Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-''light-sheet'', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-''light-sheet'' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.     

Registration and Visualization of Correlative Super-Resolution Microscopy Data

We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.     

Visualization and Analysis of Hepatitis C Virus Structural Proteins at Lipid Droplets by Super-Resolution Microscopy

Cytosolic lipid droplets are central organelles in the Hepatitis C Virus (HCV) life cycle. The viral capsid protein core localizes to lipid droplets and initiates the production of viral particles at lipid droplet–associated ER membranes. Core is thought to encapsidate newly synthesized viral RNA and, through interaction with the two envelope proteins E1 and E2, bud into the ER lumen. Here, we visualized the spatial distribution of HCV structural proteins core and E2 in vicinity of small lipid droplets by three-color 3D super-resolution microscopy. We observed and analyzed small areas of colocalization between the two structural proteins in HCV-infected cells with a diameter of approximately 100 nm that might represent putative viral assembly sites.     

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the ''mislocalization'' phenomenon.     

A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images.     

Recent Developments in Correlative Super-Resolution Fluorescence Microscopy and Electron Microscopy

The recently developed correlative super-resolution fluorescence microscopy (SRM) and electron microscopy (EM) is a hybrid technique that simultaneously obtains the spatial locations of specific molecules with SRM and the context of the cellular ultrastructure by EM. Although the combination of SRM and EM remains challenging owing to the incompatibility of samples prepared for these techniques, the increasing research attention on these methods has led to drastic improvements in their performances and resulted in wide applications. Here, we review the development of correlative SRM and EM (sCLEM) with a focus on the correlation of EM with different SRM techniques. We discuss the limitations of the integration of these two microscopy techniques and how these challenges can be addressed to improve the quality of correlative images. Finally, we address possible future improvements and advances in the continued development and wide application of sCLEM approaches.     

CellProfiler: Novel Automated Image Segmentation Procedure for Super-Resolution Microscopy

Background

Super resolution (SR) microscopy enabled cell biologists to visualize subcellular details up to 20 nm in resolution. This breakthrough in spatial resolution made image analysis a challenging procedure. Direct and automated segmentation of SR images remains largely unsolved, especially when it comes to providing meaningful biological interpretations.

Results

Here, we introduce a novel automated imaging analysis routine, based on Gaussian, followed by a segmentation procedure using CellProfiler software (www.cellprofiler.org). We tested this method and succeeded to segment individual nuclear pore complexes stained with gp210 and pan-FG proteins and captured by two-color STED microscopy. Test results confirmed accuracy and robustness of the method even in noisy STED images of gp210.

Conclusions

Our pipeline and novel segmentation procedure may benefit end-users of SR microscopy to analyze their images and extract biologically significant quantitative data about them in user-friendly and fully-automated settings.

     

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass.     

ConfocalVR: Immersive Visualization for Confocal Microscopy

ConfocalVR is a virtual reality (VR) application created to improve the ability of researchers to study the complexity of cell architecture. Confocal microscopes take pictures of fluorescently labeled proteins or molecules at different focal planes to create a stack of two-dimensional images throughout the specimen. Current software applications reconstruct the three-dimensional (3D) image and render it as a two-dimensional projection onto a computer screen where users need to rotate the image to expose the full 3D structure. This process is mentally taxing, breaks down if you stop the rotation, and does not take advantage of the eye's full field of view. ConfocalVR exploits consumer-grade VR systems to fully immerse the user in the 3D cellular image. In this virtual environment, the user can (1) adjust image viewing parameters without leaving the virtual space, (2) reach out and grab the image to quickly rotate and scale the image to focus on key features, and (3) interact with other users in a shared virtual space enabling real-time collaborative exploration and discussion. We found that immersive VR technology allows the user to rapidly understand cellular architecture and protein or molecule distribution. We note that it is impossible to understand the value of immersive visualization without experiencing it first hand, so we encourage readers to get access to a VR system, download this software, and evaluate it for yourself. The ConfocalVR software is available for download at http://www.confocalvr.com, and is free for nonprofits.     

Advantages of Single-Molecule Real-Time Sequencing in High-GC Content Genomes

Next-generation sequencing has become the most widely used sequencing technology in genomics research, but it has inherent drawbacks when dealing with high-GC content genomes. Recently, single-molecule real-time sequencing technology (SMRT) was introduced as a third-generation sequencing strategy to compensate for this drawback. Here, we report that the unbiased and longer read length of SMRT sequencing markedly improved genome assembly with high GC content via gap filling and repeat resolution.     

Nuclear Transport and Accumulation of Smad Proteins Studied by Single-Molecule Microscopy

Nuclear translocation of stimulated Smad heterocomplexes is a critical step in the signal transduction of transforming growth factor β (TGF-β) from transmembrane receptors into the nucleus. Specifically, normal nuclear accumulation of Smad2/Smad4 heterocomplexes induced by TGF-β1 is involved in carcinogenesis. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-β1 remains obscure. By combining a high-speed single-molecule tracking microscopy and Förster resonance energy transfer technique, we tracked the entire TGF-β1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complexes in live cells, with a high single-molecule localization precision of 2 ms and <20 nm. Our single-molecule Förster resonance energy transfer data have revealed that in TGF-β1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, we found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-β1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-β1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, we believe that the determined TGF-β1-dependent transport configurations and efficiencies for the basal-state Smad or stimulated Smad heterocomplexes elucidate the basic molecular mechanism to understand their nuclear transport and accumulation.     

Super-Resolution Imaging of Plasmodesmata Using Three-Dimensional Structured Illumination Microscopy

We used three-dimensional structured illumination microscopy (3D-SIM) to obtain subdiffraction (“super-resolution”) images of plasmodesmata (PD) expressing a green fluorescent protein-tagged viral movement protein (MP) in tobacco (Nicotiana tabacum). In leaf parenchyma cells, we were able to resolve individual components of PD (neck and central cavities) at twice the resolution of a confocal microscope. Within the phloem, MP-green fluorescent protein filaments extended outward from the specialized pore-PD that connect sieve elements (SEs) with their companion cells (CCs) along the tubular sieve element reticulum (SER). The SER was shown to interconnect individual pore-PD at the SE-CC interface. 3D-SIM resolved fine (less than 100 nm) endoplasmic reticulum threads running into individual pore-PD as well as strands that crossed sieve plate pores, structurally linking SEs within a file. Our data reveal that MP entering the SE from the CC may remain associated with the SER. Fluorescence recovery after photobleaching experiments revealed that this MP pool is relatively immobile compared with the membrane probe 3,3’-dihexyloxacarbocyanine iodide, suggesting that MP may become sequestered by the SER once it has entered the SE. The advent of 3D-SIM offers considerable potential in the subdiffraction imaging of plant cells, bridging an important gap between confocal and electron microscopy.Fluorescence-based imaging has revolutionized cell biology, allowing the localization of proteins to specific cells and organelles (Shaner et al., 2007; Frigault et al., 2009). However, conventional fluorescence microscopy is limited by the diffraction of light to approximately 200 nm in the lateral (x-y) plane and to about 500 nm in the axial (z) plane (Fernandez-Suarez and Ting, 2008; Huang et al., 2009). This is because light traveling through a lens cannot be focused to a point, only to an airy disc with a diameter of about half the wavelength of the visible emitted light (Huang et al., 2009). Confocal laser scanning microscopy has produced improvements in axial resolution due to the removal of out-of-focus flare, but it is also limited by diffraction (Huang et al., 2009). Thus, objects closer than about 200 nm cannot be resolved but appear merged into one. Many subcellular structures of interest to cell biologists lie below this resolution limit and have remained below the diffraction barrier. Such structures can be seen but not resolved.Recently, major innovations in biological imaging have broken the diffraction barrier. These include photoactivation localization microscopy (PALM) and stimulated emission and depletion (STED; for review, see Fernandez-Suarez and Ting, 2008; Huang et al., 2009). Most subdiffraction or “super-resolution” approaches have improved resolution in either the lateral (x-y) plane or the axial (z) plane, but usually not both (Schermelleh et al., 2008). Many of the structures of interest within plant cells lie some distance from the cell wall, posing problems for some super-resolution approaches (e.g. PALM) where the subject of interest must lie close to the coverslip (Huang et al., 2009). Recently, Schermelleh et al. (2008) described a subdiffraction multicolor imaging protocol using three-dimensional structured illumination microscopy (3D-SIM). In this method, objects beyond the diffraction limit are illuminated with multiple interfering beams of light transmitted through a series of diffraction gratings, producing a resolution of 100 nm in x-y and 200 nm in z (Schermelleh et al., 2008; Huang et al., 2009). These substantial increases in resolution are significant for plant cell imaging. The thickness of the plant cell wall is typically in the region of about 700 nm, allowing limited optical sectioning capacity with a confocal microscope (about 500 nm in z). A further advantage of 3D-SIM is that it permits the imaging of conventional fluorescent reporters and dyes that are compatible with confocal imaging, allowing a direct correlation of 3D-SIM and confocal images (Schermelleh et al., 2008).The phloem of higher plants is a major conduit for the long-distance transport of solutes (Oparka and Turgeon, 1999) and also functions as a “superhighway” for macromolecular trafficking (Lucas and Lee, 2004; Kehr and Buhtz, 2008; Lee and Cui, 2009). However, the phloem is difficult to image with conventional optical microscopy (Knoblauch and van Bel, 1998; Oparka and Turgeon, 1999; van Bel et al., 2002). Sieve elements (SEs), the conducting cells of the phloem, are enucleate yet contain a plethora of proteins and RNAs associated with long-distance signaling and defense (van Bel and Gaupels, 2004; Lee and Cui, 2009). Many of these macromolecules are synthesized in the companion cell (CC) and passed into the SE via the specialized pore-plasmodesmata (PD) that connect the two cell types (Oparka and Turgeon, 1999; van Bel et al., 2002). Pore-PD have been suggested to be a major “lifeline” from CC to SE (van Bel et al., 2002), but the exact nature of this pathway remains unresolved.Our current understanding of PD substructure is derived largely from electron microscope studies (Roberts, 2005). Such methods are time-consuming and do not permit facile protein localization within PD. Recent proteomics approaches have been successful in identifying new proteins associated with PD (Maule, 2008). Localization of these proteins with confocal microscopy results in the appearance of discrete punctae at the cell wall, consistent with the location of pit fields (Faulkner et al., 2008), but does not pinpoint specific protein locations within PD. In general, there is a growing gap between proteomics studies of plant organelles, including PD, and the ability to ascribe accurate addresses to these proteins (Millar et al., 2009; Moore and Murphy, 2009). The advent of 3D-SIM prompted us to explore the potential of subdiffraction imaging in plant cells, with a view to obtaining improved florescence resolution of PD. We used 3D-SIM to examine PD in a transgenic tobacco (Nicotiana tabacum) line expressing the viral movement protein (MP) of Tobacco mosaic virus (TMV) fused to GFP. Using a specific antibody to callose, a wall constituent located at the PD collar, we were able to resolve clearly the structure of single, simple PD in epidermal cells at 100-nm resolution, discriminating between the neck region of the pore and the central cavity to which it connects (Roberts and Oparka, 2003; Faulkner et al., 2008). 3D-SIM also revealed details of the central cavities of complex PD seen previously only with the electron microscope (Ding et al., 1992; Ehlers and Kollmann, 2001; Faulkner et al., 2008).Using 3D-SIM, we were able to image PD sequentially from the epidermis to the phloem within vascular bundles, producing unparalleled images of sieve plate pores and the specialized pore-PD that connect SEs with their CCs. In the SEs, MP was no longer restricted to the central cavities of PD but became distributed along the SE parietal layer, connecting all the pore-PD along the SE-CC interface. We were able to detect fine threads of MP-GFP that extended for up to 40 μm along the SE and also crossed individual sieve plate pores. Fluorescence recovery after photobleaching (FRAP) experiments revealed that this MP-GFP pool was relatively immobile within the SE parietal layer, suggesting that the SE may sequester TMV MP on or within the sieve element reticulum (SER).Our data reveal that 3D-SIM is especially suited to the subdiffraction imaging of plant cells and yields spatial information not previously possible with conventional fluorescence-based imaging. The unique optical sectioning capacity of 3D-SIM and the ability to produce multicolor imaging with conventional fluorophores offer enormous potential in plant cell biology.     

Spatial Covariance Reconstructive (SCORE) Super-Resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy has become a powerful tool to resolve structural information that is not accessible to traditional diffraction-limited imaging techniques such as confocal microscopy. Stochastic optical reconstruction microscopy (STORM) and photoactivation localization microscopy (PALM) are promising super-resolution techniques due to their relative ease of implementation and instrumentation on standard microscopes. However, the application of STORM is critically limited by its long sampling time. Several recent works have been focused on improving the STORM imaging speed by making use of the information from emitters with overlapping point spread functions (PSF). In this work, we present a fast and efficient algorithm that takes into account the blinking statistics of independent fluorescence emitters. We achieve sub-diffraction lateral resolution of 100 nm from 5 to 7 seconds of imaging. Our method is insensitive to background and can be applied to different types of fluorescence sources, including but not limited to the organic dyes and quantum dots that we demonstrate in this work.     

Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy

In multicellular organisms, single-fluorophore imaging is obstructed by high background. To achieve a signal/noise ratio conducive to single-molecule imaging, we adapted reflected light-sheet microscopy (RLSM) to image highly opaque late-stage Drosophila embryos. Alignment steps were modified by means of commercially available microprisms attached to standard coverslips. We imaged a member of the septate-junction complex that was used to outline the three-dimensional epidermal structures of Drosophila embryos. Furthermore, we show freely diffusing single 10 kDa Dextran molecules conjugated to one to two Alexa647 dyes inside living embryos. We demonstrate that Dextran diffuses quickly (∼6.4 μm²/s) in free space and obeys directional movement within the epidermal tissue (∼0.1 μm²/s). Our single-particle-tracking results are supplemented by imaging the endosomal marker Rab5-GFP and by earlier reports on the spreading of morphogens and vesicles in multicellular organisms. The single-molecule results suggest that RLSM will be helpful in studying single molecules or complexes in multicellular organisms.     

Tightening the Knot in Phytochrome by Single-Molecule Atomic Force Microscopy

A growing number of proteins have been shown to adopt knotted folds. Yet the biological roles and biophysical properties of these knots remain poorly understood. We used protein engineering and atomic force microscopy to explore the single-molecule mechanics of the figure-eight knot in the chromophore-binding domain of the red/far-red photoreceptor, phytochrome. Under load, apo phytochrome unfolds at forces of ∼47 pN, whereas phytochrome carrying its covalently bound tetrapyrrole chromophore unfolds at ∼73 pN. These forces are not unusual in mechanical protein unfolding, and thus the presence of the knot does not automatically indicate a superstable protein. Our experiments reveal a stable intermediate along the mechanical unfolding pathway, reflecting the sequential unfolding of two distinct subdomains in phytochrome, potentially the GAF and PAS domains. For the first time (to the best of our knowledge), our experiments allow a direct determination of knot size under load. In the unfolded chain, the tightened knot is reduced to 17 amino acids, resulting in apparent shortening of the polypeptide chain by 6.2 nm. Steered molecular-dynamics simulations corroborate this number. Finally, we find that covalent phytochrome dimers created for these experiments retain characteristic photoreversibility, unexpectedly arguing against a dramatic rearrangement of the native GAF dimer interface upon photoconversion.     

Real-Time Visualization of Joint Cavitation

Cracking sounds emitted from human synovial joints have been attributed historically to the sudden collapse of a cavitation bubble formed as articular surfaces are separated. Unfortunately, bubble collapse as the source of joint cracking is inconsistent with many physical phenomena that define the joint cracking phenomenon. Here we present direct evidence from real-time magnetic resonance imaging that the mechanism of joint cracking is related to cavity formation rather than bubble collapse. In this study, ten metacarpophalangeal joints were studied by inserting the finger of interest into a flexible tube tightened around a length of cable used to provide long-axis traction. Before and after traction, static 3D T1-weighted magnetic resonance images were acquired. During traction, rapid cine magnetic resonance images were obtained from the joint midline at a rate of 3.2 frames per second until the cracking event occurred. As traction forces increased, real-time cine magnetic resonance imaging demonstrated rapid cavity inception at the time of joint separation and sound production after which the resulting cavity remained visible. Our results offer direct experimental evidence that joint cracking is associated with cavity inception rather than collapse of a pre-existing bubble. These observations are consistent with tribonucleation, a known process where opposing surfaces resist separation until a critical point where they then separate rapidly creating sustained gas cavities. Observed previously in vitro, this is the first in-vivo macroscopic demonstration of tribonucleation and as such, provides a new theoretical framework to investigate health outcomes associated with joint cracking.     

Tilting and Wobble of Myosin V by High-Speed Single-Molecule Polarized Fluorescence Microscopy

Myosin V is biomolecular motor with two actin-binding domains (heads) that take multiple steps along actin by a hand-over-hand mechanism. We used high-speed polarized total internal reflection fluorescence (polTIRF) microscopy to study the structural dynamics of single myosin V molecules that had been labeled with bifunctional rhodamine linked to one of the calmodulins along the lever arm. With the use of time-correlated single-photon counting technology, the temporal resolution of the polTIRF microscope was improved ∼50-fold relative to earlier studies, and a maximum-likelihood, multitrace change-point algorithm was used to objectively determine the times when structural changes occurred. Short-lived substeps that displayed an abrupt increase in rotational mobility were detected during stepping, likely corresponding to random thermal fluctuations of the stepping head while it searched for its next actin-binding site. Thus, myosin V harnesses its fluctuating environment to extend its reach. Additional, less frequent angle changes, probably not directly associated with steps, were detected in both leading and trailing heads. The high-speed polTIRF method and change-point analysis may be applicable to single-molecule studies of other biological systems.     

Corrected Super-Resolution Microscopy Enables Nanoscale Imaging of Autofluorescent Lung Macrophages

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.