Joint tagging assisted fluctuation nanoscopy enables fast high‐density super‐resolution imaging

In fluctuation‐based optical nanoscopy, investigating high‐density labeled subcellular structures with high fidelity has been a significant challenge. In this study, based on super‐resolution radial fluctuation (SRRF) microscopy, the joint tagging (JT) strategy is employed to enable fast high‐density nanoscopic imaging and tracking. In fixed cell experiment, multiple types of quantum dots with distinguishable fluorescence spectra are jointly tagged to subcellular microtubules. In each spectral channel, the decrease in labeling density guarantees the high‐fidelity super‐resolution reconstruction using SRRF microscopy. Subsequently, the combination of all spectral channels achieves high‐density super‐resolution imaging of subcellular microtubules with a resolution of ~62 nm using JT assisted SRRF technique. In the live‐cell experiment, 3‐channel JT is utilized to track the dynamic motions of high‐density toxin‐induced lipid clusters for 1 minute, achieving the simultaneous tracking of many individual toxin‐induced lipid clusters spatially distributed significantly below the optical diffraction limit in living cells.      

Multicomposite super‐resolution microscopy: Enhanced Airyscan resolution with radial fluctuation and sample expansions

Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm.     

Inside Back Cover: In vivo full‐field functional optical hemocytometer (J. Biophotonics 2/2018)

Full‐field functional optical hemocytometer (FFOH), based on the absorption intensity fluctuation modulation (AIFM) effect, is in vivo label‐free image method for capillaries of near‐transparent live biological specimens. FFOH can provide a flow video, flow velocity measurement and RBC count, simultaneously. The zebrafish experimental result shows the potential to study the physiological mechanisms of the blood circulation systems. Further details can be found in the article by Fuli Zhang et al. ( e201700039 )

     

Efficient super‐resolution volumetric imaging by radial fluctuation Bayesian analysis light‐sheet microscopy

Various computational super‐resolution methods are available based on the analysis of fluorescence fluctuation behind acquired frames. However, dilemmas often exist in the balance of fluorophore characteristics, computation cost, and achievable resolution. Here we present an approach that uses a super‐resolution radial fluctuations (SRRF) image to guide the Bayesian analysis of fluorophore blinking and bleaching (3B) events, allowing greatly accelerated localization of overlapping fluorophores with high accuracy. This radial fluctuation Bayesian analysis (RFBA) approach is also extended to three dimensions for the first time and combined with light‐sheet fluorescence microscopy, to achieve super‐resolution volumetric imaging of thick samples densely labeled with common fluorophores. For example, a 700‐nm thin Bessel plane illumination is developed to optically section the Drosophila brain, providing a high‐contrast 3D image of rhythmic neurons. RFBA analyzes 30 serial volumes to reconstruct a super‐resolved 3D image at 4‐times higher resolutions (~70 and 170 nm), and precisely resolve the axon terminals. The computation is over 2‐orders faster than conventional 3B analysis microscopy. The capability of RFBA is also verified through dual‐color imaging of cell nucleus in live Drosophila brain. The spatial co‐localization patterns of the nuclear envelope and DNA in a neuron deep inside the brain can be precisely extracted by our approach.     

Front Cover: A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution (J. Biophotonics 2/2018)

A new type of high‐throughput imaging flow cytometer (>20 000 cells s^‐1) based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. FACED imaging flow cytometers enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. It critically empowers largescale and deep characterization of single cells and their heterogeneity with high statistical power— an ability to become increasingly critical in single‐cell analysis adopted in a wide range of biomedical and life‐science applications. Further details can be found in the article by Wenwei Yan et al. ( e201700178 )

     

Inside Cover: Fiber‐based fluorescence lifetime imaging of recellularization processes on vascular tissue constructs (J. Biophotonics 9/2018)

Tissue autofluorescence provides fluorescence lifetime contrast between acellular tissue and that containing newly seeded cells. Fiber‐based fluorescence lifetime imaging (FLIm) can be used for tracking recellularization of engineered vascular grafts and potential matrix remodeling at large scale, without compromising sample integrity. FLIm cellular contrast was verified in a subset of samples seeded with eGFP‐labelled cells. Results suggests fiberbased FLIm is a suitable tool for monitoring recellularization of engineered tissue nondestructively. Further details can be found in the article by Alba Alfonso‐Garcia, Jeny Shklover, Benjamin E. Sherlock, et al. ( e201700391 ).

     

Back Cover: Non‐invasive optical method for real‐time assessment of intracorneal riboflavin concentration and efficacy of corneal cross‐linking (J. Biophotonics 7/2018)

We disclose a theranostic device for performing image‐guided riboflavin/UV‐A corneal cross‐linking. The device determines treatment efficacy by real time monitoring of riboflavin concentration in the corneal stroma. The study shows efficacy of the device in eye bank human donor tissues. Further details can be found in the article by Giuseppe Lombardo et al. ( e201800028 )

     

Super‐beacons: Open‐source probes with spontaneous tuneable blinking compatible with live‐cell super‐resolution microscopy

Localization‐based super‐resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non‐fluorescent states. These transitions are commonly regulated by high‐intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single‐molecule self‐quenching as a mechanism to generate spontaneous photoswitching. To demonstrate this principle, we developed a new class of DNA‐based open‐source super‐resolution probes named super‐beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live‐cell compatible super‐resolution microscopy without high‐illumination or toxic imaging buffers is revealed by imaging interferon inducible transmembrane proteins (IFITMs) at sub‐100 nm resolutions.     

Nucleocytoplasmic transport of intrinsically disordered proteins studied by high‐speed super‐resolution microscopy

Both natively folded and intrinsically disordered proteins (IDPs) destined for the nucleus need to transport through the nuclear pore complexes (NPCs) in eukaryotic cells. NPCs allow for passive diffusion of small folded proteins while barricading large ones, unless they are facilitated by nuclear transport receptors. However, whether nucleocytoplasmic transport of IDPs would follow these rules remains unknown. By using a high‐speed super‐resolution fluorescence microscopy, we have measured transport kinetics and 3D spatial locations of transport routes through native NPCs for various IDPs. Our data revealed that the rules executed for folded proteins are not well followed by the IDPs. Instead, both large and small IDPs can passively diffuse through the NPCs. Furthermore, their diffusion efficiencies and routes are differentiated by their content ratio of charged (Ch) and hydrophobic (Hy) amino acids. A Ch/Hy‐ratio mechanism was finally suggested for nucleocytoplasmic transport of IDPs.     

Inside Front Cover: Enhanced volumetric imaging in 2‐photon microscopy via acoustic lens beam shaping (J. Biophotonics 2/2018)

Two‐photon microscopy is the tool of choice for fluorescence imaging of deep tissues with high resolution, but can be limited in three‐dimensional acquisition speed and penetration depth. In this work, these issues are addressed by using an acoustic optofluidic lens capable of ultrafast beam shaping on a pixel basis. Driving the lens with different phase profiles enables high‐speed volumetric imaging, or enhanced signal‐to‐background for deeper penetration. Further details can be found in the article by Simonluca Piazza et al. ( e201700050 )

     

Inside Cover: Optimized perfusion‐based CUBIC protocol for the efficient whole‐body clearing and imaging of rat organs (J. Biophotonics 5/2018)

Optical tissue clearing is a method allowing post‐mortem deep imaging of organs in three dimensions. By optimizing the CUBIC clearing protocol, the authors provide rapid and simple approach to clear the entire adult rat organism within as little as four days, which is accompanied by the variety of its staining and imaging techniques. The image was captured with polarizers and demonstrates transparent rodent heart with thread‐like crystals of clearing reagent. Further details can be found in the article by Pawe? Matryba et al. ( e201700248 ).

     

Back Cover: Characterization and application of porous gold nanoparticles as 2‐photon luminescence imaging agents: 20‐fold brighter than gold nanorods (J. Biophotonics 2/2018)

Gold nanoparticles serve as imaging contrast agents useful for two‐photon nonlinear microscopy of biological cells and tissues. In this study, 100‐nm‐sized gold particles with a multitude of nanopores embedded inside have been physically synthesized and investigated for the plasmonic enhancement in two‐photon luminescence. Exhibiting remarkable potential for two‐photon imaging, the porous gold nanoparticles boost near‐infrared light absorption substantially and allow emission signals 20 times brighter than gold nanorods being currently used as typical imaging agents. Further details can be found in the article by Joo H. Park et al. ( e201700174 )

     

Inside Back Cover: Multiphoton dynamic imaging of the effect of chronic hepatic diseases on hepatobiliary metabolism in vivo (J. Biophotonics 9/2018)

In vivo multiphoton imaging was used to map changes in hepatobiliary metabolism in liver fibrosis (left column) and hepatocellular carcinoma (right column). The top row shows the maps of kinetic rate constant of the uptake and esterase processing while the bottom row shows that of bile canalicular excretion of xenobiotics. Further details can be found in the article by Chih‐Ju Lin, Sheng‐Lin Lee, Wei‐Hsiang Wang, et al. ( e201700338 ).

     

Front Cover: Dual‐wavelength hybrid optoacoustic‐ultrasound biomicroscopy for functional imaging of large‐scale cerebral vascular networks (J. Biophotonics 9/2018)

We present a hybrid dual‐wavelength optoacoustic and ultrasound bio‐microscope capable of rapid transcranial visualization of morphology and oxygenation status of large‐scale cerebral vascular networks. Imaging of entire cortical vasculature in mice is achieved with single capillary resolution and complemented by simultaneously acquired pulse‐echo ultrasound microscopy scans of the mouse skull. The new approach holds potential to facilitate studies into neurological and vascular abnormalities of the brain. Further details can be found in the article by Johannes Rebling, Héctor Estrada, Sven Gottschalk, et al. ( e201800057 ).

     

Front Cover: Photoluminescence intensity ratio of Eu‐conjugated lactates—A simple optical imaging technique for biomarker analysis for critical diseases (J. Biophotonics 5/2018)

Eu³⁺integrated photoluminescence intensity ratio (PLIR) approach for optical detection of lactates in blood serum, plasma and confocal imaging of brain tissues has very high potential for exploitation of this technique in both in vitro monitoring and in vivo bioimaging applications for the detection of biomarkers in various diseases states. This image is diagrammatic representation of fact that the overall PLIR is higher with more lactates conjugated with Eu³⁺ ions. Further details can be found in the article by Tarun Kakkar et al. ( e201700199 ).

     

Inside Front Cover: Increasing fluorescence lifetime for resolution improvement in stimulated emission depletion nanoscopy (J. Biophotonics 5/2019)

A STED‐FLIM system is developed to observe the changes of fluorescence lifetime. The pictures show increased lifetime of fluorescent microspheres samples with laser illumination time in both confocal and STED imaging modes. Due to the saturation power of fluorophores is correlated with fluorescence lifetime, the lifetime increase is beneficial for the reduction of the saturation power, indicating the same imaging resolution can be achieved in a lower depletion power. Further details can be found in the article by Lu‐Wei Wang, Yue Chen, Wei Yan, et al. ( e201800315 ).

     

Inside Cover: Noninvasive,high‐speed,near‐infrared imaging of the biomolecular distribution and molecular mechanism of embryonic development in fertilized fish eggs (J. Biophotonics 4/2018)

The biomaterial distribution and its molecular mechanism of embryonic development in Japanese medaka fish were visualized without staining using high‐speed near‐infrared imaging. It was a remarkable achievement to visualize the structures of eyes, lipid bilayer membranes, micelles, and water structural variations at the interface of different substances. Furthermore, insights on lipid metabolism and membrane functions were obtained from the biased distribution of lipoproteins and the presence of unsaturated fatty acids in the egg membrane. Further details can be found in the article by Mika Ishigaki ( e201700115 )

     

Inside Cover: Multi‐ATOM: Ultrahigh‐throughput single‐cell quantitative phase imaging with subcellular resolution (J. Biophotonics 7/2019)

A new quantitative phase imaging (QPI) modality, coined multi‐ATOM, can now capture and process enormous amount of quantitative phase single‐cell images (>700,000 cells) at a ultrahigh throughput without compromising sub‐cellular resolution. It could empower label‐free single‐cell analysis where large‐scale and cost‐effective screening is necessary. Further details can be found in the article by Kelvin C. M. Lee, Andy K. S. Lau, Anson H. L. Tang, et al. ( e201800479 ).

     

Back Cover: Large‐depth‐of‐field full‐field optical angiography (J. Biophotonics 5/2019)

A large‐depth‐of‐field full‐field optical angiography (LD‐FFOA) method is developed to expand the depth‐of‐field (DOF). The contrast pyramid fusion algorithm is used to fuse 10 FFOA images at different focus depth. Cover images of mouse ear shows LD‐FFOA image has higher contrast and more detailed features. The LD‐FFOA method solves the defocused problem caused by the limited DOF of lens, the curved surface and uneven thickness of the sample. Further details can be found in the article by Mingyi Wang, Nanshou Wu, Hongheng Huang, et al. ( e201800329 ).