Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.
Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in <80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.
The immunological synapse is a complex milieu of signaling proteins and cytoskeletal elements. The cytolytic synapse was originally described as having a "bulls-eye" like structure with a ring of actin and adhesion molecules surrounding a central secretory domain1-4. However, we now know that it consists of microscopic domains of active signaling that require continuous dynamic cytoskeletal reorganization for function5-11. Much of the information that we now have about the synapse has been derived from microscopy, and immunologists have been early adopters of cutting-edge imaging technology.
One such novel technology is super-resolution microscopy. Conventional light microscopy is spatially limited by the diffraction barrier of light, which sets the lower limit of resolution for all fluorescence microscopy, including confocal, at approximately 200 nm. In recent years, several techniques have been developed that allow resolution below the diffraction barrier. These include stimulation emission depletion microscopy (STED), structured illumination microscopy (SIM), stochastically resolved microscopy (STORM), and photoactivatable light microscopy (PALM). These techniques have been reviewed in detail elsewhere12-15, but are outlined below. Subdiffraction limited resolution is generated in unique ways in each system. The selection of a super-resolution technique, therefore, should be dictated by the experiment and experimental system of interest.
STED super resolution is achieved using a high-intensity torroidal depletion beam that selectively "silences" fluorescence around each fluorophore of interest following excitation, resulting in subdiffraction limited fluorescence microscopy16-18. One advantage of STED is that image acquisition is rapid and requires relatively little post-processing. While dye selection is dictated by the spectral position of the depletion beam, which in the commercially available system is situated at 592 nm, several commercially available dyes are available that make combinations of two fluorophores possible. In addition, commonly used fluorescent reporters such as GFP can be imaged, making live cell experiments possible19,20.
We have previously used STED to identify and quantify regions of F-actin hypodensity that are utilized by NK cells for degranulation21,22. We propose that STED is a good choice for imaging the immune synapse due to its relatively flexible availability of fluorophores and superior improvement in resolution in the x-y axis. In addition, on the commercially available STED system utilized for these experiments, the use of a high-speed (12,000 Hz) resonance scanner allows for rapid acquisition of images with minimal damage to samples. Limited flexibility in dye selection is considered a disadvantage of STED12, however dual color STED is relatively straightforward with several commercially available fluorophores. The integration of STED with a laser scanning confocal microscope also allows for additional confocal imaging in combination with STED, so while STED is limited to two channels, additional structures can be imaged in confocal with resolution of approximately 200 nm (E. Mace, unpublished observations). While we describe the use of STED for imaging immune cells, this technology is applied to a variety of cell types, including neural cells, and for visualizing a variety of cell structures23-26.
SIM uses a different approach to generate subdiffraction-limited images. By visualizing known periodic excitation patterns, information can then be obtained about the unknown structure being studied following mathematical transformation27. This yields an increase in resolution to ~100 nm laterally28,29. The advantage of SIM is that it is compatible with all standard confocal dyes and probes, however the disadvantage is that it is much slower to acquire images and these require lengthy post-processing12. This also limits its use for live cell imaging.
Finally, super-resolution images can be generated by stochastic photo-switching of fluorophores. This approach is exploited in photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). By scanning multiple camera frames and localizing randomly activated molecules that are turned "on" and "off" over time, images with 20-30 nm resolution are generated from accumulated frames30-32 . The trade-off for this resolution is the time required to acquire images.
Here we show, in detail, the protocol for preparing and imaging dual color samples in STED. In this system, excitation is with a pulsed, tunable, white light laser. Due to the nature of the pulsed excitation beam, time gating of detection is made possible and further increases resolution. In addition, the system is equipped with gadolinium hybrid (HyD) detectors, which are more sensitive than conventional photomultiplier tubes, thus allowing for lower laser power requirements. The depletion beam for STED is applied continuously and is tuned to 592 nm, which will dictate the choice of dyes available for two color STED. Commonly used dye combinations generally include one excitable by 488 nm (such as Alexa Fluor 488, Oregon Green, DyLights green, or Chromeo 488) and one excitable by 458 nm (such as Pacific Orange or Horizon V500). Thus, while detection of the two dyes will be in a similar range (and both are accessible by the depletion laser), excitation will occur with different wavelengths. With a tunable white light laser and tunable detectors, maximizing signal while eliminating spectral overlap is made fairly easy. As such, we have had good success with combinations of commercially available dyes, such as Pacific Orange and Alexa Fluor 488 (used here). Our protocol is tailored towards and describes the evaluation of human NK cells as that represents the historical focus of our laboratory. We are specifically utilizing the NK92 cell line in this example as that is one we have regularly applied in our experimental work21,33.
1. Coat Coverslips with Antibody
2. Activate NK Cells on Coverslips; Fix and Permeabilize
3. Stain Cells
4. Mount Coverslips on Slides
5. Experimental Setup
6. Optimization of Settings
7. Image Acquisition
8. Deconvolution
Clearly, a primary goal of super-resolution imaging will be an improvement over conventional confocal microscopy. However, there are some common pitfalls that may lead to suboptimal resolution. These require that each experiment be optimized individually. In our representative experiment, we are imaging the F-actin network in an NK cell activated by antibody bound to glass. Common causes of (and corrections for) a lack of improved resolution of STED over confocal are as follows:
By achieving the correct balance of pixel dwell time, excitation laser power, and depletion laser power, an image with improved resolution and sufficient information can be generated (Figure 1c). Resolution can be further improved by the use of deconvolution (Figure 1d). When acquisition is optimized, deconvolution will improve resolution both qualitatively and quantitatively and sub-100 nm resolution should be routinely attainable.
Figure 1. Optimization of acquisition and common pitfalls of STED imaging. NK92 cells were activated on anti-CD18 and -NKp30 coated glass for 20 min then fixed, permeabilized and stained for F-actin with Phalloidin Alexa Fluor 488. a) An example of loss of image information due to under-sampling. b) An example of loss of resolution due to bleaching/over-sampling c) conditions optimized d) optimized conditions lead to greater improvement in resolution with deconvolution. Scale bar = 5 μm.
The improvement in resolution over confocal will be somewhat dependent upon factors which cannot be controlled. These factors include minor aberrations in cover slip thickness and inconsistencies in mounting media. It is important to keep the temperature and humidity in the imaging room as consistent as possible, and the STED beam should be realigned approximately every 60 min. As mentioned in Procedures, use of VECTASHIELD mounting medium must be avoided, as this is not compatible with STED. In addition to which, one should always use #1.5 cover slips, and if available, use those which have been verified to a specific thickness.
One modification of the approach described here is to image additional channels in confocal, using fluorophores that emit at a longer wavelength than the STED beam. In this way, one can image up to four channels (two in confocal, two in STED). If taking this approach, however, the channels with fluorophores emitting above the STED depletion laser will need to be imaged first, as application of the STED beam will deplete photons in these channels. One advantage to this technique is the application of time gating, which will also improve resolution in confocal by eliminating emission from photons with short lifetimes34. In particular, the use of time gating, the timing of emission detectors to correspond with pulsed excitation STED, will decrease background fluorescence from reflection off coverslip glass when imaging close to it. Even in an experiment not suitable for STED, if using a pulsed excitation source, time gating can be a useful tool for improving resolution in confocal.
There are various modifications that can be utilized to improve resolution in STED. One is to decrease the size of the pinhole from the standard 1 Airy unit, although this will also decrease the amount of light reaching the sample. This can be compensated for by increasing laser power or gain. Another is to increase line average, which will increase the amount of information gathered for each photon, improving resolution. Again, however, this may be at the cost of photobleaching of the sample, so a balance will need to be struck between resolution and bleaching. Similarly, use of fluorescent proteins such as GFP will require careful optimization to avoid bleaching. This may be accomplished by decreasing STED laser power if necessary. Longer time points will also allow for greater photon recovery and reduce bleaching. Correction for photobleaching should be accounted for when analyzing live STED.
Of course, imaging in 3 dimensions in STED is also possible, and will also give an improvement over conventional confocal imaging. This is particularly true if it is done in combination with deconvolution, although care should be taken to correct for drift that occurs during imaging multiple planes in the z-axis. If using Huygens software to deconvolve, this correction is obtained using the “stabilize image” feature. Using this approach, resolution in the z-axis will be improved. This is a great improvement over conventional confocal imaging, which has poor axial resolution, and even over just STED itself, which also has relatively poor z-axis resolution. While acquiring multiple stacks in STED, care must be taken to avoid bleaching of the sample, and if necessary one can reduce line averaging or laser power intensity in order to do so. Again, it should be noted that if imaging other fluorophores that are not suitable for STED, application of the depletion beam in the first sequential scan would prevent emission from these channels. Therefore, a mixed STED/confocal approach (when using confocal scanning in channels that emit at a wavelength greater than 592 nm) will unfortunately not be suitable for 3D.
To summarize, we have chosen STED as an approach due to its relative ease of application and improvement in resolution over standard confocal imaging. For imaging the immune synapse, it has proven an effective and valuable technique that allows us to see details in F-actin architecture not possible at resolution over 200 nm. While many of these details seem subtle, they can have a profound effect on NK cell function. Thus, we are applying the latest nanoscopic imaging technology to deriving information that is critical for maintaining human health.
The authors have nothing to disclose.
We thank Geoff Daniels for technical assistance. This work was funded by R01 AI067946 to J.S.O.
#1.5 cover slips | VWR | 48393-172 | |
BD Cytofix/Cytoperm | BD Biosciences | 554722 | |
Bovine serum albumin | Sigma | A2153 | |
Cotton tipped applicator | Fisher Scientific | S450941 | |
Falcon centrifuge tubes (50 ml) | VWR | 352070 | |
Fetal calf serum (FCS) (500 mL) | Atlantic Biologicals | S11050 | |
Goat anti-rabbit Pacific Orange | Life Technologies | P31584 | |
Laboratory tissue wipers | VWR | 82003-820 | |
Nail polish | VWR | 100491-940 | |
NK-92 cells | ATCC | CRL-2407 | |
Phalloidin Alexa Fluor 488 | Life Technologies | A12379 | |
Phosphate buffered saline | Life Technologies | 14190250 | |
Prolong anti-fade reagent | Life Technologies | P7481 | |
Purified anti-CD18 | Biolegend | 301202 | |
Purified anti-NKp30 | Biolegend | 325202 | |
Purified anti-perforin | Biolegend | 308102 | |
RPMI 1640 medium (500 mL) | Life Technologies | 11875-093 | |
Saponin from Quillaja bark | Sigma | S4521 | |
Super PAP pen | Life Technologies | 008899 | |
Triton X-100 | Electron Microscopy Sciences | 22142 | |
Material Name | Company | Catalogue Number | Comments (optional) |
Huygens deconvolution software | SVI | Contact company | |
Leica SP8 TCS STED microscope | Leica Microsystems | Contact company |