Mapping solar magnetic fields from the photosphere to the base of the corona.

Routine ultraviolet imaging of the Sun's upper atmosphere shows the spectacular manifestation of solar activity; yet, we remain blind to its main driver, the magnetic field. Here, we report unprecedented spectropolarimetric observations of an active region plage and its surrounding enhanced network, showing circular polarization in ultraviolet (Mg ii h & k and Mn i) and visible (Fe i) lines. We infer the longitudinal magnetic field from the photosphere to the very upper chromosphere. At the top of the plage chromosphere, the field strengths reach more than 300 G, strongly correlated with the Mg ii k line core intensity and the electron pressure. This unique mapping shows how the magnetic field couples the different atmospheric layers and reveals the magnetic origin of the heating in the plage chromosphere.

Super-resolution fluorescence microscopy by stepwise optical saturation.

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a [Formula: see text]-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples.

Skeletal cell YAP and TAZ combinatorially promote bone development.

The functions of the paralogous transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in bone are controversial. Each has been observed to promote or inhibit osteogenesis in vitro, with reports of both equivalent and divergent functions. Their combinatorial roles in bone physiology are unknown. We report that combinatorial YAP/TAZ deletion from skeletal lineage cells, using Osterix-Cre, caused an osteogenesis imperfecta-like phenotype with severity dependent on allele dose and greater phenotypic expressivity with homozygous TAZ vs. YAP ablation. YAP/TAZ deletion decreased bone accrual and reduced intrinsic bone material properties through impaired collagen content and organization. These structural and material defects produced spontaneous fractures, particularly in mice with homozygous TAZ deletion and caused neonatal lethality in dual homozygous knockouts. At the cellular level in vivo, YAP/TAZ ablation reduced osteoblast activity and increased osteoclast activity, in an allele dose-dependent manner, impairing bone accrual and remodeling. Transcriptionally, YAP/TAZ deletion and small-molecule inhibition of YAP/TAZ interaction with the transcriptional coeffector TEAD reduced osteogenic and collagen-related gene expression, both in vivo and in vitro. These data demonstrate that YAP and TAZ combinatorially promote bone development through regulation of osteoblast activity, matrix quality, and osteoclastic remodeling.-Kegelman, C. D., Mason, D. E., Dawahare, J. H., Horan, D. J., Vigil, G. D., Howard, S. S., Robling, A. G., Bellido, T. M., Boerckel, J. D. Skeletal cell YAP and TAZ combinatorially promote bone development.

Saturation-compensated measurements for fluorescence lifetime imaging microscopy.

Fluorophore saturation is the key factor limiting the speed and excitation range of fluorescence lifetime imaging microscopy (FLIM). For example, fluorophore saturation causes incorrect lifetime measurements when using conventional frequency-domain FLIM at high excitation powers. In this Letter, we present an analytical theoretical description of this error and present a method for compensating for this error in order to extract correct lifetime measurements in the limit of fluorophore saturation. We perform a series of simulations and experiments to validate our methods. The simulations and experiments show a 13.2× and a 2.6× increase in excitation range, respectively. The presented method is based on algorithms that can be easily applied to existing FLIM setups.

Super-sensitivity multiphoton frequency-domain fluorescence lifetime imaging microscopy.

We present a series of experiments that demonstrate a super-sensitive chemical imaging technique based on multiphoton frequency-domain fluorescence lifetime imaging microscopy (MPM-FD-FLIM) that shows a 2× improvement in imaging speed compared to the theoretical limit of conventional MPM-FD-FLIM. Additionally, this technique produces unprecedented sensitivity over a large range of fluorescence lifetimes. These results are achieved through simple modifications to data analysis in a conventional MPM-FD-FLIM microscope and are based on an analytical model describing the signal-to-noise ratio (SNR) of a MPM-FD-FLIM system [J. Opt. Soc. Am. A33, B1 (2016)]. Here we experimentally validate this model.

Investigation of signal-to-noise ratio in frequency-domain multiphoton fluorescence lifetime imaging microscopy.

Multiphoton microscopy (MPM) combined with fluorescence lifetime imaging microscopy (FLIM) has enabled three-dimensional quantitative molecular microscopy in vivo. The signal-to-noise ratio (SNR), and thus the imaging rate of MPM-FLIM, which is fundamentally limited by the shot noise and fluorescence saturation, has not been quantitatively studied yet. In this paper, we investigate the SNR performance of the frequency-domain (FD) MPM-FLIM with two figures of merit: the photon economy in the limit of shot noise, and the normalized SNR in the limit of saturation. The theoretical results and Monte Carlo simulations find that two-photon FD-FLIM requires 50% fewer photons to achieve the same SNR as conventional one-photon FLIM. We also analytically show that the MPM-FD-FLIM can exploit the DC and higher harmonic components generated by nonlinear optical mixing of the excitation light to improve SNR, reducing the required number of photons by an additional 50%. Finally, the effect of fluorophore saturation on the experimental SNR performance is discussed.

Photophysical characterization of sickle cell disease hemoglobin by multi-photon microscopy.

The photophysical properties of human sickle cell disease (SCD) Hemoglobin (Hb) is characterized by multi-photon microscopy (MPM). The intrinsic two-photon excited fluorescence (TPEF) signal associated with extracted hemoglobin was investigated and the solidified SCD variant (HbS) was found to demonstrate broad emission peaking around 510 nm when excited at 800 nm. MPM is used to dynamically induce and image HbS gelling by photolysis of deoxygenated HbS. For comparison, photolysis conditions were applied to a healthy variant of human hemoglobin (HbA) and found to remain in solution not forming fibers. The use of this signal to study the mechanism of HbS polymerization associated with the sickling of SCD erythrocytes is discussed.

Label-free and depth resolved optical sectioning of iron-complex deposits in sickle cell disease splenic tissue by multiphoton microscopy.

Multiphoton microscopy (MPM) imaging of intrinsic two-photon excited fluorescence (TPEF) is performed on humanized sickle cell disease (SCD) mouse model splenic tissue. Distinct morphological and spectral features associated with SCD are identified and discussed in terms of diagnostic relevance. Specifically, spectrally unique splenic iron-complex deposits are identified by MPM; this finding is supported by TPEF spectroscopy and object size to standard histopathological methods. Further, iron deposits are found at higher concentrations in diseased tissue than in healthy tissue by all imaging methods employed here including MPM, and therefore, may provide a useful biomarker related to the disease state. These newly characterized biomarkers allow for further investigations of SCD in live animals as a means to gain insight into the mechanisms impacting immune dysregulation and organ malfunction, which are currently not well understood.

Automated bacterial identification by angle resolved dark-field imaging.

We propose and demonstrate a dark-field imaging technique capable of automated identification of individual bacteria. An 87-channel multispectral system capable of angular and spectral resolution was used to measure the scattering spectrum of various bacteria in culture smears. Spectra were compared between various species and between various preparations of the same species. A 15-channel system was then used to prove the viability of bacterial identification with a relatively simple microscope system. A simple classifier was able to identify four of six bacterial species with greater than 90% accuracy in bacteria-by-bacteria testing.