Long-term in vivo three-photon imaging reveals region-specific differences in healthy and regenerative oligodendrogenesis.

The generation of new myelin-forming oligodendrocytes in the adult central nervous system is critical for cognitive function and regeneration following injury. Oligodendrogenesis varies between gray and white matter regions, suggesting that local cues drive regional differences in myelination and the capacity for regeneration. However, the layer- and region-specific regulation of oligodendrocyte populations is unclear due to the inability to monitor deep brain structures in vivo. Here we harnessed the superior imaging depth of three-photon microscopy to permit long-term, longitudinal in vivo three-photon imaging of the entire cortical column and subcortical white matter in adult mice. We find that cortical oligodendrocyte populations expand at a higher rate in the adult brain than those of the white matter. Following demyelination, oligodendrocyte replacement is enhanced in the white matter, while the deep cortical layers show deficits in regenerative oligodendrogenesis and the restoration of transcriptional heterogeneity. Together, our findings demonstrate that regional microenvironments regulate oligodendrocyte population dynamics and heterogeneity in the healthy and diseased brain.

Long-term in vivo three-photon imaging reveals region-specific differences in healthy and regenerative oligodendrogenesis.

The generation of new myelin-forming oligodendrocytes in the adult CNS is critical for cognitive function and regeneration following injury. Oligodendrogenesis varies between gray and white matter regions suggesting that local cues drive regional differences in myelination and the capacity for regeneration. Yet, the determination of regional variability in oligodendrocyte cell behavior is limited by the inability to monitor the dynamics of oligodendrocytes and their transcriptional subpopulations in white matter of the living brain. Here, we harnessed the superior imaging depth of three-photon microscopy to permit long-term, longitudinal in vivo three-photon imaging of an entire cortical column and underlying subcortical white matter without cellular damage or reactivity. Using this approach, we found that the white matter generated substantially more new oligodendrocytes per volume compared to the gray matter, yet the rate of population growth was proportionally higher in the gray matter. Following demyelination, the white matter had an enhanced population growth that resulted in higher oligodendrocyte replacement compared to the gray matter. Finally, deep cortical layers had pronounced deficits in regenerative oligodendrogenesis and restoration of the MOL5/6-positive oligodendrocyte subpopulation following demyelinating injury. Together, our findings demonstrate that regional microenvironments regulate oligodendrocyte population dynamics and heterogeneity in the healthy and diseased brain.

Roadmap on Label-Free Super-Resolution Imaging.

Label-free super-resolution (LFSR) imaging relies on light-scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super-resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state-of-the-art in this field, and to discuss the resolution boundaries and hurdles which need to be overcome to break the classical diffraction limit of the LFSR imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction-limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super-resolution capability which are based on understanding resolution as an information science problem, on using novel structured illumination, near-field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere-assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.

Two-photon PSF-engineered image scanning microscopy.

We present two-photon fluorescence image scanning microscopy (ISM) with engineered excitation and detection point-spread-functions enabling 3D imaging in a single 2D scan. This demonstration combines excitation using a holographic multispot array of focused femtosecond pulses with a high-efficiency single-helix PSF phase mask detection. Camera detection along with a multiview reconstruction algorithm allows volumetric imaging of biological samples over a depth of field spanning more than 1500 nm with an axial resolution of better than 400 nm. The nonlinear two-photon process improves sectioning and the inherent longer wavelengths increase the penetration depth in scattering samples. Our method extends the performance of 3D ISM towards thicker biological samples.

Material anisotropy as a degree of freedom in optical design.

We present an approach for the design of refractive optical elements using materials degrees of freedom that are accessible via engineered materials. Starting from first principles and an unconstrained general material, we specify homogeneous refractive lenses that focus light with diffraction-limited resolution resulting from a tailored anisotropic refractive index. We analyze the performance, physical feasibility, and advantages over isotropic lenses. Materials degrees of freedom enable new flexibility for imaging system designs with lower complexity expanding the existing aspheric and graded index paradigms.

Thermal expansion feedback for wave-front shaping.

Focusing inside scattering media is a challenging task with a variety of applications in biomedicine. State of the art methods mostly require invasive feedback inside or behind the sample, limiting practical use. We present a technique for dynamic control and focusing inside scattering media that combines two powerful methods: optical coherence tomography (OCT) and wave-front shaping (WFS). We use OCT as a non-invasive feedback for WFS optimization of a separate, penetrating laser. Energy absorbed in the sample, creates thermal expansions that are used for the feedback mechanism. By maximizing thermal deformations within a selected focal region, we demonstrate enhanced focusing of light through scattering media beyond the ballistic regime and within the penetration range of OCT.

Lock-in detection of photoacoustic feedback signal for focusing through scattering media using wave-front shaping.

Wave-front shaping techniques enable focusing and imaging through scattering media. Unfortunately, most approaches require invasive feedback inside or behind the sample, or use of spatial correlations (memory effect) limiting the application to specific types of samples. Recent approaches overcome these limitations by taking advantage of acoustic waves via the photoacoustic (PA) effect or via photon tagging. We present a fully analog signal processing lock-in scheme for PA detection to improve focusing through scattering media and to efficiently extract nonlinear photoacoustic signals towards wave-front optimization. Our implementation improves PA feedback performance in terms of SNR, speed, and resolution.

Super resolution methodology based on temperature dependent Raman scattering.

The recent advances in far-field super-resolution (SR) microscopy rely on, and therefore are limited by the ability to control the fluorescence of label molecules. We suggest a new, label-free, far-field SR microscopy based on temperature dependence of Raman scattering. Here, we present simulation and experimental characterization of the method. In an ultrafast pump-probe scheme, a spatial temperature profile is optically excited throughout the diffraction-limited spot; the Raman spectrum is probed with an overlapping laser. Thermally induced shifts, recorded in a specific spectral region of interest (ROI), enable spatial discrimination between areas of different temperature. Our simulations show spatial resolution that surpasses the diffraction limit by more than a factor of 2. Our method is compatible with material characterization in ambient, vacuum and liquid, thin and thick samples alike.

Super-resolution in label-free photomodulated reflectivity.

We demonstrate a new, label-free, far-field super-resolution method based on an ultrafast pump-probe scheme oriented toward nanomaterial imaging. A focused pump laser excites a diffraction-limited spatial temperature profile, and the nonlinear changes in reflectance are probed. Enhanced spatial resolution is demonstrated with nanofabricated silicon and vanadium dioxide nanostructures. Using an air objective, resolution of 105 nm was achieved, well beyond the diffraction limit for the pump and probe beams and offering a novel kind of dedicated nanoscopy for materials.

Intracoronary monocyte chemoattractant protein 1 and vascular endothelial growth factor levels are associated with necrotic core, calcium and fibrous tissue atherosclerotic plaque components: an intracoronary ultrasound radiofrequency study.

AIMS: To investigate the relationship between various serum biomarkers and coronary atherosclerotic plaque composition obtained by intravascular ultrasound virtual histology (IVUS-VH). METHODS: Using ELISA, we measured the serum levels of CD40 ligand, C-reactive protein, monocyte chemoattractant protein 1 (MCP-1), metalloproteinase 9, P-selectin and vascular endothelial growth factor (VEGF) in 40 patients with manifested coronary artery disease. RESULTS: Correlation analysis between biomarkers levels, IVUS grayscale parameters and VH-defined necrotic core (NC), calcium, fibrous and fibrofatty components was performed. MCP-1 and VEGF levels correlated with the severity of area stenosis (r = 0.35, p = 0.03 and r = 0.38, p = 0.017, respectively) and inversely correlated with the remodeling index (r = -0.35, p = 0.03 and r = 0.35, p = 0.02, respectively). Higher levels of MCP-1 were associated with increased calcium (r = 0.47, p = 0.004), NC (r = 0.38, p = 0.02) and less fibrous tissue components (r = -0.34, p = 0.03), whereas VEGF had an inverse correlation with both calcium components (r = -0.37, p = 0.02) and NC (r = -0.34, p = 0.036) but was strongly associated with increased fibrous components (r = 0.47, p = 0.003). No significant correlation was noted for any of the other biomarkers. CONCLUSIONS: MCP-1 and VEGF serum levels in patients with ischemic heart disease are correlated with coronary artery plaque burden and composition.

Necrotic core and thin cap fibrous atheroma distribution in native coronary artery lesion-containing segments: a virtual histology intravascular ultrasound study.

OBJECTIVES: The aim of this study was to assess the longitudinal topographical relationships between minimal luminal area (MLA) sites and plaques with the most vulnerable characteristics using radiofrequency-based virtual histology intravascular ultrasound analysis. METHODS: We analyzed 69 native coronary artery segments with de-novo lesions (>50% stenosis) obtained from 50 patients with ischemic coronary artery disease. Maximal necrotic core (maxNC) was defined as a virtual histology intravascular ultrasound frame with the maxNC area and virtual histology-characterized thin cap fibrous atheroma was defined as a cross-section, which contained a plaque burden of more than 40%, relative necrotic core area of 10% or more, and a narrow band encircling the lumen containing relative necrotic core area of more than 10%, in three consecutive frames. RESULTS: MaxNC was present at the MLA site in only 17.4% of the segments, proximal in 52.2% (by 5.0 ± 5.4 mm), and distal to MLA in 30.4% (by 4.0 ± 5.1 mm). Non-MLA sites with maxNC (n=57) compared with MLA sites had reduced plaque burden (64.5 ± 11.2% vs. 76.0 ± 10.5%, P<0.001), increased remodeling index (1.04 ± 0.17 vs. 0.89 ± 0.15, P<0.001), less fibrotic tissue (47.7 ± 13.4% vs. 54.8 ± 13.8%, P<0.001), and higher dense calcium deposition (15.3 ± 10.8% vs. 11.9 ± 10.3%, P<0.001). Plaques containing maxNC and virtual histology-characterized thin cap fibrous atheroma were found in 23 of the non-MLA sites compared with two of the MLA sites (P<0.0001). CONCLUSIONS: In coronary artery segments with intermediate-to-severe stenosis, plaques containing maxNC are mostly located away from the MLA site and more often comprise virtual histology-characterized thin cap fibrous atheroma. Such data may carry practical implications for coronary revascularization procedures.