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.

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.

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.

[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.

WDR5 Facilitates Human Cytomegalovirus Replication by Promoting Capsid Nuclear Egress.

WD repeat-containing protein 5 (WDR5) is essential for assembling the VISA-associated complex to induce a type I interferon antiviral response to Sendai virus infection. However, the roles of WDR5 in DNA virus infections are not well described. Here, we report that human cytomegalovirus exploits WDR5 to facilitate capsid nuclear egress. Overexpression of WDR5 in fibroblasts slightly enhanced the infectious virus yield. However, WDR5 knockdown dramatically reduced infectious virus titers with only a small decrease in viral genome replication or gene expression. Further investigation of late steps of viral replication found that WDR5 knockdown significantly impaired formation of the viral nuclear egress complex and induced substantially fewer infoldings of the inner nuclear membrane. In addition, fewer capsids were associated with these infoldings, and there were fewer capsids in the cytoplasm. Restoration of WDR5 partially reversed these effects. These results suggest that WDR5 knockdown impairs the nuclear egress of capsids, which in turn decreases virus titers. These findings reveal an important role for a host factor whose function(s) is usurped by a viral pathogen to promote efficient replication. Thus, WDR5 represents an interesting regulatory mechanism and a potential antiviral target.IMPORTANCE Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 170 open reading frames and exploits numerous cellular factors to facilitate its replication. HCMV infection increases protein levels of WD repeat-containing protein 5 (WDR5) during infection, overexpression of WDR5 enhances viral replication, and knockdown of WDR5 dramatically attenuates viral replication. Our results indicate that WDR5 promotes the nuclear egress of viral capsids, the depletion of WDR5 resulting in a significant decrease in production of infectious virions. This is the first report that WDR5 favors HCMV, a DNA virus, replication and highlights a novel target for antiviral therapy.

Divide and conquer: real-time maximum likelihood fitting of multiple emitters for super-resolution localization microscopy.

Multi-emitter localization has great potential for maximizing the imaging speed of super-resolution localization microscopy. However, the slow image analysis speed of reported multi-emitter localization algorithms limits their usage in mostly off-line image processing with small image size. Here we adopt the well-known divide and conquer strategy in computer science and present a fitting-based method called QC-STORM for fast multi-emitter localization. Using simulated and experimental data, we verify that QC-STORM is capable of providing real-time full image processing on raw images with 100 µm × 100 µm field of view and 10 ms exposure time, with comparable spatial resolution as the popular fitting-based ThunderSTORM and the up-to-date non-iterative WindSTORM. This study pushes the development and practical use of super-resolution localization microscopy in high-throughput or high-content imaging of cell-to-cell differences or discovering rare events in a large cell population.

Quantitative performance evaluation of a back-illuminated sCMOS camera with 95% QE for super-resolution localization microscopy.

Scientific Complementary Metal Oxide Semiconductor (sCMOS) cameras were introduced into the market in 2009 and are now becoming a major type of commercial cameras for low-light imaging. sCMOS cameras provide simultaneously low read noise, high readout speed, and large pixel array; however, the relatively low quantum efficiency (QE) of sCMOS cameras has been a major limitation for its application in single molecule imaging, especially super-resolution localization microscopy which requires high detection sensitivity. Here we report the imaging performance of a newly released back-illuminated sCMOS camera (called Dhyana 95 from Tucsen) which is claimed to be the world's first 95% QE sCMOS camera. The imaging performance evaluation is based on a new methodology which is designed to provide paired images from two tested cameras under almost identical experimental conditions. We verified that this new 95% QE sCMOS camera is able to provide superior imaging performance over a representative front-illuminated sCMOS camera (Hamamatsu Flash 4.0 V2) and a popular back-illuminated EMCCD camera (Andor iXon 897 Ultra) in a wide signal range. We hope this study will inspire more studies on using sCMOS cameras in super-resolution localization microscopy, or even single molecule imaging. © 2017 International Society for Advancement of Cytometry.

High-power homogeneous illumination for super-resolution localization microscopy with large field-of-view.

As a wide-field imaging technique, super-resolution localization microscopy (SRLM) is theoretically capable of increasing field-of-view (FOV) without sacrificing either imaging speed or spatial resolution. There are two key factors for realizing large FOV SRLM: one is high-power illumination over the whole FOV with sufficient illumination homogeneity and the other is large FOV signal detection by a camera that has large number of pixels and sufficient detection sensitivity. However nowadays, even though the state-of-art scientific complementary metal-oxide semiconductor (sCMOS) cameras provide single molecule fluorescence signal detection ability over an FOV of more than 200 µm × 200 µm, large FOV SRLM still has not been achieved due to the lack of high-power homogeneous illumination. In this paper, we report large FOV SRLM with a high-power homogeneous illumination system. We demonstrate experimentally that our illumination system, which is based on a newly designed multimode fiber combiner, is capable of providing sufficient illumination intensity (~4.7 kW/cm2 @ 640 nm) and excellent illumination homogeneity. Compared with the reported approaches, our illumination system is advantageous in laser power scaling and square-shape illumination without beam clipping. As a result, our system makes full use of the sensor of a representative Hamamatsu Flash 4.0 V2 sCMOS camera (2048 × 2048 active pixels) and achieves a FOV as large as 221 µm × 221 µm with homogeneous spatial resolution. The flexible solution for realizing large FOV SRLM reported in this paper pushes a significant step toward the development of SRLM.

Bulky 4,6-disubstituted tetraphenylethene-naphthalimide dyad: synthesis, copolymerization, stimuli-responsive fluorescence and cellular imaging.

We report the design and synthesis of a tetraphenylethene substituted with naphthalimide at the 4, 6 positions, named NI-2TPE. NI-2TPE exhibits strong solvent-dependent emission properties with combined ICT and AIE characteristics in THF-H2O systems. This probe was used directly on test papers to distinguish normal organic solvents using their emission colours under UV light based on its AIE and ICT nature. Thanks to the vinyl group in NI-2TPE, we synthesized a copolymer of NIPAM and NI-2TPE, termed P(NIPAM-co-NI-2TPE). The resulting polymer is highly soluble and fluorescent in water (ΦF = 15.4%). Due to the well-known thermo-responsive character of NIPAM, P(NIPAM-co-NI-2TPE) exhibits an interesting fluorescence change in response to various temperatures. Due to the thermo-induced shrinking of the PNIPAM chain, the fluorescence intensity gradually increased from 20 to 34 °C. As the temperature further increased from 34 to 90 °C, the fluorescence intensity decreased sharply, which was caused by the well-known thermal effects. Furthermore, we synthesized a P(HEA-co-NI-2TPE-TPP acrylate) copolymer, in which HEA is a hydrophilic unit, TPP is a mitochondria label and NI-2TPE a fluorescent probe. The corresponding polymer probe is highly soluble in water with FLQY = 7% and we have further applied this probe as a mitochondria targeted imaging tracker in HeLa cells successfully.

Single-wavelength-controlled in situ dynamic super-resolution fluorescence imaging for block copolymer nanostructures via blue-light-switchable FRAP.

Photoswitchable fluorophores are promising in single-molecule optical devices and super-resolution fluorescence imaging, especially in single-molecule photo-activated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM). However, the scarcity of current photoswitchable fluorophores stimulates researchers to develop complicated optical systems and processing software, in accordance with the limited photoswitchable fluorescent proteins and organic fluorophores. Previous efforts to develop synthetic photoswitchable fluorophores have exhibited their promising potential in super-resolution fluorescence imaging. Here, we have designed and synthesized a fluorescence molecular switch with reversible green emission, a napthalimide-hexaarylbiimidazole conjugate (NI-N-HABI), which exhibits strong fluorescence in the emissive state, with fast thermal fading of the photochromism and spontaneous fluorescence recovery after photobleaching (FRAP) induced by blue-light. The photoswitchable fluorophore enables the red-edge wavelength of the optical response to red-shift from the initial near-UV region at less than 400 nm, to 500 nm. The relatively fast fading speed of NI-N-HABI and its sensitivity to longer blue-light irradiation (400-500 nm) have allowed simplification of the optical microscopic system from a two-wavelength laser source to a single-wavelength laser. We applied NI-N-HABI in single-wavelength-controlled in situ dynamic super-resolution fluorescence imaging for the self-assembly and solvent annealing of amphiphilic block polymers, with 50 nm of optical resolution. Single-wavelength-controlled dynamic super-resolution fluorescence imaging facilitates nanoscale optical visualization for the dynamic physical and chemical fluctuation processes of stimuli-responsive nanostructures.