We thank Dr. Kristen Verhey (University of Michigan, Ann Arbor) and Dr. Gregory Pazour (University of Massachusetts Medical School) for providing some plasmids. The project was supported by grants from the National Institutes of Health (NIH GM097037, GM116204 and GM122552 to W.Y.).

1. NIH-3T3 cell preparation for SPEED microscopy from stock
<ol>
	<li>1.5 weeks in advance of the experiment, recover a fresh culture of NIH-3T3 cells from a frozen stock by thawing at 37 &#176;C and transferring the cells to a 25 cm2 cell culture flask with 3 mL of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 110 mg/mL sodium pyruvate, 2 mM glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin.</li>
	<li>Incubate the cells at 37 &#176;C in a 5% CO2 incubator.</l...

<ol>
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This protocol describes the application of SPEED microscopy to the primary cilium, a cellular signaling organelle that is highly reliant on efficient protein transport. SPEED microscopy can provide high resolution (&#60; 10 nm) locations for fluorescently-labeled molecules as they pass through the single point illumination centered on the TZ. Previously it has been applied to study the protein trafficking through the NPC6,7,8. H...

Since stated by Ernst Abbe in 1873, the resolution of conventional light microscopy has been believed to be limited to approximately 200 nm due to light diffraction from the objective1,2. Currently, super-resolution light microscopy techniques break this limitation and allow the capture of dynamic images with sub-diffraction (&#60; 200 nm) resolution. The techniques generally fall into two broad categories: stimulated emission depletion (STED) microscopy based approaches, which generate sub-diffraction illumination volume due to nonlinear optical response of fluorophores in samples3; and photoactivated light microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)-based super-resolution techniques, which utilize mathematical functions to localize the centroids of fluorophores and then reconstitute these centroids to form super-resolution images4,5. Currently, due to the relatively uncomplicated optical setup, PALM and STORM are extensively employed by only activating a small subset of fluorophores in each frame of a long video of a biological preparation. This allows for the more accurate localization by 2D Gaussian fitting of the fluorescent spot, termed the point spread function (PSF), of fluorescently-labeled proteins in each frame of the video. The 2D location of each fluorescently-labeled molecule can then be superimposed on a single imaging plane to produce a super-resolution image of the biological preparation1,2. While these single-molecule localization, super-resolution approaches to microscopy certainly revolutionized how imaging of biological samples was performed, there are still challenges to be overcome. For example, STORM and PALM can achieve their best spatial resolutions after fixation of biological samples and thus present a static representation of the fluorescently-labeled proteins, which is a similar limitation of electron microscopy. Additionally, to achieve high spatial resolution for each fluorescently-labeled protein in live cells, samples must be imaged at very long framerates which are unable to capture protein dynamics. Therefore, it is necessary to overcome these main technical hurdles.
To obtain a high spatiotemporal resolution that is well-suited for detecting fast-moving proteins or RNAs in live cells, we have developed super-resolution SPEED microscopy in our laboratory (Figure 1)6,7,8. Several major technical advances in SPEED microscopy have previously enabled us to successfully track nucleocytoplasmic transport of small molecules, proteins, mRNA and virus through native nuclear pore complexes (NPCs)6,7,8. Briefly, the following features of SPEED microscopy will be used to track fast-moving macromolecules through sub-micrometer rotationally symmetrical structures in live cells, such as NPCs and primary cilia: (1) An inclined or a vertical illumination PSF enables the excitation of single molecules within a small diffraction-limit volume in the focal plane (Figure 1); (2) The inclined PSF can greatly avoid out-of-focus fluorescence and thus improve the signal-to-noise ratio. (3) The optical density of 100-500 kW/ cm2 in the illumination PSF allows thousands of photons to be collected from single fluorophores with fast detection speeds (&#62; 500 Hz). (4) The fast detection speed also greatly reduces the single-molecule spatial localization error (&#60; 10 nm) in determining the spatial trajectories of moving fluorescent molecules in live cells, because molecular diffusion is one of major factors causing imperfections of single-molecule localization for moving molecules. (5) Well-established 2D to 3D transformation algorithms enable us to provide 3D spatial probability density maps of transport routes for molecules in the NPC or the primary cilium. It is noteworthy that our conversion process between the Cartesian and the cylindrical coordination system is used to generate a 3D spatial probability density map rather than 3D single-molecule tracking (Figure 2). Previously, electron microscopy data have revealed that the NPC9,10 and the primary cilium11 both have a rotationally symmetrical structure. In principle, randomly diffusing molecules moving through the NPC or primary cilium should also have rotationally symmetrical distributions. As shown in Figure 2, a high number of randomly diffusing molecules inside the cylinder would generate rotationally symmetrical distributions at the cross-section view as that in the NPC, further resulting in an approximately uniform spatial distribution within each very small sub-region between two neighboring rings (Figure 2E). This uniform distribution leads that the spatial distribution along &#952; dimension in the cylindrical system is constant. Then the 3D coordinates (R, X, &#952;) can be simplified to be the 2D coordinates (R, X, constant). Actually, our conversion process between the Cartesian and the cylindrical systems is from 2D (X, Y) to 2D (R, X, constant). The constant &#952;, refers to the spatial density p in Figure 2E, is calculated by using the equation A<img alt="Equation 1" src="/files/ftp_upload/56475/56475eq1.jpg" />.
Ultimately, single-molecule tracking has broad application in biological research, thus, it is natural that a plethora of techniques will be developed to fill specific biological niches12,13,14. Such is the case with SPEED microscopy. Previously, when coupled with a 3D transformation algorithm, this technique was developed to resolve 3D transport routes of transiting molecules through the NPCs, a sub-diffraction-sized and rotationally symmetric biological structure6. In this paper, primary cilia are shown to be excellent model organelles as well. Primary cilia are cylindrical, antenna-like organelles (~125 nm radius) that project from the surface of most mammalian cells15,16,17. They are responsible for receiving external signals and transmitting an intracellular response typically associated with growth and metabolism15,16. Therefore, flux of structural proteins, recycling of transmembrane receptors, and transmission of intracellular messengers are vital responsibilities of primary cilia. At the juncture between the primary cilia and the cell body is a critical selectivity barrier, called the transition zone or TZ, through which all this protein transport must occur11,18,19,20. In addition to the gating function of the TZ, at least two transport processes, intraflagellar transport and passive diffusion, are thought to be responsible for the movement of protein through this region16,21,22. From a human health standpoint, the loss of primary cilia and subsequent deregulation of downstream signaling is characteristic of many cancers. In addition, many genetic diseases, such as Bardet-Biedl syndrome and polycystic kidney disease, are associated with defective protein transport23. Both the sub-diffraction limit size and the complex process of selective protein transport through the TZ make the primary cilia a prime target for this technique. In this methods paper, we will demonstrate the tracking of a ciliary transmembrane protein, somatostatin receptor 3 (SSTR3)24, labeled externally with Alexa Fluor 647 and a component of IFT, IFT2025, labeled with a fused GFP molecule.

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	<tbody>
		<tr height="47">
			<td height="47" style="height:47px;width:123px;">25 cm2 tissue culture dish</td>
			<td style="width:204px;">Corning</td>
			<td style="width:101px;">VV-01936-00</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Penicillin/streptomycin</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">15140122</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Fetal bovine serum</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">10438018</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">DMEM</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">10566-016</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">OPTIMEM</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">31985062</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Trypsin</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">25300054</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">Phosphate buffered saline</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:101px;">P3813-1PAK</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Transit LT1</td>
			<td style="width:204px;">Mirus</td>
			<td style="width:101px;">MIR 2300</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">35 mm glass bottom dish</td>
			<td style="width:204px;">MatTek</td>
			<td style="width:101px;">P35GCOL-0-14-C</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">AlexaFluor 647-conjugated streptavidin</td>
			<td style="width:204px;">ThermoFisher</td>
			<td style="width:101px;">S21374</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Biotin</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:101px;">B4501-100MG</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">633 nm He-Ne laser</td>
			<td style="width:204px;">Melles Griot</td>
			<td style="width:101px;">25-LHP-928-249</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">561 nm solid state laser</td>
			<td style="width:204px;">Coherent</td>
			<td style="width:101px;">OBIS 561-50 LS</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">488 nm solid state laser</td>
			<td style="width:204px;">Coherent</td>
			<td style="width:101px;">1185053</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">Inverted fluorescence microscope</td>
			<td style="width:204px;">Olympus</td>
			<td style="width:101px;">IX81</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:123px;">1.4-NA 100&#215; oil-immersion apochromatic objective</td>
			<td style="width:204px;">Olympus</td>
			<td style="width:101px;">UPLSAPO 100&#215;</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:123px;">On-chip multiplication gain charge-coupled-device camera</td>
			<td style="width:204px;">Roper Scientific</td>
			<td style="width:101px;">Cascade 128+</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">Dichroic filter</td>
			<td style="width:204px;">Semrock</td>
			<td style="width:101px;">Di01- R405/488/561/635-25x36</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:123px;">Emission filter</td>
			<td style="width:204px;">Semrock</td>
			<td style="width:101px;">NF01-405/488/561/635-25X5.0</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:123px;">Slidebook 6.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td></td>
			<td style="width:191px;">digital microscopy software</td>
		</tr>
	</tbody>
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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.

This section demonstrates the data obtained from performing SPEED microscopy at the TZ of primary cilia to study the transport route of SSTR3 connected by a ~15 nm external linker to Alexa647 (Figure 3A). It serves the dual purpose of verifying the 3D transformation algorithm. Alexa647 should only label the external surface of the primary cilium and therefore, the 3D transport route should reveal a high-density transport route at that locatio...

Watch this Scientific Journal Video about Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium at JoVE.com

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

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 ...

The overall goal of this experiment is to identify protein transport routes in primary cilia. This method can help answer key question in the cilia field, such as protein transport route differences in normal and dysfunctional cilia. The main advantage of this technique is that it has high enough resolution to see subdiffraction protein transport routes in live cells.To begin, thaw frozen stock of specially engineered of NIH-3T3 cells at 37 degrees Celsius, one and a half weeks in advanced of the experiment. Transfer the thawed cells to a 25 centimeter squared cell cultured flask containing three milliliters of pre-warmed media. Maintain the cells at 37 degrees Celsius in a 5%carbon-dioxide incubator.Culture the cells until they reach 80%confluency and then split the cells. In order to split the cells, trypsinize them with 0.25%Trypsin for two minutes at 37 degrees Celsius. Following the brief incubation, aspirate the Trypsin and replace it with two milliliters of pre-warmed culture media.Pipette the media to break up any cell clusters, then remove 75%of the cells from the flask and bring total volume of media back up to three milliliters. Split the cells at least three times before the experiment to ensure homogeneity of their cell cycle. Two days before the experiment, split the cells and plate them 60-70%confluency on a 35 millimeter glass bottom plate with 1.5 milliliters of the same culture medium.Then, one day before the experiment, chemically transfect the cells with the desired plasmid. Mix 500-1000 nanograms of the plasmid in a one to 2.5 ratio with transfection reagent and 0.25 milliliters of reduced serum media without antibiotics for 30 minutes. Once the transfection complex has formed, aspirate the media from the 35 millimeter glass bottom dish and replace it with the 0.25 millimeters transfection mixture and an extra 1.25 millimeters of reduced serum media without antibiotics and return the plate to the incubator.For cells with externally labeled SSTR3 construct, remove the media from the glass bottom dish one hour before the experiment. Wash the cells five times with one milliliter of PBS and add one milliliter of reduced serum media supplemented with one micromolar of Alexa 647 conjugated streptavidin. No more than 15 minutes before the experiment, remove media from the glass bottom dish and wash the cells five times with one milliliter of PBS.Then add one milliliter of the imaging buffer into the glass bottom dish. Affix the glass bottom plate to the stage in the microscope. Find a GFP express cell using the GFP filter set and locate a cell that is properly expressing SSTR3-GFP in a primary cilia.Once a suitable cell has been found, align the NPHP4-mCherry spot at the base of the primary cilia with the location on the imaging plane that corresponds to the laser's single point illumination. Using the snap function, in the camera tab of the focus controls window, capture an epifluorescence image of NPHP4-mCherry and SSTR3 proteins tagged with green fluorescence protein. Once the reference images are obtained, locally reduce the concentration of labeled single molecules by photobleaching the transition zone with a one milliwatt laser illumination for 20 seconds.To prepare for single molecule tracking, reduce the laser illumination power to 0.5 milliwatts for molecules labeled with Alexa 647. Set the gain and intensification to maximum and the frame rate to two milliseconds. Then click the stream button in the camera tab of the focus controls window and engage the appropriate illumination laser.Record the non-photobleached labeled single molecules as they are transported through the photobleached region of the transition zone. And remember to record no more than two minutes of video to avoid the effects of ciliary drift. After capturing the single molecules, process the videos using a 2D Gaussian fitting algorithm such as Glimpse by the Gelles lab, which precisely localizes the centroid of each single molecule's excitation point, spread function, and an encompassing area of interest.Finally, select all single molecule locations with a precision better than 10 nanometers and correct the center of the cilia based on the distribution of single molecule locations fitted with a 2D Gaussian function. As described in this video, single point illumination can be used to track an AF647 tagged SSTR3 molecule through the transition zone marked by NPHP4-mCherry. Before data analysis, superimposing the single molecule video on the wide field illumination is a useful validation step to make sure the laser is properly aligned, and that there is a adequate signal-to-noise ratio.Imaged here is a cell with a primary cilium identified by the staining. IFT20 is component of the intraflagellar transport complex which carries cargo along microtubules inside primary cilia, marked by Arl13b-mCherry. Once identified, speed microscopy is used to track individual IFT20-GFP proteins.Next, and 2D to 3D transformation algorithm is used to produce the spatial probability density distribution of the protein inside primary cilia along a single dimension. This high density region likely colocalizes with the axonemal microtubules in agreement with the known location of the intraflagellar transport route. While attempting this procedure, it's important to precisely align the sample with the laser to minimize systematic error.After watching this video, you should have a good understanding of how to do single molecule tracking with speed microscopy and you will know how to obtain single molecule transport route in primary cilia.

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8.2K vistas.  Temple University. The overall goal of this experiment is to identify protein transport routes in primary cilia. This method can help answer key question in the cilia field, such as protein transport route differences in normal and dysfunctional cilia. The main advantage of this technique is that it has high enough resolution to see subdiffraction protein transport routes in live cells.To begin, thaw frozen stock of specially engineered of NIH-3T3 cells at 37 degrees Celsius, one and a half weeks in adva...