High throughput instrument to screen fluorescent proteins under two-photon excitation

Author Posting. © Optical Society of America , 2020. This article is posted here by permission of Optical Society of America for personal use, not for redistribution. The definitive version was published in Molina, R. S., King, J., Franklin, J., Clack, N., McRaven, C., Goncharov, V., Flickinger, D., Svoboda, K., Drobizhev, M., & Hughes, T. E. High throughput instrument to screen fluorescent proteins under two-photon excitation. Biomedical Optics Express, 11(12), (2020): 7192-7203, https://doi.org/10.1364/BOE.409353.Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.National Institute of Neurological Disorders and Stroke (F31 NS108593, U01 NS094246, U24 NS109107); Howard Hughes Medical Institute

Frequency comb-referenced measurements of self- and nitrogen-broadening in the ν1 + ν3 band of acetylene

We report measurements of self- and nitrogen-pressure broadening of the P(11) line in the ν1 + ν3 combination band of acetylene at 195 739.649 5135(80) GHz by absorption of radiation emitted by an extended cavity diode laser referenced to a femtosecond frequency comb. Broadening, shift and narrowing parameters were determined at 296 K. For the most appropriate, hard collision, model in units of cm-1/atm, we find 0.146317(27), 0.047271(104) and -0.0070819(22) for the acetylene self-broadening, narrowing and shift, and 0.081129(35), 0.022940(74) and -0.0088913(25) respectively, for the nitrogen-broadening parameters. The uncertainties are expressed as one standard deviation (in parenthesis) in units of the last digit reported. These parameters are 2-3 orders of magnitude more precise than those reported in previous measurements. Similar analyses of the experimental data using soft collision and simple Voigt lineshape models were made for comparison. © 2011 Elsevier Inc. All rights reserved.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Temperature-dependent pressure broadened line shape measurements in the ν 1+ν 3 band of acetylene using a diode laser referenced to a frequency comb

Using an extended cavity diode laser referenced to a femtosecond frequency comb, the P(11) absorption line in the ν 1+ν 3 combination band of the most abundant isotopologue of pure acetylene was studied at temperatures of 296, 240, 200, 175, 165, 160, 155, and 150 K to determine pressure-dependent line shape parameters at these temperatures. The laser emission profile, the instrumental resolution, is a Lorentz function characterized by a half width at half the maximum emission (HWHM) of 8.3×10-6 cm-1 (or 250 kHz) for these measurements. Six collision models were tested in fitting the experimental data: Voigt, speed-dependent Voigt, Rautian-Sobel'man, Galatry, and two Rautian-Galatry hybrid models (with and without speed-dependence). Only the speed-dependent Voigt model was able to fit the data to the experimental noise level at all temperatures and for pressures between 3 and nearly 360 torr. The variations of the speed-dependent Voigt profile line shape parameters with temperature were also characterized, and this model accurately reproduces the observations over their entire range of temperature and pressure. © 2011 Springer-Verlag.SCOPUS: cp.jinfo:eu-repo/semantics/publishe

SPECTROSCOPY WITH COMB-REFERENCED DIODE LASERS

Author Institution: Department of Chemistry, Stony Brook University, Stony Brook, New York 11794; Homer L. Dodge Department of Physics and Astronomy, The University of Oklahoma, Norman, OK 72019-2061Extended cavity diode lasers have been stabilized by locking to components of an erbium-doped fiber laser-based frequency comb with a 250 MHz comb spacing centered at 1.5$\mu m$. We find the Allan variance of the diode laser frequency relative to the single comb component to which it is locked is of the order of a few Hz. For the system as a whole, the absolute frequency accuracy is approximately 1.5 parts in 10$^{12}$. In order to characterize the system more completely, we have recorded saturation dip absorption spectra of several transitions in the $\nu_1 + \nu_3$ combination band of acetylene near 6530 cm$^{-1}$. We find good agreement with published absolute frequency measurements for these transitions, which have been used as secondary frequency standards in the past. Aside from extremely precise saturation dip measurements such as these, comb-stabilized lasers should permit excellent measurements of Doppler-broadened lineshapes, both to compare with theory and for analytical applications. Progress along these lines will be reported at the meeting. Acknowledgments: T. J. Sears gratefully acknowledges support from a Brookhaven National Laboratory program development grant that enabled this work and also support for research at Brookhaven National Laboratory which was carried out under Contract No. DE-AC02-98CH10886 with the U.S. Department of Energy and supported by its Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences

FREQUENCY-COMB REFERENCED, SUB-DOPPLER SPECTROSCOPY OF HOT BANDS OF ACETYLENE IN THE REGION OF THE ν1+ν3\nu_1+\nu_3 COMBINATION BAND

Author Institution: Chemistry Department, Brookhaven National Laboratory, Upton, New York 11973; Department of Chemistry, Stony Brook University, Stony Brook, New York 11794; Chemistry Department, Brookhaven National Laboratory, Upton, New York 11973; Chemistry Department, Brookhaven National Laboratory, Upton, New York 11973To take full advantage of recent improvements in the accuracy obtained in collision-induced absorption line shape measurements of the acetylene $\nu_1+\nu_3$ combination band, independent and accurate knowledge of the positions of weaker, overlapping hot band transitions is required. We have used cavity-enhanced saturation-absorption spectroscopy together with a frequency-comb referenced, extended cavity diode laser to determine Doppler-free transition frequencies for the $\nu_4$ and $\nu_5$ hot band transitions in this region . A laser intensity dependent asymmetry in the sub-Doppler saturation line shape is observed, and will be discussed in terms of the intra-cavity beam geometry. Acknowledgments: Work at Brookhaven National Laboratory was carried out under Contract No. DE-AC02-98CH10886 with the U.S. Department of Energy and supported by its Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences

Argon-Induced Pressure Broadening, Shifting, and Narrowing in the CN A²Π–X²Σ⁺ (1–0) Band

Selected isolated rotational transitions in the 1–0 band of the red A²Π–X ²Σ⁺ system in CN have been recorded with transient frequency modulation spectroscopy as a function of argon pressure up to 0.2 atm at room temperature. Line shapes were fit using Fourier transforms of a parametrized time correlation function, including Doppler and velocity-dependent collisional broadening, and collisional shifts. Deviations from Voigt line shapes can be equally well fit by modeling the narrowing with a speed-dependent collision model or with a velocity-changing collisional narrowing model. Pressure broadening coefficients were observed with little rotational state dependence, in the range of 0.070–0.075 cm^–1 atm^–1. In contrast, stronger and qualitatively different rotational state dependences are observed for both pressure-dependent blue shift coefficients and the narrowing parameters. No asymmetry in the pressure broadened lines was observed

Moored Turbulence Measurements Using Pulse-Coherent Doppler Sonar

Upper-ocean turbulence is central to the exchanges of heat, momentum, and gases across the air–sea interface and therefore plays a large role in weather and climate. Current understanding of upper-ocean mixing is lacking, often leading models to misrepresent mixed layer depths and sea surface temperature. In part, progress has been limited by the difficulty of measuring turbulence from fixed moorings that can simultaneously measure surface fluxes and upper-ocean stratification over long time periods. Here we introduce a direct wavenumber method for measuring turbulent kinetic energy (TKE) dissipation rates c from long-enduring moorings using pulse-coherent ADCPs. We discuss optimal programming of the ADCPs, a robust mechanical design for use on a mooring to maximize data return, and data processing techniques including phase-ambiguity unwrapping, spectral analysis, and a correction for instrument response. The method was used in the Salinity Processes Upper-Ocean Regional Study (SPURS) to collect two year-long datasets. We find that the mooring-derived TKE dissipation rates compare favorably to estimates made nearby from a microstructure shear probe mounted to a glider during its two separate 2-week missions for O(10⁻⁸) < ϵ < O(10⁻⁵) m² s⁻³. Periods of disagreement between turbulence estimates from the two platforms coincide with differences in vertical temperature profiles, which may indicate that barrier layers can substantially modulate upper-ocean turbulence over horizontal scales of 1–10 km. We also find that dissipation estimates from two different moorings at 12.5 and at 7 m are in agreement with the surface buoyancy flux during periods of strong nighttime convection, consistent with classic boundary layer theory

Frequency Comb-Referenced Spectroscopy in the ν₁ +ν₃ Region of Acetylene

<p>By using saturation dip absorption spectroscopy with an extended cavity diode laser locked to a frequency comb, we have measured the rest frequencies of transitions in the <em>ν</em>₄= 1 and <em>ν</em>₅= 1 hot bands in the <em>ν</em>₁+ <em>ν</em>₃combination band of acetylene. The measured line frequencies are accurate to approximately 20 kHz <em>i.e. </em>approximately one part in 10¹¹. Positions of the hot-band lines quoted in the HITRAN database, which are derived from the analysis of high-resolution FTIR spectra, are of the order of 10’s of MHz in error. These measurements were undertaken because pressure broadened lineshape measurements of rotational lines in the combination band indicated that weak underlying hot band features were not correctly accounted for on the basis of their previously reported positions. As a result, measured line profiles in the band could not be accurately fit leading to errors of up to 1% in acetylene concentrations derived from the measurements. In addition, the pressure broadened P(11) line in the <em>ν</em>₁+ <em>ν</em>₃combination band has been studied as a function of varying concentration of the absorber in nitrogen. Mixture concentrations of 1, 5 and 10% at 296 K and pressures between a few Torr and one atmosphere were made and the measurements analyzed using two different speeddependent broadening models. These experiments are designed to test the additivity of contributions to pressure broadening and shift in speed-dependent line-shape modeling, <em>i.e. </em>whether the lineshape parameters follow partial pressure weighting in the binary mixtures. P(11) is relatively isolated with respect to underlying hot band transitions and neighboring transitions of the same band, but it was found that the accurate positions of underlying hot-band transitions were crucial to the successful modeling of the observed line shapes, even though these lines are typically 100-1000 times weaker than P(11) itself and are many Doppler line widths removed from the line center.</p> <p>Acknowledgments: Work at Brookhaven National Laboratory was carried out under Contract No. DE-AC02-98CH10886 with the U.S. Department of Energy and supported by its Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences.</p