Complete Emission Spectrum Research Articles

Quantitation of the physicochemical properties of myelin using Nile Red fluorescence spectroscopy.

Myelin is a vital structure that is key to rapid saltatory conduction in the central and peripheral nervous systems. Much work has been done over the decades examining the biochemical composition and morphology of myelin at the light and electron microscopic levels. Here we report a method to study myelin based on the fluorescent probe Nile Red. This lipophilic dye readily partitions into live and chemicallyfixed myelin producing bright, well-resolved images of the sheath. Using spectral confocal microscopy, a complete emission spectrum of Nile Red fluorescence can be acquired for each pixel in an image. The solvatochromic properties of Nile Red cause its emission spectrum to change depending on the polarity of its local environment. Therefore, measuring spectral shifts can report subtle changes in the physicochemical properties of myelin. We show differences in myelin polarity in central versus peripheral nervous system and in different regions of central nervous system white matter of the mouse brain, together with developmental and sex variations. This technique is also well suited for measuring subtle changes in myelin properties in live exvivo white matter specimens. We also demonstrate how light deprivation induces a myelin polarity change in adult mouse optic nerve underscoring a continuing myelin plasticity in response to axonal activity well into adulthood. The Nile Red spectroscopic method allows measurement of subtle physicochemical changes in myelin that can importantly influence its electrical properties and by extension, conduction velocities in axons.

The Hubble PanCET Program: Emission Spectrum of Hot Jupiter HAT-P-41b

Abstract We present the most complete emission spectrum for inflated hot Jupiter HAT-P-41b combining new Hubble Space Telescope Wide Field Camera/G141 spectrum from the Hubble Panchromatic Comparative Exoplanet Treasury program with archival Spitzer eclipse observations. We found a near blackbody-like emission spectrum, which is best fitted with an isothermal temperature–pressure (TP) profile, that agrees well with the dayside heat redistribution scenario assuming zero Bond albedo. The noninverted TP profile is consistent with the nondetection of near-UV/optical absorbers in the transit spectra. We do not find any evidence for significant H− opacity nor a metal-rich atmosphere. HAT-P-41b is an ideal target that sits in the transitioning parameter space between hot and ultra-hot Jupiters, and future James Webb Space Telescope observations will help us to better constrain the thermal structure and chemical composition.

High-Dimensional Immunophenotyping with 37-Color Panel Using Full-Spectrum Cytometry.

A comprehensive study of the cellular components of the immune system demands both deep and broad immunophenotyping of numerous cell subsets in an effective and practical way. Novel full-spectrum technology reveals the complete emission spectrum of each dye maximizing the amount of information that can be obtained on a single sample regarding conventional flow cytometry and provide an expanded knowledge of biological processes. In this chapter, we describe a 37-color protocol that allows to identify more than 45 different cell populations on whole blood samples of SARS-CoV-2-infected patients.

Optical phase curve of the ultra-hot Jupiter WASP-121b

We present the analysis of TESS optical photometry of WASP-121b, which reveals the phase curve of this transiting ultra-hot Jupiter. Its hotspot is located at the sub-stellar point, showing inefficient heat transport from the dayside (2870 ± 50 K) to the nightside (<2500 K at 3σ) at the altitudes probed by TESS. The TESS eclipse depth, measured at the shortest wavelength to date for WASP-121b, confirms the strong deviation from blackbody planetary emission. Our atmospheric retrieval on the complete emission spectrum supports the presence of a temperature inversion, which can be explained by the presence of VO and possibly TiO and FeH. The strong planetary emission at short wavelengths could arise from an H− continuum.

High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.

Photonic Flash Sintering of Ink-Jet-Printed Back Electrodes for Organic Photovoltaic Applications.

A study of the photonic flash sintering of a silver nanoparticle ink printed as the back electrode for organic solar cells is presented. A number of sintering settings with different intensities and pulse durations have been tested on both full-area and grid-based silver electrodes, using the complete emission spectrum of the flash lamps from UV-A to NIR. However, none of these settings was able to produce functional devices with performances comparable to those of reference cells prepared using thermally sintered ink. Different degradation mechanisms were detected in the devices with a flash-sintered back electrode. The P3HT:PCBM photoactive layer appears to be highly heat-sensitive and turned out to be severely damaged by the high temperatures generated in the silver layer during the sintering. In addition, UV-induced photochemical degradation of the functional materials was identified as another possible source of performance deterioration in the devices with grid-based electrodes. Reducing the light intensity does not provide a proper solution because in this case the Ag electrode is not sintered sufficiently. For both types of devices, with full-area and grid-based electrodes, these problems could be solved by excluding the short wavelength contribution from the flash light spectrum using a filter. Optimized sintering parameters allowed manufacture of OPV devices with performance equal to those of the reference devices. Photonic flash sintering of the top electrode in organic solar cells was demonstrated for the first time. It reveals the great potential of this sintering method for the future roll-to-roll manufacturing of organic solar cells from solution.

In vivo Thermodynamics of RTKS in the Cell Membrane: Quantitative Spectral FRET

Here, we introduce a new quantitative FRET method (qs FRET) that allows for the measurement of apparent FRET efficiency and the concentrations of donor and acceptor-tagged membrane-bound proteins in cell-derived vesicles and live cells. This new method utilizes two-photon excitation and spectral imaging technology, where the complete emission spectrum of each pixel in an image is acquired. We utilize this method to measure the equilibrium association constant of fluorescent protein-tagged Receptor Tyrosine Kinases in live cells.

Stable single-molecule lines of terrylene in polycrystalline para-dichlorobenzene at 1.5 K.

The spectroscopic properties of single terrylene (Tr) molecules are studied in a polycrystalline matrix of para-dichlorobenzene (p-DCB) at 1.5 K. Samples grown in a glass capillary show a very strong site at 597 nm, which is redshifted by more than 700 cm(-1) from the observed transition energy for Tr in p-DCB prepared as a film on a coverslip (572 nm). Each of these two sites is characterized by measuring their single-molecule spectroscopic parameters at 1.5 K. Lifetime-limited linewidths of 45±5 MHz are found for both sites. Fluorescence detection rates reach 8×10(4) count s(-1) at saturation. The spectral trails of the majority of single molecules show no spectral jumps, indicating an absence of interacting two-level systems; however, the small distribution of linewidths may indicate weak interactions with low-frequency modes. Frequency jumps are observed for 10 % of the molecules. The complete emission spectra from two different single molecules at the center of each of the two sites is presented. Debye-Waller factors of αDW=0.33±0.05 for the normal site (572 nm) and αDW=0.30±0.05 for the red site (597 nm) are reported. This new host-guest system provides a quick and easy way to obtain lifetime-limited single-molecule lines.

Visible and near infrared fluorescence spectral flow cytometry

There is a long standing interest in measuring complete emission spectra from individual cells in flow cytometry. We have developed flow cytometry instruments and analysis approaches to enable this to be done routinely and robustly. Our spectral flow cytometers use a holographic grating to disperse light from single cells onto a CCD for high speed, wavelength-resolved detection. Customized software allows the single cell spectral data to be displayed and analyzed to produce new spectra-derived parameters. We show that familiar reference and calibration beads can be employed to quantitatively assess instrument performance. We use microspheres stained with six different quantum dots to compare a virtual bandpass filter approach with classic least squares (CLS) spectral unmixing, and then use antibody capture beads and CLS unmixing to demonstrate immunophenotyping of peripheral blood mononuclear cells using spectral flow cytometry. Finally, we characterize and evaluate several near infrared (NIR) emitting fluorophores for use in spectral flow cytometry. Spectral flow cytometry offers a number of attractive features for single cell analysis, including a simplified optical path, high spectral resolution, and streamlined approaches to quantitative multiparameter measurements. The availability of robust instrumentation, software, and analysis approaches will facilitate the development of spectral flow cytometry applications.

Relative and absolute intensity calibrations of a modern broadband echelle spectrometer

We report on relative and absolute intensity calibrations of a modern broadband echelle spectrometer (type ESA 3000® trademark of LLA Instruments GmbH, Berlin) for use in the diagnostics of low-temperature plasma. This type of device measures simultaneously complete emission spectra in the spectral range from 200 to 800 nm with a spectral resolution of several picometres by using more than 90 spectral orders, causing a strongly structured efficiency function. The assumptions and approximations entering the calibration procedure under these conditions are discussed in section 3. For coping with the strongly structured efficiency function a continuum light source is needed, which covers the entire spectral range. Furthermore, the variation of its intensity must be low enough to ensure that neither statistical errors perturb the calibration in regions with low photon flux and/or low efficiency, nor local memory overflow in regions with high photon flux or high efficiency. In our case this requires that during calibration over the whole spectral range of the spectrometer the counts per pixel in one measurement vary at highest by a factor 10 to 12. Usual broadband light sources do not meet this latter requirement. We, therefore, use an uncalibrated ‘composite’ source, an adjustable combination of a standard tungsten strip lamp and a deuterium lamp, and calibrate the spectrometer in a two-step process against the tungsten strip lamp and well-known rovibrational intensity distributions in the emission spectra of NO and N2. We adjust the composite source in a way to produce a perturbation-free first approximation of an (uncalibrated) efficiency function, which is then corrected and thus calibrated by comparison with the (secondary) standards mentioned above. For absolute calibration we use the tungsten strip lamp. The uncertainty attained in this way for the relative calibration depends on the wavelength and varies between 5% and 10%. For the absolute calibration we obtained an uncertainty of 12%. We further discuss problems caused by the non-uniform spectral efficiency and dispersion of the spectrometer, which complicate the calibration procedure.

Wavelength-resolved fluorescence detector for microchip capillary electrophoresis separations

A wavelength-resolved fluorescence detector for microchip and capillary separations is developed. It consists of a xenon lamp as flexible excitation source, a fluorescence microscope, a spectrograph with exchangeable gratings (150 and 600 lines/mm) and an intensified CCD camera. In contrast to standard LIF-detection systems, this set-up facilitates tuning of excitation and emission wavelengths over the whole visible spectrum of light (350–800 nm). The detector allows to record on-line emission spectra with high repetition rates of up to 60 Hz, which are needed to monitor rapid on-chip separations with peak widths <0.5 s. In this work, the detector system is applied to the capillary electrophoretic microchip separation of three rhodamines and their impurities (excitation: 450–490 nm; emission: >500 nm). Complete emission spectra of submicromolar solutions are recorded on-line. Comparable with diode-array detection in UV/vis-spectroscopy, this detector set-up yields information-rich electropherograms. The additional dimension of information compared to standard fluorescence detection systems enables peak assignment by means of fluorescent properties of the analytes and the immediate detection of coelutions due to a change of signal ratios at chosen wavelengths (peak purity plots).

Fluorescence Emission Spectral Shift Measurements of Membrane Potential in Single Cells

Previous measurements of transmembrane potential using the electrochromic probe di-8-ANEPPS have used the excitation spectral shift response by alternating excitation between two wavelengths centered at voltage-sensitive portions of the excitation spectrum and recording at a single wavelength near the peak of the emission spectrum. Recently, the emission spectral shift associated with the change in transmembrane potential has been used for continuous membrane potential monitoring. To characterize this form of the electrochromic response from di-8-ANEPPS, we have obtained fluorescence signals from single cells in response to step changes in transmembrane potentials set with a patch electrode, using single wavelength excitation near the peak of the dye absorption spectrum. Fluorescence changes at two wavelengths near voltage-sensitive portions of the emission spectrum and shifts in the complete emission spectrum were determined for emission from plasma membrane and internal membrane. We found that the fluorescence ratio from either dual-wavelength recordings, or from opposite sides of the emission spectrum, varied linearly with the amplitude of the transmembrane potential step between −80 and +60 mV. Voltage dependence of difference spectra exhibit a crossover point near the peak of the emission spectra with approximately equal gain and loss of fluorescence intensity on each side of the spectrum and equal response amplitude for depolarization and hyperpolarization. These results are consistent with an electrochromic mechanism of action and demonstrate how the emission spectral shift response can be used to measure the transmembrane potential in single cells.

Hyperspectral imaging: a novel approach for microscopic analysis.

The usefulness of the light microscope has been dramatically enhanced by recent developments in hardware and software. However, current technologies lack the ability to capture and analyze a high-resolution image representing a broad diversity of spectral signatures in a single-pass view. We show that hyperspectral imaging offers such a technology. METHODS AND RESULTS We developed a prototype hyperspectral imaging microscope capable of collecting the complete emission spectrum from a microscope slide. A standard epifluorescence microscope was optically coupled to an imaging spectrograph, with output recorded by a CCD camera. Software was developed for image acquisition and computer display of resultant X--Y images with spectral information. Individual images were captured representing Y-wavelength planes, with the stage successively moved in the X direction, allowing an image cube to be constructed from the compilation of generated scan files. This prototype instrument was tested with samples relevant to cytogenetic, histologic, cell fusion, microarray scanning, and materials science applications. Hyperspectral imaging microscopy permits the capture and identification of different spectral signatures present in an optical field during a single-pass evaluation, including molecules with overlapping but distinct emission spectra. This instrument can reduce dependence on custom optical filters and, in future imaging applications, should facilitate the use of new fluorophores or the simultaneous use of similar fluorophores.

Temperature-dependent total emission spectra of azulene in polymers: Modeling using spectral densities

Monochromatically excited total emission spectra have been measured for the S1→S0 transition of azulene in polyethylene, polystyrene, and poly(methylmethacrylate) matrices over a temperature range from 1.4 to 100 K. The spectra in all three polymers exhibit strong zero-phonon lines (excitation of azulene vibrations only) accompanied by well-defined Stokes-shifted phonon sidebands at the lowest temperatures. As the temperature is raised the phonon bands broaden and gain relative intensity at the expense of the zero-phonon lines, and the spectra become qualitatively similar to the room-temperature liquid-phase spectra with sharp Raman lines on a broad fluorescence background. The near-origin-excited data are simulated by calculating the complete emission spectrum as a χ(3) process that assumes no artificial partitioning between “Raman” and “fluorescence.” The internal vibrations of azulene are modeled as simple undamped displaced harmonic oscillators while the intermolecular or matrix phonons are either modeled as a Brownian oscillator or treated as effective spectral densities extracted from published neutron scattering and/or low-frequency nonresonant Raman data in the same polymers. While the qualitative features of the spectra and their temperature dependence are reproduced, none of the spectral densities employed give a fully satisfactory fit to the experimental spectra. The results demonstrate the sensitivity of total emission spectra to the chromophore–matrix interactions, and suggest that the spectral densities describing these interactions are functions not only of the matrix but also of the chromophore involved.

Monochromatic Cathodoluminescence Image in the Scanning Electron Microscope

A TECHNIQUE has been developed for observing monochromatic Cathodoluminescence from semiconductors in the scanning electron microscope, enabling optical micro-analysis of materials to be carried out in a manner analogous to the electron probe X-ray microanalyser. The experimental arrangement is shown in Fig. 1. A three lens system gives a beam of electrons of 30 keV energy focused to a spot of 500 A diameter at the specimen with a current density between 1 and 100 A cm−2. The specimen, in the form of a thin platelet, is mounted on a small movable liquid nitrogen cold finger, and is cooled to 100° K during observation. The Cathodoluminescence is recorded on one of two channels—a sample of the total emitted light is collected by a quartz light pipe, the signal being fed through a photomultiplier and amplifier to the display system of the microscope, to build up a scanning micrograph. Simultaneously an image of the specimen is focused on to the entrance slit of a prism spectrometer by means of a quartz lens in the vacuum wall of the system. The output signal from the spectrometer may then be fed by means of a second photomultiplier and a preamplifier into the detecting system of the microscope, in place of the signal from the total emitted luminescence, thus enabling a scanning micrograph to be built up from radiation of any desired wavelength. (Alternatively the complete emission spectrum may be observed by feeding the amplifier output on to a chart recorder and driving the spectrometer as in conventional optical spectroscopy.)