometry employs light scattering from an excitation path, which can then be translated into size measurements, but is an emerging technology, and there is little consensus on what size of particles are within the linear range of detection for various instruments12,17,18.
Traditional light microscopy using fluorescent proteins or dyes has been one of the most heavily employed techniques for visualizing subcellular compartments, protein complexes, and signaling machinery within a cell. While this technique proves useful in visualizing the localization of complexes, the diffraction limit of traditional light microscopy (around 250-400 nm) prevents the clear resolution of proteins or structures in the typical size range of an exosome (40-150 nm)12,19,20.
Super-resolution microscopy, namely, direct stochastic optical reconstruction microscopy (dSTORM), distinguishes itself from conventional light microscopy by employing the photoswitchable properties of specific fluorophores and detecting these blinking events to reconstruct images down to nanometer precision21. Photoswitching events are collected using a high-framerate detection camera over the course of tens of thousands of individual exposures, and a point spread function is used to map with high confidence the exact location of the photoswitching fluorophore19,20,22. This allows dSTORM to bypass the diffraction limit of light microscopy. Several groups have reported the use of super-resolution techniques for visualizing and tracking exosomes and their associated proteins22,23,24,25. The final resolution depends on the biophysical properties of the fluorophore, but often ranges from +/-10-100 nm along the XY-axis, allowing single-molecule resolution.
The ability to resolve individual fluorophores at this scale on the XY-axis has revolutionized microscopy. However, there is little data on the three-dimensional (3-D) dSTORM of an exosome. Therefore, we sought to establish a standard operating procedure (SOP) for dSTORM-based visualization and characterization of purified EVs, including exosomes to nanometer precision in 3-D.

<table><tbody><tr><td>15 &amp;micro;-Slide 8 well plates</td><td>Ibidi</td><td>80827</td><td></td></tr><tr><td>1X PBS</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>1X Penicillin Streptomycin solution</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>50 mL conical tube</td><td>Thermo Fisher</td><td>339652</td><td></td></tr><tr><td>500 mL 0.22 &amp;micro;m vacuum filtration apparatus</td><td>Genesee</td><td>25-227</td><td></td></tr><tr><td>750 kDa hollow-fiber cartridge cutoff filter</td><td>Cytiva</td><td>29-0142-95</td><td></td></tr><tr><td>AKTA Flux S</td><td>Cytiva</td><td>29-0384-37</td><td></td></tr><tr><td>AKTA Start</td><td>Cytiva</td><td>29022094-ECOMINSSW</td><td></td></tr><tr><td>Anti-CD81 magnetic beads</td><td>Thermo Fisher</td><td>10616D</td><td></td></tr><tr><td>B-cubed buffer</td><td>ONI</td><td>&amp;nbsp;BCA0017</td><td></td></tr><tr><td>CellMask Red</td><td>Thermo Fisher</td><td>C10046</td><td></td></tr><tr><td>Dubelco&#039;s Modified Eagle Medium</td><td>Thermo Fisher</td><td>10566016</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>97068-085</td><td></td></tr><tr><td>Frac 30 Fraction collector</td><td>Cytiva</td><td>29022094-ECOMINSSW</td><td></td></tr><tr><td>Glycine pH=2.0</td><td>Thermo Fisher</td><td>BP381-5</td><td></td></tr><tr><td>HiTrap CaptoCore 700 Column</td><td>Cytiva</td><td>17548151</td><td></td></tr><tr><td>Molecular Biology Grade Water</td><td>Corning</td><td>9820003</td><td></td></tr><tr><td>Nanoimager</td><td>Oxford Nanoimaging</td><td>Custom</td><td></td></tr><tr><td>Paraformaldehhyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Polyethylene glycol</td><td>Thermo Fisher</td><td>BP233-1</td><td></td></tr><tr><td>RNase A</td><td>Promega</td><td>A797C</td><td></td></tr><tr><td>T175 Flasks</td><td>Genesee</td><td>25-211</td><td></td></tr><tr><td>Tetraspek microspheres</td><td>Invitrogen</td><td>T7279</td><td></td></tr><tr><td>Tris- HCl pH=7.5</td><td>Thermo Fisher</td><td>BP153-1</td><td></td></tr><tr><td>Unicorn V</td><td>Cytiva</td><td>29022094-ECOMINSSW</td><td></td></tr></tbody></table>

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.

The goal of this study was to evaluate the effectiveness of super-resolution microscopy in visualizing individual EVs with nanometer resolution in three dimensions (3-D). To analyze the shape and size of individual EVs, we employed photoswitchable dye and incubated the EVs with a far-red, membrane intercalating dye, and removed excess dye through chromatography29. The affinity-captured anti-CD81 and red-stained EVs were then viewed in the super-resolution microscope under the 640 nm excitation las...

Watch this Scientific Journal Video about Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions at JoVE.com

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

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 ...

direct-stochastic-optical-reconstruction-microscopy-extracellular

Research

JoVE Journal

Biology

3.7K Views.  The University of North Carolina at Chapel Hill School of Medicine. Dit protocol maakt gebruik van superresolutiemicroscopie, met name directe stochastische optische reconstructiemicroscopie, ook bekend als dSTORM, om de diffractielimiet te omzeilen en EV's met nanometerprecisie in drie dimensies te visualiseren. Een opmerkelijk voordeel van dSTORM is het vermogen om deeltjes onder het diffractieniveau van licht direct te visualiseren zonder stappen te beschadigen die de biochemische aard van de EV veranderen. Veel evolutionair verschillende virussen maken ge...

Video: Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

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.

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 ...

Bioengineering

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Biochemistry

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

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

Developmental Biology

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular processes in vitro. In recent years, encapsulation within GUVs has proven to be a helpful approach for reconstitution experiments in cell biology and related fields. It better mimics confinement conditions inside living cells, as opposed to conventional biochemical reconstitution. Methods for encapsulation inside GUVs are often not easy to implement, and success rates can differ significantly from lab to lab. One technique that has proven to be successful for encapsulating more complex protein systems is called continuous droplet interface crossing encapsulation (cDICE). Here, a cDICE-based method is presented for rapidly encapsulating cytoskeletal proteins in GUVs with high encapsulation efficiency. In this method, first, lipid-monolayer droplets are generated by emulsifying a protein solution of interest in a lipid/oil mixture. After being added into a rotating 3D-printed chamber, these lipid-monolayered droplets then pass through a second lipid monolayer at a water/oil interface inside the chamber to form GUVs that contain the protein system. This method simplifies the overall procedure of encapsulation within GUVs and speeds up the process, and thus allows us to confine and observe the dynamic evolution of network assembly inside lipid bilayer vesicles. This platform is handy for studying the mechanics of cytoskeleton-membrane interactions in confinement.

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

Immunology and Infection

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.

This protocol presents single-particle interferometric reflectance imaging that is designed for the multi-level and comprehensive measurements of extracellular vesicles (EV) size, EV count, EV phenotype, and EV biomarker colocalization.

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different ...

Customizable Fluorescence Microscope to Visualize Cytoskeleton Networks

Design and Building of a Customizable, Single-Objective, Light-Sheet Fluorescence Microscope for the Visualization of Cytoskeleton Networks

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the dynamics of these 3D, dense networks calls for optical sectioning, which is often associated with fluorescence confocal microscopes. However, recent developments in light-sheet fluorescence microscopy (LSFM) have established it as a cost-effective and, at times, superior alternative. To make LSFM accessible to cytoskeleton researchers less familiar with optics, we present a step-by-step beginner's guide to building a versatile light-sheet fluorescence microscope from off-the-shelf components. To enable sample mounting with traditional slide samples, this LSFM follows the single-objective light-sheet (SOLS) design, which utilizes a single objective for both the excitation and emission collection. We describe the function of each component of the SOLS in sufficient detail to allow readers to modify the instrumentation and design it to fit their specific needs. Finally, we demonstrate the use of this custom SOLS instrument by visualizing asters in kinesin-driven microtubule networks.

Author Spotlight: Advancing Knowledge in Far-From-Equilibrium Materials Through Light-Sheet Microscopy

This protocol describes in detail how to build a single-objective, light-sheet fluorescence microscope and its usage for visualizing cytoskeleton networks.

Reconstituted cytoskeleton composites have emerged as a valuable model system for studying non-equilibrium soft matter. The faithful capture of the ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

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