DecodeSTORM:A user-friendly ImageJ plug-in for quantitative data analysis in single-molecule localization microscopy

Quantitative data analysis in single-molecule localization microscopy(SMLM)is crucial for studying cellular functions at the biomolecular level.In the past decade,several quantitative methods were developed for analyzing SMLM data;however,imaging artifacts in SMLM experiments reduce the accuracy of these methods,and these methods were seldom designed as user-friendly tools.Researchers are now trying to overcome these di±culties by developing easyto-use SMLM data analysis software for certain image analysis tasks.But,this kind of software did not pay su±cient attention to the impact of imaging artifacts on the analysis accuracy,and usually contained only one type of analysis task.Therefore,users are still facing di±culties when they want to have the combined use of different types of analysis methods according to the characteristics of their data and their own needs.In this paper,we report an ImageJ plug-in called DecodeSTORM,which not only has a simple GUI for human–computer interaction,but also combines artifact correction with several quantitative analysis methods.DecodeSTORM includes format conversion,channel registration,artifact correction(drift correction and localization¯ltering),quantitative analysis(segmentation and clustering,spatial distribution statistics and colocalization)and visualization.Importantly,these data analysis methods can be combined freely,thus improving the accuracy of quantitative analysis and allowing users to have an optimal combination of methods.We believe DecodeSTORM is a user-friendly and powerful ImageJ plug-in,which provides an easy and accurate data analysis tool for adventurous biologists who are looking for new imaging tools for studying important questions in cell biology.

Modulated illumination localization microscopy-enabled sub-10 nm resolution

Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues.To break the limit of resolution caused by diffraction,researchers have made continuous advances and innovations to improve the resolution of optical microscopy since the 1990s.These contributions,however,still make sub-10nm imaging an obstacle.Here,we name a series of technologies as modulated illumination localization microscopy(MILM),which makes ultra-high-resolution imaging practical.Besides,we review the recent progress since 2017 when MINFLUX was proposed and became the inspiration and foundation for the follow-up devel-opment of MILM.This review divides MILM into two types:point-scanning and wide-field.The schematics,principles and future research directions of MILM are discussed elaborately.

Geometric phase helical PSF for simultaneous orientation and 3D localization microscopy

The 3D location and dipole orientation of light emitters provide essential information in many biological,chemical,and physical systems.Simultaneous acquisition of both information types typically requires pupil engineering for 3D localization and dual-channel polarization splitting for orientation deduction.Here we report a geometric phase helical point spread function for simultaneously estimating the 3D position and dipole orientation of point emitters.It has a compact and simpler optical configuration compared to polarization-splitting techniques and yields achromatic phase modulation in contrast to pupil engineering based on dynamic phase,showing great potential for single-molecule orientation and localization microscopy.

Super-resolution morphological dissemination of intercalating dye in single DNA molecules via binding activated localization microscopy

Fluorescent dye （YOYO-I） intercalated with single DNA molecules were investigated via bindingactivated localization microscopy （BALM） at sub-diffraction limit resolutions. Various dye-to-DNA base pair （bp） ratios were imaged using the blinking property of YOYO-1 dye under optimum BALM switching buffer conditions. Individual DNA molecules exhibited regular/irregular intercalating phenomena with respect to dye-to-DNA bp ratio. The acquired images were reconstructed into super-resolution images by applying a Gaussian fit to the centroid of the point spread function. The YOYO-1 intercalated with λ-DNA possessed a non-homogeneous region due to the different binding modes of YOYO-1 with λ-DNA. Each binding mode was imaged at the sub-diffraction limit super-resolution. The distance between homogenously localized intercalating dyes within the DNA molecules was measured to be 34nm （n= 10; dye：DNAbp= 1：100） without photocleavage in 50mmol/L β-mercaptoethylamine buffer. The results were similar to those of the theoretical values without photocleavage in the base pairs of single DNA molecules below the diffraction limit. The results paved the way for an in-depth microscopic analysis of molecular variation with single λ-DNA molecules. With this method, it should be possible to analyze the exact base pair breakdown during various stages of cell apoptosis.

Application of super-resolution fluorescence microscopy in hematologic malignancies

Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type,treatment and prognosis of hematologic malignancies.Super-resolution fluorescence microscopy(SRM)is a special kind of optical microscopy technology,which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014.With the development of SRM,many related technologies have been applied to the diagnosis and treatment of clinical diseases.It was reported that a major type of SRM technique,single molecule localization microscopy(SMLM),is more sensitive than flow cytometry(FC)in detecting cell membrane antigens'expression,thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools.Furthermore,SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases.In this paper,we mainly discuss the application of SRM in clone hematological malignancies.

Background-free three-dimensional selective imaging of anisotropic plasmonic nanoparticles

There is an increasing demand for advanced optical imaging techniques that can detect and resolve nanosize objects at a spatial resolution below the optical diffraction limit, especially in three-dimensional （3D） cellular environments. In this study, using a polarization-activated localization scheme based on the orientation-dependent properties of anisotropic plasmonic metal nanoparticles （MNPs）, ＂photoswitchable＂ imaging of single gold nanorods （AuNRs） was accomplished not only in two dimensions but also in three dimensions. Moreover, the Rayleigh scattering background arising from the congested subcellular structures was efficiently suppressed. Thus, we obtained the 3D distributions of both the position and the orientation of the AuNRs inside the cells and investigated their intemalization kinetics. To our knowledge, this is the first demonstration of the confocal-like 3D imaging of non-fluorescence nanoparticles with a high resolution and almost zero background. This technique is easy to implement and should greatly facilitate MNP studies and applications in biomedicine and biology.

Dipole and charge effects of chloroaluminum phthalocyanine revealed by local work function measurements at sub-molecular level

Work function plays a significant role in surface chemistry. Local work function provides the information of local d/pole-d/pole interaction and charge distribution between adsorbates and substrate, highlighting the local charge effect of the targeted spot which is normally smeared out in conventional average work function measurements. Chloroaluminum phthalocyanine （CIA1Pc）, an important optoelectronic molecule with a permanent dipole moment pointing from the Pc ring to the ending CI atom, adsorbed on Au（111） in either Cl-up or Cl-down configuration. Scanning tunneling microscopy/spectroscopy measurements revealed that at the centers of Cl-up and CI-down molecules, the local work functions changed oppositely with respect to the Au（111） substrate. At their Pc lobes, however, the local work functions unanimously increased due to charging effect of the indole lobes in the CIAIPc molecule.