Depth-encoded synthetic aperture optical coherence tomography of biological tissues with extended focal depth.

Optical coherence tomography (OCT) has proven to be able to provide three-dimensional (3D) volumetric images of scattering biological tissues for in vivo medical diagnostics. Unlike conventional optical microscopy, its depth-resolving ability (axial resolution) is exclusively determined by the laser source and therefore invariant over the full imaging depth. In contrast, its transverse resolution is determined by the objective's numerical aperture and the wavelength which is only approximately maintained over twice the Rayleigh range. However, the prevailing laser sources for OCT allow image depths of more than 5 mm which is considerably longer than the Rayleigh range. This limits high transverse resolution imaging with OCT. Previously, we reported a novel method to extend the depth-of-focus (DOF) of OCT imaging in Mo et al.Opt. Express 21, 10048 (2013)]. The approach is to create three different optical apertures via pupil segmentation with an annular phase plate. These three optical apertures produce three OCT images from the same sample, which are encoded to different depth positions in a single OCT B-scan. This allows for correcting the defocus-induced curvature of wave front in the pupil so as to improve the focus. As a consequence, the three images originating from those three optical apertures can be used to reconstruct a new image with an extended DOF. In this study, we successfully applied this method for the first time to both an artificial phantom and biological tissues over a four times larger depth range. The results demonstrate a significant DOF improvement, paving the way for 3D high resolution OCT imaging beyond the conventional Rayleigh range.

Fiber-based polarization-sensitive OCT of the human retina with correction of system polarization distortions.

In polarization-sensitive optical coherence tomography (PS-OCT) the use of single-mode fibers causes unpredictable polarization distortions which can result in increased noise levels and erroneous changes in calculated polarization parameters. In the current paper this problem is addressed by a new Jones matrix analysis method that measures and corrects system polarization distortions as a function of wavenumber by spectral analysis of the sample surface polarization state and deeper located birefringent tissue structures. This method was implemented on a passive-component depth-multiplexed swept-source PS-OCT system at 1040 nm which was theoretically modeled using Jones matrix calculus. High-resolution B-scan images are presented of the double-pass phase retardation, diattenuation, and relative optic axis orientation to show the benefits of the new analysis method for in vivo imaging of the human retina. The correction of system polarization distortions yielded reduced phase retardation noise, and better estimates of the diattenuation and the relative optic axis orientation in weakly birefringent tissues. The clinical potential of the system is shown by en face visualization of the phase retardation and optic axis orientation of the retinal nerve fiber layer in a healthy volunteer and a glaucoma patient with nerve fiber loss.

Collecting optical coherence elastography depth profiles with a micromachined cantilever probe.

We present an experimental setup that combines optical coherence elastography depth sensing with atomic force microscope indentation. The instrument relies on a miniaturized cantilever probe that compresses a sample with a small footprint force and simultaneously collects an optical coherence tomography (OCT) depth profile underneath the indenting point. The deflection of the cantilever can be monitored via optical fiber interferometry with a resolution of 2 nm. The OCT readout then provides depth profiles of the subsurface layer deformation with 15 nm resolution and depth range of a few millimeters.

Focus-extension by depth-encoded synthetic aperture in Optical Coherence Tomography.

We present a novel method to extend the depth-of-focus of Optical Coherence Tomography (OCT). OCT is an interferometric imaging technique that provides depth-resolved scattering information. The axial resolution in OCT is provided by the coherence gate and is invariant over the full image depth. The lateral resolution is determined by the beam parameters such as wavelength and numerical aperture. The Rayleigh range determines the depth range over which the lateral resolution can be maintained. The lateral resolution is often sacrificed to maintain relatively long Rayleigh range. In this study, we propose to use a depth-encoded synthetic aperture detection scheme to extend the depth range over which a sharp focus can be maintained beyond the Rayleigh range. An annular phase plate is inserted into the light path in the sample arm, which gives rise to three separate images in a single B-scan, corresponding to three different optical path length encoded apertures. These three images are coherently summed after phase-manipulation to reconstruct a new image with a lateral resolution that is maintained over a five times larger depth range.

Three-dimensional intracellular optical coherence phase imaging.

Quantitative phase imaging has many applications for label-free studies of the nanoscale structure and dynamics of cells and tissues. It has been demonstrated that optical coherence phase microscopy (OCPM) can provide quantitative phase information with very high sensitivity. The excellent phase stability of OCPM is obtained by use of a reflection from the microscope cover glass as a local reference field. For detailed intracellular studies a large numerical aperture (N.A.) objective is needed in order to obtain the required resolution. Unfortunately, this also means that the depth of field becomes too small to obtain sufficient power from the cover glass when the beam is focused into the sample. To address this issue, we designed a setup with a dual-beam sample arm. One beam with a large diameter (filling the 1.2 N.A. water immersion objective) enabled high-resolution imaging. A second beam with a small diameter (underfilling the same objective) had a larger depth of field and could detect the cover glass used as a local phase reference. The phase stability of the setup was quantified by monitoring the front and back of a cover glass. The standard deviation of the phase difference was 0.021 rad, corresponding to an optical path displacement of 0.9 nm. The lateral and axial dimensions of the confocal point spread function were 0.42 and 0.84 µm, respectively. This makes our dual-beam setup ideal for three-dimensional intracellular phase imaging.

High speed miniature motorized endoscopic probe for optical frequency domain imaging.

We present a miniature motorized endoscopic probe for Optical Coherence Tomography with an outer diameter of 1.65 mm and a rotation speed of 3,000-12,500 rpm. This is the smallest motorized high speed OCT probe to our knowledge. The probe has a motorized distal end which provides a significant advantage over proximally driven probes since it does not require a drive shaft to transfer the rotational torque to the distal end of the probe and functions without a fiber rotary junction. The probe has a focal Full Width at Half Maximum of 9.6 µm and a working distance of 0.47 mm. We analyzed the non uniform rotation distortion and found a location fluctuation of only 1.87° in repeated measurements of the same object. The probe was integrated in a high-speed Optical Frequency Domain Imaging setup at 1310 nm to acquire images from ex vivo pig lung tissue through the working channel of a human bronchoscope.

Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning.

We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this method, the emitted fluorescence is collected in the backward direction and is sent through a phase plate that encodes the depth information into the phase of a spectrally resolved interference pattern. We demonstrate that decoding this phase information allows for depth localization accuracy better than 4 µm over a 500 µm depth-of-field. In a high numerical aperture configuration with a much smaller depth of field, a localization accuracy of tens of nanometers can be achieved. This approach is ideally suited for miniature endoscopes, where space limitations at the endoscope tip render depth scanning difficult. We illustrate the potential for 3D visualization of complex biological samples by constructing a three-dimensional volume of the microvasculature of ex vivo murine heart tissue from a single 2D scan.

Nanocolloidal albumin-IRDye 800CW: a near-infrared fluorescent tracer with optimal retention in the sentinel lymph node.

PURPOSE: At present, the only approved fluorescent tracer for clinical near-infrared fluorescence-guided sentinel node (SN) detection is indocyanine green (ICG), but the use of this tracer is limited due to its poor retention in the SN resulting in the detection of higher tier nodes. We describe the development and characterization of a next-generation fluorescent tracer, nanocolloidal albumin-IRDye 800CW that has optimal properties for clinical SN detection. METHODS: The fluorescent dye IRDye 800CW was covalently coupled to colloidal human serum albumin (HSA) particles present in the labelling kit Nanocoll in a manner compliant with current Good Manufacturing Practice. Characterization of nanocolloidal albumin-IRDye 800CW included determination of conjugation efficiency, purity, stability and particle size. Quantum yield was determined in serum and compared to that of ICG. For in vivo evaluation a lymphogenic metastatic tumour model in rabbits was used. Fluorescence imaging was performed directly after peritumoral injection of nanocolloidal albumin-IRDye 800CW or the reference ICG/HSA (i.e. ICG mixed with HSA), and was repeated after 24 h, after which fluorescent lymph nodes were excised. RESULTS: Conjugation of IRDye 800CW to nanocolloidal albumin was always about 50% efficient and resulted in a stable and pure product without affecting the particle size of the nanocolloidal albumin. The quantum yield of nanocolloidal albumin-IRDye 800CW was similar to that of ICG. In vivo evaluation revealed noninvasive detection of the SN within 5 min of injection of either nanocolloidal albumin-IRDye 800CW or ICG/HSA. No decrease in the fluorescence signal from SN was observed 24 h after injection of the nanocolloidal albumin-IRDye 800CW, while a strong decrease or complete disappearance of the fluorescence signal was seen 24 h after injection of ICG/HSA. Fluorescence-guided SN biopsy was very easy. CONCLUSION: Nanocolloidal albumin-IRDye 800CW is a promising fluorescent tracer with optimal kinetic features for SN detection.

Spectroscopy and dynamics of methyl-4-hydroxycinnamate: the influence of isotopic substitution and water complexation.

High-resolution Resonance Enhanced MultiPhoton Ionization (REMPI) and Laser Induced Fluorescence (LIF) excitation spectra of jet-cooled methyl-4-hydroxycinnamate, methyl-4-OD-cinnamate, and of their water clusters have been recorded. Whereas water complexation leads to significant linewidth narrowing, isotopic substitution does for all practical purposes not influence the excited-state dynamics. In this light, we evaluate two previously proposed decay channels of the photoexcited ππ* state involving the dissociative πσ* state (analogous to phenol) and involving the optically dark nπ* state (as concluded for para-coumaric acid). To come to an unambiguous interpretation of the REMPI studies, it has been necessary to determine ionization thresholds. For methyl-4-hydroxycinnamate and its water cluster values of 8.078 and 7.636 eV have been found. Apart from the electronic excitation studies, IR absorption studies have been performed as well. These studies provide important vibrational markers for the assignment of the various conformations that are present under molecular beam conditions, and offer a direct measure of the influence of hydrogen bonding on the properties of the hydroxyl group.

Interference in acetylene intersystem crossing acts as the molecular analog of Young's double-slit experiment.

We report on an experimental approach that reveals crucial details of the composition of singlet-triplet mixed eigenstates in acetylene. Intersystem crossing in this prototypical polyatomic molecule embodies the mixing of the lowest excited singlet state (S(1)) with 3 triplet states (T(1), T(2), and T(3)). Using high-energy (157-nm) photons from an F(2) laser to record excited-state photoelectron spectra, we have decomposed the mixed eigenstates into their S(1), T(3), T(2), and T(1) constituent parts. One example of the interpretive power that ensues from the selective sensitivity of the experiment to the individual electronic state characters is the discovery and examination of destructive interference between two doorway-mediated intersystem crossing pathways. This observation of an interference effect in nonradiative decay opens up possibilities for rational coherent control over molecular excited state dynamics.

High-resolution spectroscopy of methyl 4-hydroxycinnamate and its hydrogen-bonded water complex.

The electronic structure and spectroscopic properties of the lower excited singlet states of methyl 4-hydroxycinnamate, a model for the chromophore of the photoactive yellow protein in neutral form, have been investigated using various high-resolution gas-phase spectroscopic techniques and quantum-chemical calculations. The experiments show that under our experimental conditions the molecule can adopt four conformations with similar spectroscopic properties. From the detailed assignment of the vibrationally active modes in excitation and emission spectra, it is concluded that the S(1) and S(2) states should be assigned to the V' and V pipi* states that are characterized by, respectively, small and large contributions of the HOMO --> LUMO excitation. We find that complexation with a single water molecule affects the spectroscopic properties of methyl 4-hydroxycinnamate considerably in terms of stabilization of the lowest excited singlet state but in particular with respect to the transition intensities. The latter observation is tentatively interpreted as being caused by an increase in the oscillator strength of the respective electronic transition as well as by a rise/removal of conical intersections with the pisigma* state.

Photoelectron studies on vibronic coupling in pyrazine.

Ionization pathways from the S(1) and T(1) states of pyrazine are investigated using one- and two-photon ionization of the excited state by both resonance enhanced multiphoton ionization photoelectron spectroscopy and zero electron kinetic energy pulsed field ionization techniques. For the triplet manifold, we show that two-photon ionization of T(1) is enhanced by a vibronically induced resonance for which we determine the inducing mode and the nature of the intermediate state, as well as the (3)3s(n(-1)) Rydberg state. For the singlet manifold, we identify the mode responsible for the vibronically induced intensity of a 3p Rydberg state that was previously found to greatly perturb the 1+2(') photoelectron spectrum of S(1) by a resonance at the two-photon level.

Tagging multiphoton ionization events by two-dimensional photoelectron spectroscopy.

Two-dimensional photoelectron spectroscopy has been used to supply process-specific labels to multiphoton ionization events. Employing these tags, the authors can construct excitation and photoelectron spectra along predefined excitation routes in the neutral manifold and ionization routes to the ionic manifold from one single two-dimensional photoelectron spectrum. These results offer a novel way to elucidate the vibronic and dynamic properties of excited and ionic states.

Combined experimental-theoretical study of the lower excited singlet states of paravinyl phenol, an analog of the paracoumaric acid chromophore.

The low-lying excited singlet states of paravinyl phenol (pVP) are investigated experimentally and theoretically paying attention to their similarity to excited states of paracoumaric acid, the chromophore of the photoactive yellow protein (PYP). Resonance enhanced multiphoton ionization and laser induced fluorescence spectroscopic techniques are employed to obtain supersonically cooled, vibrationally resolved excitation and emission spectra related to the lowest (1)A'(V') excited state of pVP. Comprehensive analyses of the spectral structures are carried out by means of the equation-of-motion coupled cluster singles and doubles and time dependent density functional theory methods in combination with the linear vibronic coupling model and Franck-Condon calculations. The assignments of the spectral patterns are given, mostly in terms of excitations of totally symmetric modes. Weak activity of the non-totally-symmetric modes indicates low probability of photochemical processes in the Franck-Condon region of the (1)A'(V') state. The second (1)A'(V) and third (1)A" (Ryd) excited states of pVP are characterized with regard to their electronic structure, properties, and effects of geometry relaxations. The lengthening of the double bond relevant to the trans-cis isomerization of the PYP chromophore is found for the (1)A'(V) state. A possibility of photochemical processes and strong vibronic interactions in this state can be expected. The theoretical results for the (1)A"(Ryd) state predict that dissociation with respect to the O-H bond is possible.

Ionization potentials of fluoroindoles and the origin of nonexponential tryptophan fluorescence decay in proteins.

This work reports an explanation for the unusual monoexponential fluorescence decay of 5-fluorotryptophan (5FTrp) in single-Trp mutant proteins [Broos, J.; Maddalena, F.; Hesp, B. H. J. Am. Chem. Soc. 2004, 126, 22-23] and substantially clarifies the origin of the ubiquitous nonexponential fluorescence decay of tryptophan in proteins. Our results strongly suggest that the extent of nonexponential fluorescence decay is governed primarily by the efficiency of electron transfer (ET) quenching by a nearby amide group in the peptide bond. Fluoro substitution increases the ionization potential (IP) of indole, thereby suppressing the ET rate, leading to a longer average lifetime and therefore a more homogeneous decay. We report experimental IPs for a number of substituted indoles including 5-fluoroindole, 5-fluoro-3-methylindole, and 6-fluoroindole, along with accurate ab initio calculations of the IPs for these and 20 related molecules. The results predict the IP of 5-fluorotryptophan to be 0.19 eV higher than that of tryptophan. 5-Fluoro substitution does not measurably alter the excitation-induced change in permanent dipole moment nor does it change the fluorescent state from 1La to 1Lb. In combination with electronic structure information this argues that the increased IP and the decreased excitation energy of the 1La state, together 0.3 eV, are solely responsible for the strong reduction of electron transfer quenching. 6-Fluoro substitution is predicted to increase the IP by a mere 0.09 eV. In agreement with our conclusions, the fluorescence decay curves of 6-fluorotryptophan-containing proteins are well fit using only two decay times compared to three required for Trp.