Localization microscopy at doubled precision with patterned illumination.

MINFLUX offers a breakthrough in single molecule localization precision, but is limited in field of view. Here we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micrometer-sized field of view. We show a near two-fold improvement in precision over standard localization with the same photon count on DNA-origami nanostructures and tubulin in cells, using DNA-PAINT and STORM imaging.

Volumetric structured illumination microscopy enabled by a tunable-focus lens.

We present a mechanical-scan-free method for volumetric imaging of biological tissue. The optical sectioning is provided by structured illumination, and the depth of the imaging plane is varied using an electrically tunable-focus lens. We characterize and evaluate the ability of this axial-scanning mechanism in structured illumination microscopy and demonstrate its ability to perform subcellular resolution imaging in oral mucosa ex vivo. The proposed mechanism can potentially convert any wide-field microscope to a 3D-imaging platform without the need for mechanical scanning of imaging optics and/or sample.

High-speed multicolor structured illumination microscopy using a hexagonal single mode fiber array.

Structured illumination microscopy (SIM) is a widely used imaging technique that doubles the effective resolution of widefield microscopes. Most current implementations rely on diffractive elements, either gratings or programmable devices, to generate structured light patterns in the sample. These can be limited by spectral efficiency, speed, or both. Here we introduce the concept of fiber SIM that allows for camera frame rate limited pattern generation and manipulation over a broad wavelength range. Illumination patterns are generated by coupling laser beams into radially opposite pairs of fibers in a hexagonal single mode fiber array where the exit beams are relayed to the microscope objective's back focal plane. The phase stepping and rotation of the illumination patterns are controlled by fast electro-optic devices. We achieved a rate of 111 SIM frames per second and imaged with excitation patterns generated by both 488 nm and 532 nm lasers.

Enhanced detection of oral dysplasia by structured illumination fluorescence lifetime imaging microscopy.

We demonstrate that structured illumination microscopy has the potential to enhance fluorescence lifetime imaging microscopy (FLIM) as an early detection method for oral squamous cell carcinoma. FLIM can be used to monitor or detect changes in the fluorescence lifetime of metabolic cofactors (e.g. NADH and FAD) associated with the onset of carcinogenesis. However, out of focus fluorescence often interferes with this lifetime measurement. Structured illumination fluorescence lifetime imaging (SI-FLIM) addresses this by providing depth-resolved lifetime measurements, and applied to oral mucosa, can localize the collected signal to the epithelium. In this study, the hamster model of oral carcinogenesis was used to evaluate SI-FLIM in premalignant and malignant oral mucosa. Cheek pouches were imaged in vivo and correlated to histopathological diagnoses. The potential of NADH fluorescence signal and lifetime, as measured by widefield FLIM and SI-FLIM, to differentiate dysplasia (pre-malignancy) from normal tissue was evaluated. ROC analysis was carried out with the task of discriminating between normal tissue and mild dysplasia, when changes in fluorescence characteristics are localized to the epithelium only. The results demonstrate that SI-FLIM (AUC = 0.83) is a significantly better (p-value = 0.031) marker for mild dysplasia when compared to widefield FLIM (AUC = 0.63).

An ultraviolet-curable, core-shell vaccine formed via phase separation.

One of the central challenges in the field of vaccine delivery is to develop a delivery method that maintains antigen stability while also enabling control over the system's release kinetics. Addressing these challenges would not only allow for expanded access to vaccines worldwide but would also help significantly reduce mortality rates in developing countries. In this article, we report the development of single-injection vaccine depots for achieving novel delayed burst release. Synthesized poly(ε-caprolactone) and poly(ε-caprolactone) triacrylate were used to form stationary bubbles within an aqueous solution of 10% carboxymethylcellulose. These polymeric bubbles (referred to as "polybubbles") can then be injected with an aqueous solution of cargo, resulting in the formation of a polymeric shell. The puncture resulting from cargo injection self-heals prior to ultraviolet (UV) curing. UV curing and lyophilization were shown to enhance the stability of the polybubbles. BSA- CF 488 and HIV1 gp120/41 were used as the antigen in the study as a proof-of-concept. Further endeavors to automate the production of polybubbles are underway.

Optically sectioned wide-field fluorescence lifetime imaging microscopy enabled by structured illumination.

In this paper, we demonstrate the ability of structured illumination microscopy to enhance the ability of fluorescence lifetime imaging to resolve fluorescence lifetimes in relatively thick samples that possess distinct but spectrally overlapping fluorescent layers. Structured illumination fluorescent lifetime imaging microscopy (SI-FLIM) is shown to be able to accurately reconstruct lifetime values in homogenous fluorophore samples (POPOP, NADH, and FAD) as well as accurately measure fluorescent lifetime in two layer models that are layered with NADH/FAD over POPOP, where NADH/FAD and POPOP have spectral overlap. Finally, the ability of SI-FLIM was demonstrated in a hamster cheek pouch ex vivo to show that more accurate lifetimes could be measured for each layer of interest in the oral mucosa (epithelium and submucosa).

Handheld tunable focus confocal microscope utilizing a double-clad fiber coupler for in vivo imaging of oral epithelium.

A reflectance confocal endomicroscope with double-clad fiber coupler and electrically tunable focus lens is applied to imaging of the oral mucosa. The instrument is designed to be lightweight and robust for clinical use. The tunable lens allows axial scanning through > 250 ?? ? m in the epithelium when the probe tip is placed in contact with tissue. Images are acquired at 6.6 frames per second with a field of view diameter up to 850 ?? ? m . In vivo imaging of a wide range of normal sites in the oral cavity demonstrates the accessibility of the handheld probe. In vivo imaging of clinical lesions diagnosed as inflammation and dysplasia illustrates the ability of reflectance confocal endomicroscopy to image cellular changes associated with pathology.