Name: highly multiplexed, super-resolution imaging of t cells using madstorm
Uploaded: 2017-06-24T12:30:00
Description: Watch this Scientific Journal Video about Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM at JoVE.com

We thank Xufeng Wu for access to the STORM microscope. This research was supported by the Intramural Research Program of the National Cancer Institute (NCI) Center for Cancer Research and the National Heart Lung and Blood Institute (NHLBI).

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in this protocol are toxic and carcinogenic. Please use all appropriate safety practices when performing the protocol including the use of engineering controls (fume hood, glovebox) and personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes).
1. Multiplexed Imaging of Activated T Cells
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The sequential multiplexing, drift correction, and alignment procedures in madSTORM allow precise, highly multiplexed visualization of heterogenous structures in cells.10 In addition, madSTORM avoids the limitations of multi-color STORM such as chromatic aberration and sub-optimal photoswitching/emission properties of non-far red dyes9,12. As the elution step significantly reduces steric interference from sequentially bound antibodies, mad...

A variety of super-resolution microscopy techniques have been developed to overcome the diffraction limit of light microscopy (~200 nm). Among these is a category of techniques called single molecule localization microscopy (SMLM) which includes photo-activation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). SMLM techniques share in the use of fluorophores that can be switched between on (fluorescent) and off (dark/photo-switched) states, allowing sequential localization of fluorescence from single molecules1,2,3.
Due to its compatibility with commercially available dyes and microscopes, direct STORM (dSTORM) has become a widely adopted SMLM technique4. dSTORM can routinely achieve ~10 nm localization precision, defined as the uncertainty in calculating the center of a diffraction-limited point spread function (PSF). However, despite the high precision estimated using localization algorithms5,6,7, accurate determination of the actual location of single molecules has been hampered by a number of issues. First, mechanical movement of the microscope stage during image acquisition adds significant uncertainty to localization precision. As SMLM images are obtained over thousands of time-lapse frames, nano-scale movements of the microscope stage can significantly compromise the precision of the final super-resolution image8. To compensate for stage movement during image acquisition, stage drift is commonly estimated from regression-based fitting of binned localizations from the image itself (cross correlation) or sequential localizations from fiducial markers (fiducial correction)1,9. However, these methods require optimization of multiple parameters for each image stack, and cannot account for stage movements at short time scales such as mechanical vibration. Gold nano-particles and multi-color fluorescent beads have been used as fiducial markers in SMLM, but they are not photo-stable, and result in significantly lower precision after drift correction than the nitrogen vacancy-center fluorescent nano-diamonds (FNDs) used in madSTORM10.
In addition to the diffraction limit, light microscopy is further restricted by spectral limits. Simultaneous visualization of multiple targets requires fluorescent probes with non-overlapping spectral profiles, generally restricting fluorescence-based light microscopy to 6 colors and SMLM to 2-3 colors4,11,12. Moreover, non-linear chromatic aberration causes misalignment of multicolor images, which require extensive alignment procedures using multi-colored fiducial markers8,13. To overcome these limits, previous studies have imaged multiple targets using repetitive photobleaching or chemical quenching of sequentially bound fluorophores14,15,16,17,18,19. While these methods can overcome the spectral limits of microscopy, fluorescence bleaching is known to be a toxic process20, and prolonged bleaching or quenching may cause unwanted side effects such as loss of crosslinking. Furthermore, the accumulation of fluorescent probes could lead to steric blocking of binding sites in the sample, preventing large-scale multiplexing and robust targeting of epitopes. To avoid such steric interference, a recent study achieved multiplexing using stochastic exchange of freely diffusing protein fragments21. Whereas this method allows dense labeling of cellular structures, it requires extensive biochemical preparation to isolate peptide fragments, cannot locate single molecule positions, and does not readily facilitate large-scale multiplexing using commercially available probes. We present a detailed video protocol describing the sequential binding and elution of fluorescently antibodies for multiplexed, antibody size-limited dSTORM (madSTORM) imaging, and use of fluorescent nano-diamonds to achieve precise drift correction and alignment.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">8 well coverslip chamber</td>
			<td style="width:117px;">Lab-tek</td>
			<td style="width:124px;">155409</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% Poly-L-Lysine solution</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NV-100nm Fluorescent Nano-diamond</td>
			<td>Adamas</td>
			<td>Red FND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD3&#949; antibody</td>
			<td>BD Biosciences</td>
			<td>555329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin, Fraction V</td>
			<td>KSE Scientific</td>
			<td>98-100P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Paraformaldehyde</td>
			<td>EMS</td>
			<td>15712</td>
			<td>Carcinogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin from fresh water fish skin</td>
			<td>Sigma-Aldrich</td>
			<td>G7041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 647 antibody labeling kit</td>
			<td>Thermo Fisher</td>
			<td>A20186</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>138924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M7154</td>
			<td>Highly toxic, air sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cysteamine</td>
			<td>Sigma-Aldrich</td>
			<td>30070</td>
			<td>Highly toxic&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclooctatetraene 98%</td>
			<td>Sigma-Aldrich</td>
			<td>138924</td>
			<td>Highly toxic, air sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PBS</td>
			<td>KD Medical</td>
			<td>RGF-3210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x TBS</td>
			<td>KD Medical</td>
			<td>RGF-3385</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly multiplexed, super-resolution imaging of t cells using madstorm

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

The sequential elution and staining method was used to produce the multiplexed madSTORM image of microclusters and other structures in an activated Jurkat T cell (Figure 1, check figure alignments). Each pseudo-colored image represents one round of madSTORM imaging acquired in steps 1.1.1 to 1.3.13. As described previously10, the field of view should be monitored for residual signal from previous, non-eluted antibody to avoid crosstalk...

Watch this Scientific Journal Video about Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM at JoVE.com

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

The overall goal of this imaging protocol is to visualize multiple proteins in an activated T cell, using single molecule localisation microscopy. This method can help answer key questions in the T cell activation field such as, the single molecule distribution of TC or microcluster components. The main advantage of this technique is that, it allows greater multiplexing than achieved by current supervised solution microscopy methods.To begin, directly conjugate antibodies with Alexa 647 dye, using the Alexa 647 antibody labeling kit, following the labeling protocol provided by the manufacturer. Centrifuge the labeled antibodies for five minutes at 20, 800 times G.Following centrifugation, collect the supernatant to remove the aggregated antibodies. Next, coat A12 cover slip chambers with 250 microliters of 01%poly-l-lyzine for 15 minutes.Then, aspirate the solution without rinsing and dry the chambers at 65 degree celsius for 30 minutes. Prepare dilution of 100 nanometer fluorescent nanodiamonds or FNDs, in one PBS, test various dilutions to ensure that enough FNDs will be visible in each field. Vortex the diluted FNDs for one minute.Next, sonicate the FND supernatant for 30 seconds at a high power setting. Incubate the sonicated FND supernatant in a poly-l-lyzine coated A12 cover slip chamber for 30 minutes at room temperature. After washing the chamber five times, with 1x PBS, visualize the FND coated chamber using 647 nanometer laser excitation on the turf microscope.Use a 100x objective tealed a 61 by 61 micron field of view. Ideally, four to 10 individual FNDs should be visible in the field of view, given the appropriate dilution of FNDs, with at least one FND present in each quadrant of the imaging field. Once the proper density of FNDs has been achieved, add 250 microliters of anti-CD3 antibody to each well.Incubate the chamber for one hour at 37 degree celsius or overnight at four degree celsius. Following incubation, remove the solution from the wells and add 1x PBS for 30 seconds. Repeat this wash step five times.Spin one milliliter of Jurkat T cells at 800 times G for six minutes. Jurkat T cells should ideally be at a concentration of 0.5 to one million cells per milliliter concentration before spinning. Re-suspend the cells in 300 microliters of 1x HBS solution.Meanwhile, add 150 microliters of 1x HBS solution to each well of the chamber and incubate at 37 degree celsius for 15 minutes. Then, add 50 microliters of the re-suspended Jurkat T cells to each well and incubate for three minutes at 37 degree celsius. Next, add 300 microliters of 4%paraformaldehyde to each well and incubate for 30 minutes at 37 degree celsius.After washing three times with 1x PBS, permeabilise the cells, by adding 250 microliters of 01%Triton x solution for five minutes at room temperature. Following three additional washes with 1x PBS, add 250 microliters of 1%fish gelatin solution to each well for 30 minutes at room temperature. Finally, wash the cells three more times with 1x PBS.Add 200 microliters of labeled antibody at 0.1 to 0.5 micrograms per milliliter to the fixed cells for one hour at room temperature. After washing the cells five times with 1x PBS, add one milliliter of STORM buffer and cover the chamber with a glass cover slip to limit exposure. Use clamps on the microscope stage to hold the cover slip chamber in position.This step is critical for large scale multiplexing, since the same field of view must be present after several rounds of washing and incubation. Using a low 647 nanometer laser power in turf mode, locate a stained cell with at least three FNDs in the field of view. Increase the 647 nanometer laser power and acquire the images.Typical parameters can be found in the text protocol. Wash the cells five times with 1x tris-buffered saline or 1x TBS. Then, add one milliliter of elution buffer and incubate at room temperature for one minute.Repeat the elution three times. Use the exact elution conditions and a number of elution rinses needed to remove the signal as specified for each antibody. Then, wash the cells three times with 1x TBS and add one milliliter of 1x PBS.Proper execution of this elution step, will result in removal of previously bound antibody and allow higher efficiency for subsequent antibody labeling. It is important to use TBS for washes to minimize precipitation between elution steps. Photo bleach the cells using a 647 nanometer laser at high power, with a 405 nanometer laser at two to five milliwatts to photo activate Alexa 647 dyes in the dark state.Wait until all remaining signal from non-eluted antibody is photo bleached. Typically, this takes two to five seconds of laser exposure. Acquire sample images, using the STORM settings after illusion and photo bleaching, to confirm removal of the signal.Next, add 250 microliters of 4%paraformaldehyde for 15 minutes. This prevents reverse cross-linking of fixed molecules in the cell. Wash the cells three times with 1x PBS.Repeat these steps for sequential labeling of multiple targets. Finally, perform drift correction and alignment of multiplex image stacks at described in the text protocol. The sequential illusion and standing method was used to produce the multiplex MET STORM image of microclusters and other structures in an activated Jurkat T cell.The final MET STORM image has been corrected for a drift and alignment, using average fiducial correction and FND fiducial markers. Shown here, are single molecule localisation microscopy images of a single FND localized in 30, 000 image frames before and after drift correction with average fiducial correction. Similarly, here are single molecule localisation microscopy images of an activated Jurkat T cell stained with anti-phosphorelated SLP76 antibody and FND futusial markers, before and after drift correction with average futusial correction.After watching this video, you should have a good understanding of how to perform multiplexed super resolution imaging of proteins in an activated T cell, using MET STORM. Once mastered, this technique can be done in three hours for each multiplexing round. While attempting this procedure, it's important to remember to keep the specimen in the same position on the microscope stage.This can be done using stage clamps. Don't forget that working with our modified STORM buffer, can be extremely hazardous and precautions such as, respirators and goggles should always be taken while performing this procedure.

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