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Expansion microscopy (ExM) enables sub-diffraction imaging by physically expanding labeled tissue samples. This increases the tissue volume relative to the instrument point spread function (PSF), thereby improving the effective resolution by reported factors of 4 - 20X. However, this volume increase dilutes the fluorescence signal, reducing both signal-to-noise ratio (SNR) and acquisition speed. This paper proposes and validates a method for mitigating these challenges. We overcame the limitations of ExM by developing a fast photo-stable protocol to enable scalable widefield three-dimensional imaging with ExM. We combined widefield imaging with quantum dots (QDots). Widefield imaging provides a significantly faster acquisition of a single field-of-view (FOV). However, the uncontrolled incoherent illumination induces photobleaching. We mitigated this challenge using QDots, which exhibit a long fluorescence lifetime and improved photostability. First, we developed a protocol for QDot labeling. Next, we utilized widefield imaging to obtain 3D image stacks and applied deconvolution, which is feasible due to reduced scattering in ExM samples. We show that increased transparency, which is a side-effect of ExM, enables widefield deconvolution, dramatically reducing the acquisition time for three-dimensional images compared to laser scanning microscopy. The proposed QDot labeling protocol is compatible with ExM and provides enhanced photostability compared to traditional fluorescent dyes. Widefield imaging significantly improves SNR and acquisition speed compared to conventional confocal microscopy. Combining widefield imaging with QDot labeling and deconvolution has the potential to be applied to ExM for faster imaging of large three-dimensional samples with improved SNR. 

Immunohistochemical techniques, such as immunofluorescence (IF) staining, enable microscopic imaging of local protein expression within tissue samples. Molecular profiling enabled by IF is critical to understanding pathogenesis and is often involved in complex diagnoses. A recent innovation, known asmicroscopy with ultraviolet surface excitation(MUSE), uses deep ultraviolet (≈280 nm) illumination to excite labels at the tissue surface, providing equivalent images without fixation, embedding, and sectioning. However, MUSE has not yet been integrated into traditional IF pipelines. This limits its application in more complex diagnoses that rely on protein-specific markers. This paper aims to broaden the applicability of MUSE to multiplex immunohistochemistry using quantum dot nanoparticles. We demonstrate the advantages of quantum dot labels for protein-specific MUSE imaging on both paraffin-embedded and intact tissue, significantly expanding MUSE applicability to protein-specific applications. Furthermore, with recent innovations in three-dimensional ultraviolet fluorescence microscopy, this opens the door to three-dimensional IF imaging with quantum dots using ultraviolet excitation. 

In vivo , microvasculature provides oxygen, nutrients, and soluble factors necessary for cell survival and function. The highly tortuous, densely-packed, and interconnected three-dimensional (3D) architecture of microvasculature ensures that cells receive these crucial components. The ability to duplicate microvascular architecture in tissue-engineered models could provide a means to generate large-volume constructs as well as advanced microphysiological systems. Similarly, the ability to induce realistic flow in engineered microvasculature is crucial to recapitulating in vivo -like flow and transport. Advanced biofabrication techniques are capable of generating 3D, biomimetic microfluidic networks in hydrogels, however, these models can exhibit systemic aberrations in flow due to incorrect boundary conditions. To overcome this problem, we developed an automated method for generating synthetic augmented channels that induce the desired flow properties within three-dimensional microfluidic networks. These augmented inlets and outlets enforce the appropriate boundary conditions for achieving specified flow properties and create a three-dimensional output useful for image-guided fabrication techniques to create biomimetic microvascular networks. 

Abstract Uncontrollable dendrite growth is closely related to non‐uniform reaction environments. However, there is a lack of understanding and analysis methods to probe the localized electrochemical environment (LEE). Here the effects of the LEE are investigated, including localized ion concentrations, current density, and electric potential, on metal plating/stripping dynamics and dendrite minimization. A novel in situ 3D microscopy technique is developed to image the morphology dynamics and deposition rate of Zn plating/stripping processes on 3D Zn–Mn anodes. Using the in situ 3D microscope, the electrode morphology changes during the reactions are directly imaged and Zn deposition rate maps at different time points are obtained. It is found that reaction kinetics are highly correlated to LEE and electrode morphology. To further quantify the LEE effects, the digital twin technique is employed that allows the accurate calculation of the electrochemical environments, such as localized ion concentrations, current density, and electric potential, which cannot be directly measured from experiments. It is found that the curvature of the 3D electrode surface determines the LEE and significantly influences reaction kinetics. This provides a new strategy to minimize the dendrite formation by designing and optimizing the 3D geometry of the electrode to control the LEE.