Name: Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM (Video) | JoVE Journal
Uploaded: 2017-06-24T12:30:00.000+00:00
Duration: 8 min 43 s
Description: Learn multiplexed super-resolution imaging of T cells using madSTORM to visualize multiple proteins at single molecule resolution. This method enables greater multiplexing than traditional microscopy for immunology research.

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5 minutes

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
<ol>
	<li>Directly conjugate antibodies with Alexa-647 (A647) dye using the Alexa 647 antibody labeling kit. Follow the labeling protocol provided by the manufacturer. 
	NOTE: Dialysis of antibody solution in 1x PBS is recommended before this step to increase antibody labeling efficiency.</li>
	<li>Spin labeled antibodies for 5 min at 20,800 rcf and collect supernatant to remove aggregated antibodies.</li>
	<li>Preparation of fluorescent nano-diamond-coated coverslips
	<ol>
		<li>Coat 8-well coverslip chambers (see reagent/material list) with 250 &#956;L 0.01% Poly-L-Lysine (PLL) for 15 min, aspirate, and dry at 65 &#176;C for 30 min. (No rinse before drying.)</li>
		<li>Prepare a dilution of 100 nm FNDs in 1x PBS. Test various dilutions to ensure enough FNDs are visible each field of view in step 1.2.7 (we are using a 1:200 dilution of FNDs provided by the manufacturer).</li>
		<li>Vortex the diluted FNDs for 1 min.</li>
		<li>Sonicate the FND supernatant for 30 s at a high-power setting.</li>
		<li>Incubate the sonicated FND supernatant in a PLL-coated 8-well coverslip chamber for 30 min at room temp.</li>
		<li>Wash 5x with 1x PBS and visualize the FND-coated chamber using 647 nm laser excitation on the TIRF microscope. Ideally, 4-10 individual FNDs should be visible in a field of view given the appropriate dilution of FNDs in step 1.2.2 (we are using a 100X objective with 256 x 256 camera pixel setting to yield 61 x 61 &#956;m field of view) with at least one FND present in each quadrant of the imaging field. If the FNDs appear to be clustered (i.e. FNDs with significantly higher signal than surrounding FNDs and a point spread function larger than 200 nm), centrifuge FNDs at 6,800 rcf for 1 min, and repeat the coating procedure using the FND supernatant.</li>
		<li>Add 250 &#956;l of anti-CD3 antibody (10 &#956;g/mL) in each well and incubate for 1 hr at 37 &#176;C, or overnight at 4 &#176;C.</li>
		<li>Remove solution and add 1x PBS for 30 sec. Repeat this wash step 5 times (wash 5x).</li>
	</ol>
	</li>
	<li>Activation of Jurkat T cells
	<ol>
		<li>Spin 1 ml of Jurkat T cells at 800 rcf for 6 min and resuspend in 300 &#956;l 1x HBS solution. Jurkat T cells should ideally be at a concentration of 0.5-1.0 x 106 cells/ml before spinning.&#160;1x HBS solution consists of HEPES buffer saline with 1% Bovine serum albumin as described previously22.</li>
		<li>Add 150 &#956;L of 1x HBS solution in each well and incubate at 37 &#176;C for 15 min.</li>
		<li>Add 50 &#956;L resuspended Jurkat T cells to each well and incubate for 3 min at 37 &#176;C.</li>
		<li>Add 300 &#956;L of 4% Paraformaldehyde to each well and incubate for 30 min at 37 &#176;C.</li>
		<li>Wash 3x with 1x PBS.</li>
		<li>Permeabilize cells by adding 250 &#956;L of 0.1% Triton-X solution for 5 min at room temp.</li>
		<li>Wash 3x with 1x PBS.</li>
		<li>Add 250 &#956;L 1% fish gelatin solution in 1x PBS to each well for 30 min at room temp.</li>
		<li>Wash 3x with 1x PBS.</li>
	</ol>
	</li>
	<li>Imaging activated Jurkat T cells using madSTORM
	<ol>
		<li>Add 200 &#956;L of labeled antibody at 0.1-0.5 &#956;g/mL to fixed cells for 1 hr at room temp.</li>
		<li>Wash 5x with 1x PBS.</li>
		<li>Add 1 ml of STORM buffer and cover the chamber with a glass coverslip to limit exposure to air. The STORM buffer has been described previously10. We recommend making new STORM buffer for each round of imaging and spinning the STORM buffer at 20,800 rcf for 2 min to remove precipitates.</li>
		<li>Using low 647 nm laser power in TIRF mode, locate a stained cell with at least 3 FNDs in the field of view. To aid in locating FNDs, concomitant excitation with 568 nm laser can be used to increase the brightness of the FND signal.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: The performance of averaged fiducial correction described in section 2.1 improves with inclusion of more FNDs.</li>
		<li>Increase 647 nm laser power and acquire images. We typically acquire 10,000 frames at 200 ms exposure, 125 mW 647 nm laser, 100X TIRF objective lens (1.49 NA), 100X EM gain (5 MHz at 16 bit, conversion gain 1). Details of our imaging setup have been described previously10.</li>
		<li>Wash 5x with 1x TBS.</li>
		<li>Add 1 ml of elution buffer (3.5 M MgCl2, 20 mM PIPES, 0.1% Tween-20, pH 6.5) and incubate at room temp for 1 min.</li>
		<li>Repeat elution 3 times. (exact elution conditions and the number of elution rinses needed to remove the signal must be checked for each antibody used).</li>
		<li>Wash 3x with 1x TBS and add 1 ml of 1x PBS.</li>
		<li>Photobleach using 647 nm laser at high power (125 mW) with 405 nm laser at 2-5 mW to photo-activate A647 dyes in dark state. Wait until all remaining signal from non-eluted antibody is photobleached. Typically this takes 2-5 s of laser exposure. Acquiring sample images (~100 frames) using the STORM settings in 1.3.5 after elution and photobleaching is recommended to confirm removal of signal. 
		We typically remove 99.8% of signal using this method. We recommend testing the elution efficiency of each antibody before use in madSTORM as shown previously10.</li>
		<li>Add 250 &#956;L 4% Paraformaldehyde for 15 min. This prevents reverse crosslinking of fixed molecules in the cell.</li>
		<li>Wash 3x with 1x PBS.</li>
		<li>Repeat steps 1.3.1-1.3.12 for sequential labeling of multiple targets. Figure 1 shows a composite image of multiple molecules in an activated Jurkat T cell acquired using madSTORM.</li>
	</ol>
	</li>
</ol>
2. Drift Correction and Alignment of Multiplexed Image Stacks
<ol>
	<li>Drift correction of madSTORM image stacks
	<ol>
		<li>Open images using ImageJ. We recommend converting the image files to a TIFF format before opening in ImageJ. Use rectangle ROI to select a single FND. Play the time lapse to make sure that the FND is visible in every frame of the image stack. Use Run Analysis in the ThunderSTORM plugin to localize the FND. For the averaged fiducial correction method described in 2.1.5, it is important that a single FND is localized in each image frame (i.e. number of identified peaks should be equal to the number of frames).</li>
		<li>If the FND is not identified in every frame, select another FND. If multiple peaks are located in a single frame, remove the peak that deviates the most from previous peaks.</li>
		<li>Repeat 2.1.2 for each FND in the image stack. Keep in mind that some FNDs should not be included in the drift correction process so they can serve as an independent gauge for drift correction precision as described in step 2.1.7.</li>
		<li>For faster, less precise drift correction, use either the cross correlation or fiducial correction algorithms included in ThunderSTORM to correct the FND images. Typically, we use 100 bins, 5 magnification, and 0.1 trajectory smoothing factor for cross correlation and 10 max distance, 0.1 min marker visibility ratio, and 0.01 trajectory smoothing factor for fiducial correction. Cross correlation and fiducial correction will yield a correction file which can be applied to other FNDs to test the drift correction precision.</li>
		<li>For more stringent, more precise drift correction, use the averaged fiducial correction (AFC) algorithm as described previously10. This process does not require optimization of parameters and yields higher localization precision than predicted by SMLM localization algorithms. However, averaged drift correction requires at least 4 FNDs and for the FNDs to be localized in every frame of the image stack. AFC drift correction performs better with inclusion of more FNDs because a large number of localized FNDs will lessen the distorting effect of an outlier peak in a given frame.</li>
		<li>Localize all point spread functions within the image stack using ThunderSTORM as described previously10. We typically use pixel size of 100 nm, photoelectrons per A/D count of 4.28, base level A/D count of 100, EM gain of 100, PSF: Integrated Gaussian localization, 5 pixel fitting radius, maximum likelihood fitting method, and 1.3 pixel initial sigma.</li>
		<li>Apply the drift correction factor acquired from FNDs to localizations from the entire image stack. Visually inspect the final image to ensure proper drift correction (Figure 2). The resulting drift correction factor should be independently tested on FNDs not included in the AFC procedure to measure the level of drift correction precision. The precision of drift-corrected, independent FNDs should be close to the average precision/uncertainty value calculated from the localization algorithm utilized in step 2.1.6.</li>
		<li>Repeat steps 2.1.1-2.1.7 for the madSTORM image stacks of sequentially labeled antibodies. Keep in mind, that localizing the same set of FNDs in each madSTORM image stack is critical for the following alignment procedure.</li>
	</ol>
	</li>
	<li>Alignment of madSTORM images
	<ol>
		<li>Calculate the centroid position of the drift corrected FNDs in each madSTORM image. To do this, find the mean x and y axis positions for each localized FND, and average the mean x and y positions of all FNDs in each madSTORM image. The detailed algorithm for this step has been described previously10. Align sequential madSTORM images using the centroid positions of FND fiducial markers. To perform the alignment, calculate the offset between two localized madSTORM images by subtracting the centroid position of FNDs in the second madSTORM image from the first. Then subtract the offset amount from all localizations in the second madSTORM image.</li>
		<li>We recommend using the first madSTORM image as a reference image and to repeat this alignment step between the first and third, first and fourth, etc. Test the alignment precision by measuring the offset between FNDs not included in the alignment process. Large offsets may indicate that different sets of FNDs were used when calculating the centroid positions of sequential madSTORM images.</li>
		<li>If so, steps 2.2.1-2.2.2 should be repeated using the same set of FNDs to perform alignment. 
		NOTE: Custom codes for both the AFC drift correction and alignment procedures are provided.</li>
	</ol>
	</li>
</ol>

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</ol>

We have nothing to disclose.

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><tbody><tr><td>8 well coverslip chamber</td><td>Lab-tek</td><td>155409</td><td></td></tr><tr><td>0.1% Poly-L-Lysine solution</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>NV-100 nm Fluorescent Nano-diamond</td><td>Adamas</td><td>Red FND</td><td></td></tr><tr><td>anti-CD3&amp;epsilon; antibody</td><td>BD Biosciences</td><td>555329</td><td></td></tr><tr><td>Bovine serum albumin, Fraction V</td><td>KSE Scientific</td><td>98-100P</td><td></td></tr><tr><td>Triton-X</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>10% Paraformaldehyde</td><td>EMS</td><td>15712</td><td>Carcinogen</td></tr><tr><td>Gelatin from fresh water fish skin</td><td>Sigma-Aldrich</td><td>G7041</td><td></td></tr><tr><td>Alexa 647 antibody labeling kit</td><td>Thermo Fisher</td><td>A20186</td><td></td></tr><tr><td>Magnesium Chloride hexahydrate</td><td>Sigma-Aldrich</td><td>138924</td><td></td></tr><tr><td>PIPES</td><td>Sigma-Aldrich</td><td>P6757</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M7154</td><td>Highly toxic, air sensitive</td></tr><tr><td>Cysteamine</td><td>Sigma-Aldrich</td><td>30070</td><td>Highly toxic</td></tr><tr><td>Cyclooctatetraene 98%</td><td>Sigma-Aldrich</td><td>138924</td><td>Highly toxic, air sensitive</td></tr><tr><td>10x PBS</td><td>KD Medical</td><td>RGF-3210</td><td></td></tr><tr><td>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

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...

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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Research

JoVE Journal

Immunology and Infection

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

Test Samples for Optimizing STORM Super-Resolution Microscopy

High-resolution intravital imaging with enhanced contrast up to 120 µm depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 µm depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

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...

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

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

Imaging the Human Immunological Synapse

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.

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

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image analysis technique.

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

This report describes an automated protocol for multiplex immunofluorescence (mIF) assays on an automated slide stainer. It demonstrates the high reproducibility of spatial proteomics on standard high-throughput tissue autostainers and whole-slide fluorescence imagers, which is ideal for custom mIF assays across a large number of tissue slides in translational research.

High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research

Learn multiplexed super-resolution imaging of T cells using madSTORM to visualize multiple proteins at single molecule resolution. This method enables greater multiplexing than traditional microscopy for immunology research.

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

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