A complication of Fourier domain optical coherence tomography (OCT) methods, such as spectral domain and swept
source OCT, is the complex conjugate ambiguity due to inverse Fourier transform of real-valued data. As a result, the
image is symmetric to the zero plane, and only half of the theoretical imaging depth range is used to avoid overlapping
"mirror images" that confuse the image. We have previously demonstrated harmonic detection in a video-rate spectral
domain OCT system using a high speed line scan camera. Harmonic detection removes the complex conjugate
ambiguity by providing the real and imaginary components of the spectral interferogram. In this work, we show that
harmonic detection is easily applied to swept source OCT to remove the complex conjugate ambiguity while maintaining the imaging performance of the original swept source instrument. Harmonic detection swept source OCT allows simultaneous experimental determination of the real and imaginary components of each spectral interferogram without the need to measure consecutive A-scans at differing phase. This harmonic detection swept source optical coherence tomography system exhibits 110 dB sensitivity, up to 55 dB dynamic range, ≥ 50 dB complex conjugate rejection, and operates at the full 16 kHz sweep rate of the swept source laser for real-time video rate imaging.
Real-time video-rate imaging using harmonically detected Fourier domain OCT is demonstrated using an 800 nm light
source and a silicon line scan camera. At an imaging rate of 11.7 B-scans (1024 pixels × 256 pixels) per second, the
measured complex conjugate artifact suppression is 30-35 dB, the sensitivity is 121 dB, and the dynamic range is about
60 dB.
The two-harmonic FD-OCT method, where the quadrature components of the spectral interferogram are obtained by
simultaneous acquisition of the first and second harmonics of the phase-modulated interferogram, is used for complex-conjugate-
resolved imaging of biological samples. The method is implemented using sampling of the phase modulated
interferogram with an integrating detector array followed by digital demodulation at the first and second harmonics. A
complex conjugate rejection ratio as high as 70 dB is achieved.
Fourier domain optical coherence tomography (FD-OCT) is an interferometric imaging technique that allows imaging to depths of a few mm in scattering biological tissues with high resolution of the order of 1-10 μm. However, the usefulness of FD-OCT is limited by background and autocorrelation interference terms that reduce the sensitivity and by phase ambiguity that halves the useful imaging depth range. These limitations can be overcome by obtaining the full, complex spectral interferogram. Simultaneous detection of the imaginary and real terms is obtained by phase modulating the reference arm of the interferometer and detecting at the first and second harmonics. A mathematical derivation of harmonically detected FD-OCT and experimental measurements showing that phase ambiguity artifacts can be suppressed by up to 70 dB are presented. The method provides efficient suppression of the complex conjugate, dc, and autocorrelation artifacts and has low sensitivity to phase noise. Beyond the removal of artifacts, the ability to obtain the full, complex interferogram is key to the development of spectrally resolved FD-OCT which would add depth-resolved spectroscopic detail to the structural information.
Backscatter gas imaging uses laser absorption spectroscopy to detect the presence of a gas by illuminating a region with light from an infrared laser and imaging the returned light. Contrast can be enhanced by comparing the back-scattered intensity on and off the absorption feature. Wavelength modulation spectroscopy can provide just such a capability, but the detector signal must be processed with a lock-in amplifier, which is incompatible or prohibitively expensive with most array detectors. Images can be recorded using a single photodiode by spatially modulating the laser or the detected image. This paper describes initial experiments to demonstrate the feasibility of a combined wavelength- and spatially- modulated gas imager. It is based on a single near-infrared laser, a single detector, lock-in detection, and a commercial micromirror array. The gases imaged include water vapor, mono-deuterated water vapor, acetylene and hydrogen cyanide. Doppler imaging is demonstrated using heterodyne detection and spatial image modulation.
The population lifetime of the amide I vibration (v10 fundamental, ca. 1650 cm-1) in the protein myoglobin in D2O has been determined by picosecond infrared pump- probe spectroscopy using the Stanford mid-infrared free electron laser to be 1.3 +/- 0.2 ps. In a glass forming mixture of deuterated glycerol and D2O, the vibrational lifetime was found to increase from 1.3 +/- 0.2 ps at 310 K to 1.8 +/- 0.2 ps at 10 K. In addition to determining the time-scale of vibrational relaxation, we also observed multi-level vibrational excitation which has implications regarding the anharmonicity and homogeneous linewidth of the mode.
A free-electron laser (FEL) microscope has been constructed to perform spatially and spectrally resolved pump/probe experiments in single living cells. Picosecond infrared FEL pulses are absorbed by the sample and rapidly converted to heat. Excitation of localized fluorescent reporter molecules using a UV/VIS probe beam leads to thermally induced alterations in the radiative signal. Fluorescence-detected infrared (FDIR) spectra are generated from regions proximal to reporter molecules by varying the FEL pump wavelength. Sub- wavelength spatial resolution is a composite function of media thermal properties and probe selectivity.
Ultra-fast time-resolved infrared spectroscopy is discussed as a tool for investigating protein dynamics. Due to continuing developments in laser technology, researchers have recently begun to take advantage of the utility of vibrational spectroscopies as structural probes in studying the fast timescale dynamics of complex macromolecular systems. The structural complexity of proteins necessitates a wide range of infrared frequencies and timescales in order to obtain a detailed understanding of their conformational dynamics. Free electron lasers are uniquely well suited to this task, as designs are possible which provide ultra-short coherent light pulses throughout the infrared spectral region. Studies planned for the Stanford Picosecond FEL Center on reaction dynamics in the trans-membrane cytochrome oxidase proteins and protein folding in polypeptides are described. These serve as examples of the contributions free electron lasers will make in the fields of biochemistry and biophysics.
William Woodruff, R. Dyer, Oloef Einarsdottir, Kristen Peterson, Page Stoutland, K. Bagley, Graham Palmer, Jon Schoonover, David Kliger, Robert Goldbeck, Timothy Dawes, Jean-Louis Martin, J.-C. Lambry, Stephen Atherton, Stefan Hubig
Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate which is formed within 20 ps after photodissociation of CO from cytochrome a3 of reduced cytochrome oxidase. This intermediate decays with the same halflife (ca. 1 microsecond(s)) as the post- photodissociation CuB+-CO species previously observed by time-resolved infrared. The transient UV-Vis spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CuB to Fea3 when CO binds to CuB, forming a cytochrome a3 species with axial ligation which differs from the reduced unliganded enzyme. The time-resolved magnetic circular dichroism results suggest that this transient is high spin and therefore five coordinate. Thus it is inferred that the ligand from CuB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed 'ligand shuttle' activity of the CuB/Fea3 pair. The authors suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme. More detail on this work is presented elsewhere.
Time-resolved infrared spectroscopy (TRIRS) has been employed to study the reactions of small molecules with the cytochrome a3-CuB site of cytochrome c oxidase (CcO). All phases of these reactions have been investigated, from ultrafast phenomena (hundreds of femtoseconds) to relatively slow processes (milliseconds). The ligation dynamics immediately following photodissociation have been studied using a TRIRS technique with time resolution of less than 1 ps. The rate of photoinitiated transfer of CO from Fea32+ to CuB+ was measured directly by monitoring the development of the transient CuB+-CO absorption. The development of a stationary CuB+ spectrum which is constant until the CO dissociates from CuB+ occurs in less than 1 ps, indicating that the photoinitiated transfer of CO is remarkably fast. This unprecedented ligand transfer rate has profound implications with regard to the structure and dynamics of the cytochrome a3-CuB site, the functional architecture of the protein and coordination dynamics in general. The photodissociation and recombination of CN- has also been studied using a real-time TRIR technique. The CN- recombination rate of 430 s-1 is consistent with a recombination pathway similar to the one we have previously proposed for CO, in which a long-lived barrier to recombination is formed by the binding of an endogenous ligand L to Fea32+. The authors suggest the rate determining step for CN- recombination is the thermal dissociation of the Fea32+-L bond.
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