Super-resolution vibrational imaging using expansion stimulated Raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously due to spectral overlap. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, we describe a new technique called Molecule Anchorable Gel-enabled Nanoscale Imaging of Fluorescence and stImulatEd Raman Scattering microscopy (MAGNIFIERS), that integrates SRS microscopy with expansion microscopy (ExM). ExM is a powerful strategy providing significant improvement in imaging resolution by physical magnification of hydrogel-embedded preserved biological specimens. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. We used MAGNIFIERS to visualize nanoscale features in a label-free manner with C-H vibration of proteins, lipids and DNA in a broad range of biological specimens, from mouse brain, liver and kidney to human lung organoid. In addition, we applied MAGNIFIERS to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, we used MAGNIFIERS to demonstrate 8-color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super-resolution label-free chemical imaging, high-resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.

Nanoscale Imaging and Analysis of Bone Pathologies

Understanding the properties of bone is of both fundamental and clinical relevance. The basis of bone’s quality and mechanical resilience lies in its nanoscale building blocks (i.e., mineral, collagen, non-collagenous proteins, and water) and their complex interactions across length scales. Although the structure–mechanical property relationship in healthy bone tissue is relatively well characterized, not much is known about the molecular-level origin of impaired mechanics and higher fracture risks in skeletal disorders such as osteoporosis or Paget’s disease. Alterations in the ultrastructure, chemistry, and nano-/micromechanics of bone tissue in such a diverse group of diseased states have only been briefly explored. Recent research is uncovering the effects of several non-collagenous bone matrix proteins, whose deficiencies or mutations are, to some extent, implicated in bone diseases, on bone matrix quality and mechanics. Herein, we review existing studies on ultrastructural imaging—with a focus on electron microscopy—and chemical, mechanical analysis of pathological bone tissues. The nanometric details offered by these reports, from studying knockout mice models to characterizing exact disease phenotypes, can provide key insights into various bone pathologies and facilitate the development of new treatments.

EGFR transactivates RON to drive oncogenic crosstalk

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation – arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.

Enhanced super-resolution microscopy by extreme value based emitter recovery

Super-resolution localization microscopy allows visualization of biological structure at nanoscale resolution. However, the presence of heterogeneous background can degrade the nanoscale resolution by tens of nanometers and introduce significant image artifacts. Here we investigate and validate an efficient approach, referred to as extreme value-based emitter recovery (EVER), to accurately recover the distorted fluorescent emitters from heterogeneous background. Through numerical simulation and biological experiments, we validated the accuracy of EVER in improving the fidelity of the reconstructed super-resolution image for a wide variety of imaging characteristics. EVER requires no manual adjustment of parameters and has been implemented as an easy-to-use ImageJ plugin that can immediately enhance the quality of reconstructed super-resolution images. This method is validated as an efficient way for robust nanoscale imaging of samples with heterogeneous background fluorescence, such as thicker tissue and cells.

Dual energy X-ray beam ptycho-fluorescence imaging

X-ray ptychography and X-ray fluorescence are complementary nanoscale imaging techniques, providing structural and elemental information, respectively. Both methods acquire data by scanning a localized beam across the sample. X-ray ptychography processes the transmission signal of a coherent illumination interacting with the sample, to produce images with a resolution finer than the illumination spot and step size. By enlarging both the spot and the step size, the technique can cover extended regions efficiently. X-ray fluorescence records the emitted spectra as the sample is scanned through the localized beam and its spatial resolution is limited by the spot and step size. The requisites for fast ptychography and high-resolution fluorescence appear incompatible. Here, a novel scheme that mitigates the difference in requirements is proposed. The method makes use of two probes of different sizes at the sample, generated by using two different energies for the probes and chromatic focusing optics. The different probe sizes allow to reduce the number of acquisition steps for the joint fluorescence–ptychography scan compared with a standard single beam scan, while imaging the same field of view. The new method is demonstrated experimentally using two undulator harmonics, a Fresnel zone plate and an energy discriminating photon counting detector.

A fluorescent amyloid sensor for quantitative super-resolution imaging of amyloid fibril assembly.

Many soluble proteins can self-assemble into macromolecular structures called amyloids, a subset of which are implicated in a range of neurodegenerative disorders. The nanoscale size and structural heterogeneity of prefibrillar and early aggregates, as well as mature amyloid fibrils, pose significant challenges for the quantification of amyloid species, identification of their cellular interaction partners and for elucidation of the molecular basis for cytotoxicity. We report a fluorescent amyloid sensor AmyBlink-1 and its application in super-resolution imaging of amyloid structures. AmyBlink-1 exhibits a 5-fold increase in ratio of the green (thioflavin T) to red (Alexa Fluor 647) emission intensities upon interaction with amyloid fibrils. Using AmyBlink-1, we performed nanoscale imaging of four different types of amyloid fibrils, achieving a resolution of ~30 nm. AmyBlink-1 enables molecular-level visualization and subsequent quantification of morphological features, such as the length and skew of individual amyloid aggregates formed at different times along the amyloid assembly pathway.

Nanoscale Imaging of Biomolecules using Molecule Anchorable Gel-enabled Nanoscale In-situ Fluorescence Microscopy

Expansion microscopy (ExM) is a powerful imaging strategy that offers a low-cost solution for nanoimaging with conventional microscopes by physically and isotropically magnifying preserved biological specimens embedded in a cross-linked water-swellable hydrogel. Current ExM protocols require prior treatment with specialized reactive anchoring chemicals to link specific labels and biomolecule classes to the gel. In addition, most techniques reportedly use strong Proteinase K to digest endogenous epitopes to enable expansion and are limited by using mechanically fragile gel formulas to expand specimens by at most 4.5× linearly. Here we describe a new ExM framework, Molecule Anchorable Gel-enabled Nanoscale In-situ Fluorescence MicroscopY (MAGNIFY), that uses a mechanically sturdy gel that enables broad retention of nucleic acids, proteins, and lipids without the need for a separate anchoring step. MAGNIFY expands biological specimens up to 11× and facilitates imaging of cells and tissues with effectively ~25-nm-resolution using an ∼280-nm diffraction-limited objective lens on conventional optical microscopes or with ~13 nm-resolution if combined with Super-resolution Optical Fluctuation Imaging (SOFI). Further, MAGNIFY generalizes well across a broad range of biological specimens, providing insight into nanoscopic subcellular structures including synaptic proteins from mouse brain, podocyte foot processes in human kidney, and defects in cilia and basal bodies in drug-treated human lung organoids. MAGNIFY provides a novel advance that expands the precision, utility, accessibility, and generality of subcellular nanoscopy.

Quantifying nanodiamonds biodistribution in whole cells with correlative iono-nanoscopy

Correlative imaging and quantification of intracellular nanoparticles with the underlying ultrastructure is crucial for understanding cell-nanoparticle interactions in biological research. However, correlative nanoscale imaging of whole cells still remains a daunting challenge. Here, we report a straightforward nanoscopic approach for whole-cell correlative imaging, by simultaneous ionoluminescence and ultrastructure mapping implemented with a highly focused beam of alpha particles. We demonstrate that fluorescent nanodiamonds exhibit fast, ultrabright and stable emission upon excitation by alpha particles. Thus, by using fluorescent nanodiamonds as imaging probes, our approach enables quantification and correlative localization of single nanodiamonds within a whole cell at sub-30 nm resolution. As an application example, we show that our approach, together with Monte Carlo simulations and radiobiological experiments, can be employed to provide unique insights into the mechanisms of nanodiamond radiosensitization at the single whole-cell level. These findings may benefit clinical studies of radio-enhancement effects by nanoparticles in charged-particle cancer therapy.