Measuring changes in blood volume fraction during induced gingivitis of healthy and unhealthy populations using hyperspectral spatial frequency domain imaging: a clinical study.

This investigation aimed to quantitatively measure the changes in inflammation of subjects with healthy and unhealthy gums during a period of induced gingivitis. A total of 30 subjects (15 healthy, 15 with gum inflammation) were enlisted and given oral exams by a dental hygienist. Baseline measurements were acquired before a 3-week period of oral hygiene abstinence. The lobene modified gingival index scoring was used for inflammation scoring and hyperspectral spatial frequency domain imaging was used to quantitatively measure oxy- and deoxygenated blood volume fraction at two time points: at Baseline and after 3 weeks of oral hygiene abstinence. We found that abstaining from oral hygiene causes a near proportional increase in oxygenated and deoxygenated blood volume fraction for healthy individuals. For individuals who started the study with mild to moderate gingivitis, increases in blood volume were mainly due to deoxygenated blood.

Spectral imaging of normal, hydrated, and desiccated porcine skin using polarized light.

Significance: Spectroscopic and structural imaging of tissue layers is important for investigating tissue health. However, investigating superficial tissue is difficult using optical imaging, due to the convolved absorption and backscatter of light from deeper layers. Aim: This report investigates the effects of hydration and desiccation of ex vivo porcine skin on the reflectance of polarized light at different wavelengths (light-emitting diodes). Approach: We developed a spectroscopic polarized imaging system to investigate submicron changes in tissue structures. By separating polarized from depolarized backscattered light, submicron structural changes in subsurface and deeper tissue layers can be separated and monitored. Results: The results demonstrate that (1) polarized light reflectance is about 2%, consistent with ∼6 scattering events, on average; (2) there was little wavelength dependence to the reflectance of polarized light; (3) increased hydration leads to a modest increase in total reflectance (from 0.8 to 0.9), whereas desiccation had little effect; however, hydration did not affect polarized reflectance, but desiccation slightly lowered polarized reflectance. Conclusions: Higher scattering from the reticular dermis was likely due to swelling of collagen fiber bundles in the dermal layers, which increased fibril spacing. The epidermal skin surface showed little change due to the stratum corneum resisting desiccation and maintaining hydration.

Multimodal hyperspectral fluorescence and spatial frequency domain imaging for tissue health diagnostics of the oral cavity.

A multimodal, hyperspectral imaging system was built for diagnostics of oral tissues. The system, termed Hyperspectral-Fluorescence-Spatial Frequency Domain Imaging (Hy-F-SFDI), combines the principles of spatial frequency domain imaging, quantitative light fluorescence, and CIELAB color measurement. Hy-F-SFDI employs a compact LED projector, excitation LED, and a 16 channel hyperspectral camera mounted on a custom platform for tissue imaging. A two layer Monte Carlo approach was used to generate a reference table for quick tissue analysis. To demonstrate the clinical capabilities of Hy-F-SFDI, we used the system to quantify gingival tissue hemoglobin volume fraction, detect caries, bacterial activity, and measure tooth color of a volunteer at different time points. Hy-F-SFDI was able to measure quantitative changes in tissue parameters.

Patterned-illumination second harmonic generation microscopy of collagen fibrils in rat scleras.

We developed a patterned-illumination second harmonic generation (PI-SHG) microscopy, which combines the principle of structured illumination reconstruction with SHG microscopy for label-free super-resolution imaging. We confirmed that PI-SHG microscopy can achieve 1.59-time resolution improvement compared to conventional SHG microscopy by imaging nanowire samples. We further demonstrated three-dimensional PI-SHG microscopy in imaging ex vivo collagen fibrils in rat scleras.

Imaging neuronal structure dynamics using 2-photon super-resolution patterned excitation reconstruction microscopy.

Visualizing fine neuronal structures deep inside strongly light-scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution but often suffer from limited imaging depth, long imaging time or high light fluence requirements. Here, we present two-photon super-resolution patterned excitation reconstruction (2P-SuPER) microscopy for 3-dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 µm in living brain tissue with approximately 100 nm spatial resolution. We confirmed 2P-SuPER resolution using fluorescence nanoparticle and quantum dot phantoms and imaged spiny neurons in acute brain slices. We induced hippocampal plasticity and showed that 2P-SuPER can resolve increases in dendritic spine head sizes on CA1 pyramidal neurons following theta-burst stimulation of Schaffer collateral axons. 2P-SuPER further revealed nanoscopic increases in dendritic spine neck widths, a feature of synaptic plasticity that has not been thoroughly investigated due to the combined limit of resolution and penetration depth in existing imaging technologies.

Stochastic fluorescence switching of nucleic acids under visible light illumination.

We report detailed characterizations of stochastic fluorescence switching of unmodified nucleic acids under visible light illumination. Although the fluorescent emission from nucleic acids under the visible light illumination has long been overlooked due to their apparent low absorption cross section, our quantitative characterizations reveal the high quantum yield and high photon count in individual fluorescence emission events of nucleic acids at physiological concentrations. Owing to these characteristics, the stochastic fluorescence switching of nucleic acids could be comparable to that of some of the most potent exogenous fluorescence probes for localization-based super-resolution imaging. Therefore, utilizing the principle of single-molecule photon-localization microscopy, native nucleic acids could be ideal candidates for optical label-free super-resolution imaging.

Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast.

Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging.

Super-resolution spectroscopic microscopy via photon localization.

Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm-a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.

Subsurface Super-resolution Imaging of Unstained Polymer Nanostructures.

Optical imaging has offered unique advantages in material researches, such as spectroscopy and lifetime measurements of deeply embedded materials, which cannot be matched using electron or scanning-probe microscopy. Unfortunately, conventional optical imaging cannot provide the spatial resolutions necessary for many nanoscopic studies. Despite recent rapid progress, super-resolution optical imaging has yet to be widely applied to non-biological materials. Herein we describe a method for nanoscopic optical imaging of buried polymer nanostructures without the need for extrinsic staining. We observed intrinsic stochastic fluorescence emission or blinking from unstained polymers and performed spatial-temporal spectral analysis to investigate its origin. We further applied photon localization super-resolution imaging reconstruction to the detected stochastic blinking, and achieved a spatial resolution of at least 100 nm, which corresponds to a six-fold increase over the optical diffraction limit. This work demonstrates the potential for studying the static heterogeneities of intrinsic polymer molecular-specific properties at sub-diffraction-limited optical resolutions.

Super-resolution two-photon microscopy via scanning patterned illumination.

We developed two-photon scanning patterned illumination microscopy (2P-SPIM) for super-resolution two-photon imaging. Our approach used a traditional two-photon microscopy setup with temporally modulated excitation to create patterned illumination fields. Combing nine different illuminations and structured illumination reconstruction, super-resolution imaging was achieved in two-photon microscopy. Using 2P-SPIM we achieved a lateral resolution of 141 nm, which represents an improvement by a factor of 1.9 over the corresponding diffraction limit. We further demonstrated super-resolution cellular imaging by 2P-SPIM to image actin cytoskeleton in mammalian cells and three-dimensional imaging in highly scattering retinal tissue.

Investigating femtosecond-laser-induced two-photon photoacoustic generation.

We investigated two-photon absorption-based photoacoustic generation and compared it with corresponding photoluminescence emission. Experimental results revealed expected quadratic dependences on the incident optical fluence in both photoacoustic and photoluminescence processes. We also investigated the influence of optical scattering on the generation of two-photon photoacoustic and photoluminescence signals and found that photoacoustic signals attenuated more slowly than photoluminescence signals when the optical scattering coefficient was increased, which was attributed to a weaker ultrasonic attenuation than that the optical attenuation in the scattering medium. Finally, we showed three-dimensional two-photon photoacoustic imaging.

Second harmonic imaging of plants tissues and cell implosion using two-photon process in ZnO nanoparticles.

The optical properties of colloidal ZnO nanoparticle (NP) solutions, with size ranging from several nm to around 200 nm, have been tailored to have high optical nonlinearity for bioimaging with no auto-fluorescence above 750 nm and minimal auto-fluorescence below 750 nm. The high second harmonic conversion efficiency enables selective tissue imaging and cell tracking using tunable near-infrared femtosecond laser source ranging from 750-980 nm. For laser energies exceeding the two-photon energy of the bandgap of ZnO (half of 3.34 eV), the SHG signal greatly decreases and the two-photon emission becomes the dominant signal. The heat generated due to two-photon absorption within the ZnO NPs enable selective cell or localized tissue destruction using excitation wavelength ranging from 710-750 nm.