Field-dependent deep learning enables high-throughput whole-cell 3D super-resolution imaging.

Single-molecule localization microscopy in a typical wide-field setup has been widely used for investigating subcellular structures with super resolution; however, field-dependent aberrations restrict the field of view (FOV) to only tens of micrometers. Here, we present a deep-learning method for precise localization of spatially variant point emitters (FD-DeepLoc) over a large FOV covering the full chip of a modern sCMOS camera. Using a graphic processing unit-based vectorial point spread function (PSF) fitter, we can fast and accurately model the spatially variant PSF of a high numerical aperture objective in the entire FOV. Combined with deformable mirror-based optimal PSF engineering, we demonstrate high-accuracy three-dimensional single-molecule localization microscopy over a volume of ~180 × 180 × 5 µm3, allowing us to image mitochondria and nuclear pore complexes in entire cells in a single imaging cycle without hardware scanning; a 100-fold increase in throughput compared to the state of the art.

Long noncoding RNA LINC01882 ameliorates aGVHD via skewing CD4+ T cell differentiation toward Treg cells.

Acute graft-versus-host disease (aGVHD) is a severe T cell-mediated immune response after allogeneic hematopoietic stem cell transplantation (allo-HSCT), the molecular mechanisms remain to be elucidated and novel treatments are necessary to be developed. In the present study, we found that the expression of long noncoding RNA (lncRNA) LINC01882 decreased significantly in the peripheral blood CD4+ T lymphocytes of patients with aGVHD than non-aGVHD patients. In addition, lncRNA LINC01882 overexpression promoted Treg differentiation but exhibited no effects on Th17 percentages, while its knockdown resulted in opposite effects. Mechanistically, lncRNA LINC01882 could competitively bind with let-7b-5p to prevent the degradation of its target gene smad2, which acts as a promoter in Treg differentiation. Furthermore, the mice cotransplanted with LINC01882-overexpressed CD4+ T cells with PBMCs had a lower histological GVHD score and higher survival rate compared with control mice. In conclusion, our study discloses a novel LINC01882/let-7b-5p/smad2 pathway in the modulation of aGVHD and indicates that lncRNA LINC01882 could be a promising biomarker and therapeutic target for patients with aGVHD.

dSTORM-Based Single-Cell Protein Quantitative Analysis Can Effectively Evaluate the Degradation Ability of PROTACs.

As an emerging therapeutic strategy, proteolysis-targeting chimeras (PROTACs) have been proven to be superior to traditional drugs in many aspects. However, due to their unique mechanism of action, existing methods for evaluating the degradation still have many limitations, which seriously restricts the development of PROTACs. In this methodological study, using direct stochastic optical reconstruction microscopy (dSTORM)-based single-cell protein quantitative analysis, we systematically investigated the dynamic degradation characteristics of FLT3 protein during PROTACs treatment. We found that the distribution of FLT3 varies between FLT3-ITD mutation and FLT3-WT cells. PROTACs had an obvious time-course effect on protein degradation and present two distinct phases; this provided a basis for deciding when to evaluate protein degradation. High concentrations of PROTACs were more effective than long-time administration because a higher Dmax was achieved. Two-color dSTORM-based colocalization analysis efficiently detected the proportion of ternary complexes, making it very useful in screening PROTACs. Taken together, our findings show that the dSTORM method is an ideal tool for evaluating PROTACs and will accelerate the development of new PROTACs.

Localization of the WD repeat-containing protein 5 to the Virion Assembly Compartment Facilitates Human Cytomegalovirus Assembly.

We previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WD repeat-containing protein 5 (WDR5) to facilitate capsid nuclear egress. Here, we further show that HCMV infection results in WDR5 localization in a juxtanuclear region, and that its localization to this cellular site is associated with viral replication and late viral gene expression. Furthermore, WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 co-immunoprecipitated with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain WDR5 accumulation in the vAC during infection. WDR5 fractionated with virions either in the presence or absence of Triton X-100 and was present in purified viral particles, suggesting that WDR5 was incorporated into HCMV virions. Thus, WDR5 localized to the vAC and was incorporated into virions, raising the possibility that in addition to capsid nuclear egress, WDR5 could also participate in cytoplasmic HCMV virion morphogenesis.Importance Human cytomegalovirus (HCMV) has a large (∼235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are reorganized to establish the virion assembly compartment (vAC), which has been shown to necessary for efficient assembly of progeny virions. We previously reported that WDR5 facilitates HCMV nuclear egress. Here, we show that WDR5 is localized to the vAC and incorporated into virions, perhaps contributing to efficient virion maturation. Thus, findings in this study identified a potential role for WDR5 in HCMV assembly in the cytoplasmic phase of virion morphogenesis.

ResNet-based image inpainting method for enhancing the imaging speed of single molecule localization microscopy.

Single molecule localization microscopy (SMLM) is a mainstream method in the field of super-resolution fluorescence microscopy that can achieve a spatial resolution of 20∼30 nm through a simple optical system. SMLM usually requires thousands of raw images to reconstruct a super-resolution image, and thus suffers from a slow imaging speed. Recently, several methods based on image inpainting have been developed to enhance the imaging speed of SMLM. However, these image inpainting methods may also produce erroneous local features (or called image artifacts), for example, incorrectly joined or split filaments. In this study, we use the ResNet generator, a network with strong local feature extraction capability, to replace the popularly-used U-Net generator to minimize the image artifact problem in current image inpainting methods, and develop an image inpainting method called DI-STORM. We validate our method using both simulated and experimental data, and demonstrate that DI-STORM has the best acceleration capability and produces the least artifacts in the repaired images, as compared with VDSR (the simplest CNN-based image inpainting method in SMLM) and ANNA-PALM (the best GAN-based image inpainting method in SMLM). We believe that DI-STORM could facilitate the application of deep learning-based image inpainting methods for SMLM.

Pushing the colorimetry camera-based fluorescence microscopy to low light imaging by denoising and dye combination.

Colorimetry camera-based fluorescence microscopy (CCFM) is a single-frame imaging method for observing multiple biological events simultaneously. Compared with the traditional multi-color fluorescence microscopy methods based on sequential excitation or spectral splitting, the CCFM method simplifies multi-color fluorescence imaging experiments, while keeping a high spatial resolution. However, when the level of the detected fluorescence signal decreases, the image quality, the demosaicking algorithm precision, and the discrimination of fluorescence channels on the colorimetry camera will also decrease. Thus, CCFM has a poor color resolution under a low signal level. For example, the crosstalk will be higher than 10% when the signal is less than 100 photons/pixel. To solve this problem, we developed a new algorithm that combines sCMOS noise correction with demosaicking, and a dye selection method based on the spectral response characteristics of the colorimetry camera. By combining the above two strategies, low crosstalk can be obtained with 4 ∼ 6 fold fewer fluorescence photons, and low light single-frame four-color fluorescence imaging was successfully performed on fixed cos-7 cells. This study expands the power of the CCFM method, and provides a simple and efficient way for various bioimaging applications in low-light conditions.

Real-time image resolution measurement for single molecule localization microscopy.

Recent advancements in single molecule localization microscopy (SMLM) have demonstrated outstanding potential applications in high-throughput and high-content screening imaging. One major limitation to such applications is to find a way to optimize imaging throughput without scarifying image quality, especially the homogeneity in image resolution, during the imaging of hundreds of field-of-views (FOVs) in heterogeneous samples. Here we introduce a real-time image resolution measurement method for SMLM to solve this problem. This method is under the heuristic framework of overall image resolution that counts on localization precision and localization density. Rather than estimating the mean localization density after completing the entire SMLM process, this method uses the spatial Poisson process to model the random activation of molecules and thus determines the localization density in real-time. We demonstrate that the method is valid in real-time resolution measurement and is effective in guaranteeing homogeneous image resolution across multiple representative FOVs with optimized imaging throughput.

Single-shot multi-color fluorescence microscopy via a colorimetry camera.

Multi-color fluorescence microscopy presents highly detailed biological samples interactively. However, current multi-color methods suffer from an intricate optical setup, complicated image analysis, or a long acquisition time. To address these issues, here we develop a simple multi-color method based on a customized colorimetry camera to enable the detection of multiple structures from single-shot acquisition. The unfiltered channel (W pixels) and color channels (R, G, B, and NIR pixels) in this customized camera simultaneously provide a broad detection wavelength range and high detection sensitivity. We built a simple optical setup by replacing the monochrome camera in a basic fluorescence microscopy system with a colorimetry camera, and developed effective image analysis procedures to reconstruct a multi-color image from a single frame of a raw image. We demonstrated single-shot four-color wide-field fluorescence imaging on fixed cos-7 cells with < 5% cross talk, which is comparable to the best reported values. Our method greatly simplifies both the optical system and image analysis in the widely used method of multi-color fluorescence microscopy, thus offering an effective and easy way to study multiple objects at the same time.

Influence of drift correction precision on super-resolution localization microscopy.

Super-resolution localization microscopy (SRLM) breaks the diffraction limit successfully and improves the resolution of optical imaging systems by nearly an order of magnitude. However, SRLM typically takes several minutes or longer to collect a sufficient number of image frames that are required for reconstructing a final super-resolution image. During this long image acquisition period, system drift should be tightly controlled to ensure the imaging quality; thus, several drift correction methods have been developed. However, it is still unclear whether the performance of these methods is able to ensure sufficient image quality in SRLM. Without a clear answer to this question, it is hard to choose a suitable drift correction method for a specific SRLM experiment. In this paper, we use both theoretical analysis and simulation to investigate the relationship among drift correction precision, localization precision, and position estimation precision. We propose a concept of relative localization precision for evaluating the effect of drift correction on imaging resolution, which would help to select an appropriate drift correction method for a specific experiment.

Anterograde Viral Tracer Herpes Simplex Virus 1 Strain H129 Transports Primarily as Capsids in Cortical Neuron Axons.

The features of herpes simplex virus 1 (HSV-1) strain 129 (H129), including natural neurotropism and anterograde transneuronal trafficking, make it a potential tool for anterograde neural circuitry tracing. Recently anterograde polysynaptic and monosynaptic tracers were developed from H129 and have been applied for the identification of novel connections and functions of different neural circuitries. However, how H129 viral particles are transported in neurons, especially those of the central nervous system, remains unclear. In this study, we constructed recombinant H129 variants with mCherry-labeled capsids and/or green fluorescent protein (GFP)-labeled envelopes and infected the cortical neurons to study axonal transport of H129 viral particles. We found that different types of viral particles were unevenly distributed in the nucleus, cytoplasm of the cell body, and axon. Most H129 progeny particles were unenveloped capsids and were transported as capsids rather than virions in the axon. Notably, capsids acquired envelopes at axonal varicosities and terminals where the sites forming synapses are connected with other neurons. Moreover, viral capsids moved more frequently in the anterograde direction in axons, with an average velocity of 0.62 ± 0.18 µm/s and maximal velocity of 1.80 ± 0.15 µm/s. We also provided evidence that axonal transport of capsids requires the kinesin-1 molecular motor. These findings support that H129-derived tracers map the neural circuit anterogradely and possibly transsynaptically. These data will guide future modifications and improvements of H129-based anterograde viral tracers.IMPORTANCE Anterograde transneuronal tracers derived from herpes simplex virus 1 (HSV-1) strain 129 (H129) are important tools for mapping neural circuit anatomic and functional connections. It is, therefore, critical to elucidate the transport pattern of H129 within neurons and between neurons. We constructed recombinant H129 variants with genetically encoded fluorescence-labeled capsid protein and/or glycoprotein to visualize viral particle movement in neurons. Both electron microscopy and light microscopy data show that H129 capsids and envelopes move separately, and notably, capsids are enveloped at axonal varicosity and terminals, which are the sites forming synapses to connect with other neurons. Superresolution microscopy-based colocalization analysis and inhibition of H129 particle movement by inhibitors of molecular motors support that kinesin-1 contributes to the anterograde transport of capsids. These results shed light into the mechanisms for anterograde transport of H129-derived tracer in axons and transmission between neurons via synapses, explaining the anterograde labeling of neural circuits by H129-derived tracers.

Two-color super-resolution localization microscopy via joint encoding of emitter location and color.

Multi-color super-resolution localization microscopy (SRLM) provides great opportunities for studying the structural and functional details of biological samples. However, current multi-color SRLM methods either suffer from medium to high crosstalk, or require a dedicated optical system and a complicated image analysis procedure. To address these problems, here we propose a completely different method to realize multi-color SRLM. This method is built upon a customized RGBW camera with a repeated pattern of filtered (Red, Green, Blue and Near-infrared) and unfiltered (White) pixels. With a new insight that RGBW camera is advantageous for color recognition instead of color reproduction, we developed a joint encoding scheme of emitter location and color. By combing this RGBW camera with the joint encoding scheme and a simple optical set-up, we demonstrated two-color SRLM with ∼20 nm resolution and < 2% crosstalk (which is comparable to the best-reported values). This study significantly reduces the complexity of two-color SRLM (and potentially multi-color SRLM), and thus offers good opportunities for general biomedical research laboratories to use multi-color SRLM, which is currently mastered only by well-trained researchers.

Characterizing and correcting camera noise in back-illuminated sCMOS cameras.

With promising properties of fast imaging speed, large field-of-view, relative low cost and many others, back-illuminated sCMOS cameras have been receiving intensive attention for low light level imaging in the past several years. However, due to the pixel-to-pixel difference of camera noise (called noise non-uniformity) in sCMOS cameras, researchers may hesitate to use them in some application fields, and sometimes wonder whether they should optimize the noise non-uniformity of their sCMOS cameras before using them in a specific application scenario. In this paper, we systematically characterize the impact of different types of sCMOS noise on image quality and perform corrections to these types of sCMOS noise using three representative algorithms (PURE, NCS and MLEsCMOS). We verify that it is possible to use appropriate correction methods to push the non-uniformity of major types of camera noise, including readout noise, offset, and photon response, to a satisfactory level for conventional microscopy and single molecule localization microscopy. We further find out that, after these corrections, global read noise becomes a major concern that limits the imaging performance of back-illuminated sCMOS cameras. We believe this study provides new insights into the understanding of camera noise in back-illuminated sCMOS cameras, and also provides useful information for future development of this promising camera technology.

Accelerating multi-emitter localization in super-resolution localization microscopy with FPGA-GPU cooperative computation.

The real-time multi-emitter localization method is essential for advancing high-throughput super-resolution localization microscopy (HT-SRLM). In the past decade, the graphics processing unit (GPU) computation has been dominantly used to accelerate the execution speed of the multi-emitter localization method. However, if HT-SRLM is combined with a scientific complementary metal-oxide-semiconductor (sCMOS) camera working at full frame rate, real-time image processing is still difficult to achieve using this acceleration approach, thus resulting in a massive data storage challenge and even system crash. Here we take advantage of the cooperative acceleration power of field programming gate array (FPGA) computation and GPU computation, and propose a method called HCP-STORM to enable real-time multi-emitter localization. Using simulated images, we verified that HCP-STORM is capable of providing real-time image processing for raw images from a representative Hamamatsu Flash 4 V3 sCMOS camera working at full frame rate (that is, 2048×2048 pixels @ 10 ms exposure time). Using experimental images, we prove that HCP-STORM is 25 times faster than QC-STORM and 295 times faster than ThunderSTORM, with a small but acceptable degradation in image quality. This study shows the potential of FPGA-GPU cooperative computation in accelerating multi-emitter localization, and pushes a significant step toward the maturity of HT-SRLM technology.

High-precision 3D drift correction with differential phase contrast images.

Single molecule localization microscopy (SMLM) usually requires long image acquisition time at the order of minutes and thus suffers from sample drift, which deteriorates image quality. A drift estimation method with high precision is typically used in SMLM, which can be further combined with a drift compensation device to enable active microscope stabilization. Among all the reported methods, the drift estimation method based on bright-field image correlation requires no extra sample preparation or complicated modification to the imaging setup. However, the performance of this method is limited by the contrast of bright-field images, especially for the structures without sufficient features. In this paper, we proposed to use differential phase contrast (DPC) microscopy to enhance the image contrast and presented a 3D drift correction method with higher precision and robustness. This DPC-based drift correction method is suitable even for biological samples without clear morphological features. We demonstrated that this method can achieve a correction precision of < 6 nm in both the lateral direction and axial direction. Using SMLM imaging of microtubules, we verified that this method provides a comparable drift estimation performance as redundant cross-correlation.

Diagnostic and Therapeutic Value of Hsa_circ_0002594 for T Helper 2-Mediated Allergic Asthma.

INTRODUCTION: Circular RNAs (circRNAs) are an endogenous mircoRNA sponge that could act as potential biomarkers for the diagnosis and treatment of diseases. However, the role of circRNAs in asthma is far from clear. OBJECTIVE: The aim of this study is to assess the diagnostic and therapeutic value of hsa_circ_0002594 for T helper (Th) 2-mediated allergic asthma. METHODS: The expression profiles of hsa_circ_0002594 in CD4+ T cells were revealed by circRNA microarray. Hsa_circ_0002594 expression was confirmed via quantitative real-time PCR (qRT-PCR) in asthmatic patients and healthy subjects. Hsa_circ_0002594 levels were compared between subgroups. The clinical diagnostic abilities and therapeutic response of hsa_circ_0002594 were evaluated. The analyses utilized included a student's t test, nonparametric tests, Spearman's rank-order correlation, Fisher's exact test, and the generation of receiver operating characteristic (ROC) curves. RESULTS: Hsa_circ_0002594 was upregulated and positively correlated with fraction of exhaled nitric oxide while negatively correlated with methacholine dose producing a decrease of 20% from baseline in forced expiratory volume in the first second (PD20) in CD4+ T cells of asthma. Furthermore, hsa_circ_0002594 expression was higher in subgroups with a family history, skin pricking test (SPT)-positive, or Th2-high. The hsa_circ_0002594-high subgroup was more frequently associated with Th2-high biomarker profiles and positive SPT. Hsa_circ_0002594 was decreased after inhaled corticosteroids (ICS) treatment. ROC curve analyses of hsa_circ_0002594 showed high area under the curve values in the presence of ICS or not. CONCLUSIONS: Our data suggested that hsa_circ_0002594 was upregulated in CD4+ T cells and might have potential value in the diagnosis and treatment of Th2-mediated allergic asthma.

A labeling strategy with effective preservation of fluorophores for expansion single-molecule localization microscopy (Ex-SMLM).

Expansion microscopy (ExM) significantly improves the resolution of conventional diffraction-limited optical microscopy by using physically expanding biological samples. Combining ExM with single-molecule localization microscopy (SMLM) could further enhance the resolving power of SMLM, which is typically in the order of 20-30 nm. However, to make this combination successful, we need to solve three key issues related to sample preparation, including mainly hydrogel shrinking in an ionic photoswitching buffer, fluorescence photobleaching due to a free-radical reaction and reduced labelling efficiency from protease digestion. Re-embedding polyacrylamide gel or using an improved photoswitching buffer with a low ionic strength is able to minimize or even solve the hydrogel shrinking problem, while the development of post-expansion labelling approaches avoids fluorescence bleaching. However, the preservation of protein epitopes (which determines the labelling efficiency) remains to be challenging. In this paper, we propose to tackle this challenge by introducing the highly selective and stable biotin-streptavidin interaction into the post-expansion labelling strategy. After upgrading the popular immunolabelling linkage scheme from Epitope-Primary antibody-Secondary antibody-Fluorophores to Epitope-Primary antibody-Secondary antibody-Biotin-Streptavidin-Fluorophores, we were able to label protein epitopes with biotin, which was stable during the expansion process, and thus avoid the troublesome problem in preserving protein epitopes or antibodies. We demonstrate that combining Ex-SMLM with the new post-expansion linkage scheme enables new possibilities in resolving the detailed arrangement of Nup133 proteins in the nuclear pore complex, which helps researchers to observe a clearer structure. This study provides new opportunities for studying the ultrastructural details of subcellular organelles or even biomacromolecules, using the conventional SMLM system.

Clinical characteristics and predictors of mortality in young adults with severe COVID-19: a retrospective observational study.

BACKGROUND AND OBJECTIVE: Little is yet known whether pathogenesis of COVID-19 is different between young and elder patients. Our study aimed to investigate the clinical characteristics and provide predictors of mortality for young adults with severe COVID-19. METHODS: A total of 77 young adults with confirmed severe COVID-19 were recruited retrospectively at Tongji Hospital. Clinical characteristics, laboratory findings, treatment and outcomes were obtained from electronic medical records. The prognostic effects of variables were analyzed using logistic regression model. RESULTS: In this retrospective cohort, non-survivors showed higher incidence of dyspnea and co-existing laboratory abnormalities, compared with young survivals in severe COVID-19. Multivariate logistic regression analysis showed that lymphopenia, elevated level of d-dimer, hypersensitive cardiac troponin I (hs-CTnI) and high sensitivity C-reactive protein (hs-CRP) were independent predictors of mortality in young adults with severe COVID-19. Further analysis showed that severely young adults with two or more factors abnormalities above would be more prone to death. The similar predictive effect of above four factors had been observed in all-age patients with severe COVID-19. CONCLUSION: Lymphopenia, elevated level of d-dimer, hs-CTnI and hs-CRP predicted clinical outcomes of young adults with severe COVID-19.

Pentraxin 3 promotes airway inflammation in experimental asthma.

BACKGROUND: Pentraxin 3 (PTX3) regulates multiple aspects of innate immunity and tissue inflammation. Recently, it has been reported that PTX3 deficiency enhances interleukin (IL)-17A-dominant pulmonary inflammation in an ovalbumin (OVA)-induced mouse asthma model. However, whether PTX3 treatment would provide protection against allergic airway inflammation has not been clearly elucidated. The goal of this study was to further investigate the effect of recombinant PTX3 administration on the phenotype of asthma. METHODS: C57BL/6 J mice were sensitized and challenged with OVA to induce eosinophilic asthma model, as well as sensitized with OVA plus LPS and challenged with OVA to induce neutrophilic asthma model. We evaluated effect of recombinant PTX3 on asthma phenotype through both asthma models. The bronchoalveolar lavage fluid (BALF) inflammatory cells and cytokines, airway hyperresponsiveness, and pathological alterations of the lung tissues were assessed. RESULTS: In both eosinophilic and neutrophilic asthma models, PTX3 treatment provoked airway hyperresponsiveness, concomitant with increased inflammatory cytokines IL-4, IL-17, eotaxin, and transforming growth factor (TGF)-ß1 and aggravated airway accumulation of inflammatory cells, especially eosinophils and neutrophils. In histological analysis of the lung tissue, administration of PTX3 promoted inflammatory cells infiltration, mucus production, and collagen deposition. In addition, PTX3 also significantly enhanced STAT3 phosphorylation in lung tissue. CONCLUSION: Our results show that exogenous PTX3 can exacerbate multiple asthmatic features by promoting both eosinophils and neutrophils lung infiltration and provide new evidence to better understand the complex role of PTX3 in allergic airway inflammation.

[Research progress of non-coding RNA in regulating the function of T cells in asthma].

Bronchial asthma (i.e. asthma) is a chronic inflammatory disease characterized by airway inflammatory response, hyperresponsiveness and airway remodeling, in which T cells play a vital role, especially T helper cells (Th cells). Non-coding RNAs (ncRNAs) are the RNAs that do not encode proteins, mainly including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), which are widely found in eukaryotic genomes and participate in the regulation of various biological processes. Previous studies have shown that ncRNAs play an important role in the activation and transformation of T cells and other biological processes in asthma. The specific molecular mechanism and clinical application are worth in-depth discussion. This article reviewed the research progress in regulation of miRNAs, lncRNAs and circRNAs on T cells in asthma in recent years.

Direct Observation of Nanoparticles within Cells at Subcellular Levels by Super-Resolution Fluorescence Imaging.

Direct observation of nanoparticles with high spatial resolution at subcellular levels is of great importance to understand the nanotoxicology and promote the biomedical applications of nanoparticles. Super-resolution fluorescence microscopy can break the diffraction resolution limit to achieve spatial resolution of tens of nanometers, making it ideal for highly accurate observation of nanoparticles in the cellular world. In this study, we introduced the employment of super-resolution fluorescence imaging for monitoring nanoparticles within cells. Carbocyanine dyes Alexa Flour 647 labeled mesoporous silica nanoparticles (designated as MSNs-AF647) were constructed as the super-resolution imaging nanoplatform in this work as proof of concept. The MSNs-AF647 were incubated with Hela cells, and the nanoparticles within cells were further monitored by super-resolution fluorescence microscopy. The fluorescence images of MSNs-AF647 within cells captured with the super-resolution fluorescence microscopy showed a much higher spatial resolution than that obtained using conventional fluorescence microscopy, showing that super-resolution fluorescence images can provide more accurate information to locate the nanoparticles at the subcellular levels. Moreover, other functional molecules can be easily loaded into the MSNs-AF647 super-resolution imaging nanoplatform, which suggested that super-resolution fluorescence imaging can further be applied to various bioimaging-related areas, such as imaging-guided therapy, with the aid of the MSNs-AF647 nanoplatform. This study demonstrates that super-resolution fluorescence microscopy offers a highly accurate method to study nanoparticles in the cellular world. We anticipate this strategy may further be applied to research areas such as studying the nanotoxicology and optimization of nanoparticle-based bioprobes or drugs by designing new nanostructured materials with multifunctional properties based on MSNs-AF647.