Comparing ultrafast excited state quenching of flavin 1,N-ethenoadenine dinucleotide and flavin adenine dinucleotide by optical spectroscopy and DFT calculations.

Flavins are photoenzymatic cofactors often exploiting the absorption of light to energize photoinduced redox chemistry in a variety of contexts. Both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are used for this function. The study of these photoenzymes has been facilitated using flavin analogs.

Studying nucleic envelope and plasma membrane mechanics of eukaryotic cells using confocal reflectance interferometric microscopy.

Mechanical stress on eukaryotic nucleus has been implicated in a diverse range of diseases including muscular dystrophy and cancer metastasis. Today, there are very few non-perturbative methods to quantify nuclear mechanical properties. Interferometric microscopy, also known as quantitative phase microscopy (QPM), is a powerful tool for studying red blood cell biomechanics.

Quantitative phase microscopy of red blood cells during planar trapping and propulsion.

Red blood cells (RBCs) have the ability to undergo morphological deformations during microcirculation, such as changes in surface area, volume and sphericity. Optical waveguide trapping is suitable for trapping, propelling and deforming large cell populations along the length of the waveguide. Bright field microscopy employed with waveguide trapping does not provide quantitative information about structural changes.

Automated fluorescence intensity and gradient analysis enables detection of rare fluorescent mutant cells deep within the tissue of RaDR mice.

Homologous recombination (HR) events are key drivers of cancer-promoting mutations, and the ability to visualize these events in situ provides important information regarding mutant cell type, location, and clonal expansion. We have previously created the Rosa26 Direct Repeat (RaDR) mouse model wherein HR at an integrated substrate gives rise to a fluorescent cell. To fully leverage this in situ approach, we need better ways to quantify rare fluorescent cells deep within tissues.

Speckle illumination holographic non-scanning fluorescence endoscopy.

Optical sectioning endoscopy such as confocal endoscopy offers capabilities to obtain three-dimensional (3D) information from various biological samples by discriminating between the desired in-focus signals and out-of-focus background. However, in general confocal images are formed through point-by-point scanning and the scanning time is proportional to the 3D space-bandwidth product. Recently, structured illumination endoscopy has been utilized for optically sectioned wide-field imaging, but it still needs axial scanning to acquire images from different depths of focal plane.

Near-common-path interferometer for imaging Fourier-transform spectroscopy in wide-field microscopy.

Imaging Fourier-transform spectroscopy (IFTS) is a powerful method for biological hyperspectral analysis based on various imaging modalities, such as fluorescence or Raman. Since the measurements are taken in the Fourier space of the spectrum, it can also take advantage of compressed sensing strategies. IFTS has been readily implemented in high-throughput, high-content microscope systems based on wide-field imaging modalities.

Modeling the depth-sectioning effect in reflection-mode dynamic speckle-field interferometric microscopy.

Unlike most optical coherence microscopy (OCM) systems, dynamic speckle-field interferometric microscopy (DSIM) achieves depth sectioning through the spatial-coherence gating effect. Under high numerical aperture (NA) speckle-field illumination, our previous experiments have demonstrated less than 1 μm depth resolution in reflection-mode DSIM, while doubling the diffraction limited resolution as under structured illumination. However, there has not been a physical model to rigorously describe the speckle imaging process, in particular explaining the sectioning effect under high illumination and imaging NA settings in DSIM.

Non-axial-scanning multifocal confocal microscopy with multiplexed volume holographic gratings.

Confocal imaging techniques offer an optical sectioning capability to acquire three-dimensional information from various volumetric samples by discriminating the desired in-focus signals from the out-of-focus background. However, confocal, in general, requires a point-by-point scan in both the lateral and axial directions to reconstruct three-dimensional images. In addition, axial scanning in confocal is slower than scanning in lateral directions.

A soft X-ray spectroscopic perspective of electron localization and transport in tungsten doped bismuth vanadate single crystals.

Doped BiVO is a promising photoelectrochemical water splitting anode, whose activity is hampered by poor charge transport. Here we use a set of X-ray spectroscopic methods to probe the origin and nature of localized electron states in W:BiVO. Furthermore, using the polarized nature of the X-rays, we probe variations in the electronic structure along the crystal axes.

Saturation Dynamics Measures Absolute Cross Section and Generates Contrast within a Neuron.

The intensity required to optically saturate a chromophore is a molecular property that is determined by its absorption cross section (σ) and the excited state lifetime. We present an analytical description of such a system and show that fluorescence around the onset of saturation is characterized by product of absorption cross section and lifetime. Using this approach we formulate a generalized method for measuring the multiphoton cross section of fluorophores and use it to obtain the absolute three-photon cross-section spectra of tryptophan.

Talbot multi-focal holographic fluorescence endoscopy for optically sectioned imaging.

A wide-field multi-plane endoscopic system incorporating multiplexed volume holographic gratings and Talbot illumination to simultaneously acquire optically sectioned fluorescence images of tissue structures from different depths is presented. The proposed endoscopic system is configured such that multiple Talbot-illumination planes occur inside a volumetric sample and serve as the input focal planes for the subsequent multiplexed volume holographic imaging gratings. We describe the design, implementation, and experimental data demonstrating this endoscopic system's ability to obtain optically sectioned multi-plane fluorescent images of tissue samples in wide-field fashion without scanning in lateral and axial directions.

In-line digital holographic imaging in volume holographic microscopy.

A dual-plane in-line digital holographic imaging method incorporating volume holographic microscopy (VHM) is presented to reconstruct objects in a single shot while eliminating zero-order and twin-image diffracted waves. The proposed imaging method is configured such that information from different axial planes is acquired simultaneously using multiplexed volume holographic imaging gratings, as used in VHM, and recorded as in-line holograms where the corresponding reference beams are generated in the fashion of Gabor's in-line holography. Unlike conventional VHM, which can take axial intensity information only at focal depths, the proposed method digitally reconstructs objects at any axial position.

Speckle-based volume holographic microscopy for optically sectioned multi-plane fluorescent imaging.

Structured illumination microscopy has been widely used to reconstruct optically sectioned fluorescence images in wide-field fashion; however, it still requires axial scanning to obtain multiple depth information of a volumetric sample. In this paper, a new imaging scheme, called speckle-based volume holographic microscopy system, is presented. The proposed system incorporates volumetric speckle illumination and multiplexed volume holographic gratings to acquire multi-plane images with optical sectioning capability, without any axial scanning.

Talbot holographic illumination nonscanning (THIN) fluorescence microscopy.

Optical sectioning techniques offer the ability to acquire three-dimensional information from various organ tissues by discriminating between the desired in-focus and out-of-focus (background) signals. Alternative techniques to confocal, such as active structured illumination, exist for fast optically sectioned images, but they require individual axial planes to be imaged consecutively. In this article, an imaging technique (THIN), by utilizing active Talbot illumination in 3D and multiplexed holographic Bragg filters for depth discrimination, is demonstrated for imaging in vivo 3D biopsy without mechanical or optical axial scanning.

Rosa26-GFP direct repeat (RaDR-GFP) mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues.

Reassignment of scattered emission photons in multifocal multiphoton microscopy.

Multifocal multiphoton microscopy (MMM) achieves fast imaging by simultaneously scanning multiple foci across different regions of specimen. The use of imaging detectors in MMM, such as CCD or CMOS, results in degradation of image signal-to-noise-ratio (SNR) due to the scattering of emitted photons. SNR can be partly recovered using multianode photomultiplier tubes (MAPMT).

Three dimensional HiLo-based structured illumination for a digital scanned laser sheet microscopy (DSLM) in thick tissue imaging.

Laser sheet based microscopy has become widely accepted as an effective active illumination method for real time three-dimensional (3D) imaging of biological tissue samples. The light sheet geometry, where the camera is oriented perpendicular to the sheet itself, provides an effective method of eliminating some of the scattered light and minimizing the sample exposure to radiation. However, residual background noise still remains, limiting the contrast and visibility of potentially interesting features in the samples.

Improving signal-to-noise ratio of structured light microscopy based on photon reassignment.

In this paper, we report a method for 3D visualization of a biological specimen utilizing a structured light wide-field microscopic imaging system. This method improves on existing structured light imaging modalities by reassigning fluorescence photons generated from off-focal plane excitation, improving in-focus signal strength. Utilizing a maximum likelihood approach, we identify the most likely fluorophore distribution in 3D that will produce the observed image stacks under structured and uniform illumination using an iterative maximization algorithm.

Full-field phase modulation characterization of liquid-crystal spatial light modulator using digital holography.

A direct quantitative phase measurement method to characterize intrinsic phase modulation from an entire active area of transmissive twisted-nematic liquid-crystal spatial light modulator (TN-LCSLM) is presented using digital holography (DH). The change in birefringence of liquid crystal material with respect to addressed gray scale produces phase modulation of wavefront transmitted through TN-LCSLM. Existing methods for phase modulation characterization of LCSLM mainly provides point measurement on its total active region.

Unfolded-state dynamics and structure of protein L characterized by simulation and experiment.

While several experimental techniques now exist for characterizing protein unfolded states, all-atom simulation of unfolded states has been challenging due to the long time scales and conformational sampling required. We address this problem by using a combination of accelerated calculations on graphics processor units and distributed computing to simulate tens of thousands of molecular dynamics trajectories each up to approximately 10 mus (for a total aggregate simulation time of 127 ms). We used this approach in conjunction with Trp-Cys contact quenching experiments to characterize the unfolded structure and dynamics of protein L.

Quasi-physical phase compensation in digital holographic microscopy.

In digital holographic microscopy, if an optical setup is well aligned, the phase curvature introduced by the microscope objective (MO) together with the illuminating wave to the object wave is a spherical phase curvature. It can be physically compensated by introducing the same spherical phase curvature in the reference beam. Digital holographic microscopy setups based on the Michelson interferometric configuration with MO and an adjustable lens are presented, which can well perform the quasi-physical phase compensation during the hologram recording.