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To test the significance of diurnal changes in choroidal, Haller's and Sattler's layer thickness in healthy subjects using spatial analysis of three-dimensional (3D) 1060-nm optical coherence tomography (OCT) scans.Automatically generated choroidal, Haller's and Sattler's layer thickness maps were statistically analyzed for 19 healthy subjects at two time points (8 a.m. and 6 p.m.) that represent the currently proposed ChT peak and nadir. All subjects were imaged by high-speed 1060-nm OCT over a 36° × 36° field of view. Spatial distribution of layer thickness was analyzed using the Early Treatment Diabetic Retinopathy Study (ETDRS) grid.The choroid was significantly thicker at 8 a.m. than at 6 p.m. (p0,0125, paired t-test, Bonferroni correction). Diurnal variation of mean choroidal thickness (ChT) for all ETDRS subfields was 12 μm. Haller's layer thickness showed no significant diurnal variation (P0.0125), but Sattler's layer was thicker in the morning than in late afternoon (P0.0125).Our measurements indicate that diurnal ChT variation may exist, but is less relevant than previously proposed by studies using single location imaging. Sattler's layer shows diurnal variation in line with ChT.

The combination of photoacoustic tomography (PAT) with optical coherence tomography (OCT) has seen steady progress over the past few years. With the benchtop and semi-benchtop configurations, preclinical and clinical results have been demonstrated, paving the way for wider applications using dual modality PAT/OCT systems. However, as for the most updated semi-benchtop PAT/OCT system which employs a Fabry-Perot polymer film sensor, it is restricted to only human palm imaging due to the limited flexibility of the probe. The passband limit of the polymer film sensor further restricts the OCT source selection and reduces the sensitivity of the combined OCT system. To tackle these issues, we developed an articulated PAT/OCT probe for both preclinical and clinical applications. In the probe design, the sample arm of OCT sub-system and the interrogation part of the PAT sub-system are integrated into one compact unit. The polymer film sensor has a quick release function so that before each OCT scan, the sensor can be taken off to avoid the sensitivity drop and artefacts in OCT. The holding mechanism of the sensor is also more compact compared to previous designs, permitting access to uneven surfaces of the subjects. With the help of the articulated probe and a patient chair, we are able to perform co-registered imaging on human subjects on both upper and lower extremities while they are at rest positions. An increase in performance characteristics is also achieved. Patients with skin diseases are currently being recruited to test its clinical feasibility.

We present a novel akinetic optical ultrasound sensor, consisting of a rigid, fiber-coupled Fabry-P´erot interferometer (FPI) with a central opening. The sensing principle relies exclusively on the detection of pressure-induced changes of the refractive index in the liquid located between the cavity mirrors. This enables resonance-free, inherently linear signal detection over a large bandwidth. We demonstrate that the sensor allows to realize exceedingly low noise equivalent pressure (NEP) values of 2 Pa over a 20 MHz measurement bandwidth, while maintaining a large full field of view of 2,7mm × 1,3mm as well as a flat frequency response. Imaging tests on phantoms and biological tissue show the suitability of the XARION-sensor for optical resolution photoacoustic microscopy (OR-PAM) applications. Transparent in axial direction, the sensor facilitates the implementation of highly sensitive fast-scanning reflection-mode OR-PAM setups, as well as easy integration with other imaging modalities such as confocal microscopy or OCT.

PURPOSE To demonstrate high-speed, high axial resolution optical coherence tomography (OCT) at 1060 nm with penetration to the sclera. The clinical feasibility of dense, high-speed sampling for higher levels of detail at the macula and optic nerve head is explored with respect to motion artifacts. METHODS A three-dimensional (3D) OCT system making use of a high-speed camera operating at 47,000 depth scans/s was developed. The 1010- to 1080-nm wavelength band leads to 6.7 microm effective axial resolution and enables the acquisition of retinal and choroidal 130 Megavoxel volumes of human subjects within 7 seconds. Motion artifacts were reduced by numeric postprocessing techniques. RESULTS Drift motion artifacts could be suppressed within fields up to 38 degrees x 38 degrees (approximately 1 cm(2)) using acquisition speeds of up to 74 frames/s at 512 x 512 pixel/frame. This isotropic OCT sampling of the human retina in vivo allowed reconstruction of the retinal microvasculature solely on vessel reflectivity, without the use of contrast agents, and revealed three interconnected capillary meshworks. Simultaneously in the choroid, the structure of the choriocapillaris, Sattler's layer, and Haller's layer were differentiated, and the choroidal-scleral interface was clearly delineated in densely sampled narrow- and wide-angle scans (>38 degrees). At the optic nerve head, the 3D fine structure of the lamina cribrosa and the circle of Zinn-Haller were visualized. CONCLUSIONS OCT almost centered within the 1060-nm water transmission window significantly profits from lower scattering and allows investigation of the retina and choroid at an unprecedented combination of penetration and high speed at high resolution and may provide superior clinical feasibility to commercial 800-nm devices.

We present an ultrahigh resolution dual modality optical resolution photoacoustic microsopy (OR-PAM) and spectral domain optical coherence microscopy (SD-OCM) system. The ultrahigh sub-micron lateral resolution is provided by the high numerical aperture of the objective lens used while the ultrahigh axial resolution is provided by the broadband OCT laser that covers 107 nm with a central wavelength of 840 nm. The synchronized simultaneous acquisition for the two modalities is achieved using a 40MHz FPGA. 2D-scanning is realized by two orthogonal translation stages (PI, 400 nm resolution). The transversal resolution of the system is 0.5 μm, the axial resolutions are 30 μm (PAM) and 4 μm (OCM), respectively. The values have been determined experimentally using nanospheres (diameter 10-200nm). For a demonstration of the imaging capability we present images from thin slices of different biological samples as well as in vivo imaging in the zebrafish embryo.

Optical coherence tomography (OCT) has become an established diagnostic tool for the clinical assessment of retinal pathology but correlation of acquired signals with retinal substructures has often been ambiguous. In the monkey retina we have now obtained ultrahigh resolution (UHR) OCT images with 1.4 microm axial x 3 microm transverse resolution from perfusion-fixed eye cups of Macaca fascicularis and optimized the identification of retinal anatomy by correction of spatial artefacts in correlated histology. After resin embedding, serial semithin sections were obtained that corresponded to OCT transects. The direct overlay of features identified in histological sections with corresponding OCT locations was limited by non-linear tissue shrinkage due to dehydration and sectioning stress. In the present study, these misalignments were further corrected by using polygonal spline morphing based on corresponding unequivocal landmarks. The geometric normalization then allowed detailed comparison of both profiles including delicate sublayers of photoreceptor inner- and outer segments. Such correlation will facilitate the extraction of structural information from in vivo ultrahigh resolution OCT images in clinical and experimental applications.

Novel ultra-broad bandwidth light sources enabling unprecedented sub-2 microm axial resolution over the 400 nm-1700 nm wavelength range have been developed and evaluated with respect to their feasibility for clinical ultrahigh resolution optical coherence tomography (UHR OCT) applications. The state-of-the-art light sources described here include a compact Kerr lens mode locked Ti:sapphire laser (lambdaC = 785 nm, delta lambda = 260 nm, P(out) = 50 mW) and different nonlinear fibre-based light sources with spectral bandwidths (at full width at half maximum) up to 350 nm at lambdaC = 1130 nm and 470 nm at lambdaC = 1375 nm. In vitro UHR OCT imaging is demonstrated at multiple wavelengths in human cancer cells, animal ganglion cells as well as in neuropathologic and ophthalmic biopsies in order to compare and optimize UHR OCT image contrast, resolution and penetration depth.

Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors’ knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

Detection of acoustic waves is the cornerstone of photoacoustic tomography (PAT). Detection has conventionally been performed mechanically using ultrasonic transducers, or optically by interferometric techniques. We propose an interferometric detection scheme using low coherence interferometry (LCI) and discuss the challenges, advantages and limitations of applying this technique to photoacoustics.

The dispersion mismatch between sample and reference arm in frequency-domain OCT can be used to iteratively suppress complex conjugate artifacts and thereby increase the imaging range. We propose a fast dispersion encoded full range (DEFR) algorithm that detects multiple signal components per iteration. The influence of different dispersion levels on the reconstruction quality is analyzed for in vivo retinal tomograms at 800 nm. Best results have been achieved with about 30 mm SF11, with neglectable resolution decrease due to finite resolution of the spectrometer. Our fast DEFR algorithm achieves an average suppression ratio of 55 dB and converges within 5 to 10 iterations. The processing time on non-dedicated hardware was 5 to 10 seconds for tomograms with 512 depth scans and 4096 sampling points per depth scan. Application of DEFR to the more challenging 1060 nm wavelength region is demonstrated by introducing an additional optical fibre in the sample arm.

A novel non-invasive in vivo multimodal optical coherence tomography (OCT)/photoacoustic tomography (PAT) imaging system capable of obtaining structural and functional information simultaneously has been demonstrated in skin. A 1060 nm OCT system acquiring 47k depth-scans/s with ~ 7 μm axial and ~ 20 μm transverse resolutions has been incorporated into a backward-mode PA system based on a planar, optically-transparent Fabry-Perot interferometer (FPI) sensor. In this study, the excitation wavelength was set to 670 nm and a focused laser beam at 1550 nm was used as the sensor interrogation beam. OCT and PAT images were obtained sequentially and the coregistered images were combined to form the final 3D image. OCT/PAT images obtained in vivo from the skin of a hairless mouse and human palmar skin demonstrated the ability of this multimodal imaging system to provide complementary structural and functional information from deeper depths with increased contrast.

Visualization of cell migration during chemotaxis using spectral domain optical coherence tomography (OCT) requires non-standard processing techniques. Stripe artefacts and camera noise floor present in OCT data prevent detailed computer-assisted reconstruction and quantification of cell locomotion. Furthermore, imaging artefacts lead to unreliable results in automated texture based cell analysis. Here we characterize three pronounced artefacts that become visible when imaging sample structures with high dynamic range, e.g. cultured cells: (i) time-varying fixed-pattern noise; (ii) stripe artefacts generated by background estimation using tomogram averaging; (iii) image modulations due to spectral shaping. We evaluate techniques to minimize the above mentioned artefacts using an 800 nm optical coherence microscope. Effect of artefact reduction is shown exemplarily on two cell cultures, i.e. Dictyostelium on nitrocellulose substrate, and retinal ganglion cells (RGC-5) cultured on a glass coverslip. Retinal imaging also profits from the proposed processing techniques. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

In this study, depth resolved measurements of absorption profiles in the wavelength range of 800 nm with a bandwidth of 140 nm are demonstrated using high speed spectroscopic frequency domain OCT (SOCT). With proper calibration, SOCT is able to extract absolute, depth resolved absorption profiles over the whole wavelength range at once without the need of tuning and performing measurements at single wavelengths sequentially. In addition, high acquisition speed in excess of 20 kHz allows to measure absorption dynamics with 50 µs time resolution, which might be useful for the investigation of pharmacokinetics or pharmacodynamics. SOCT of ∼600 µm thick single- and multilayered, weakly scattering phantoms with varying absorption in the range of 10–60 cm-1, equivalent to blood absorption in capillaries, is presented. SOCT measurements are compared with those using a spectrometer in transmission mode. For Indocyanine Green (ICG), a dynamic absorption measurement is demonstrated.

Ultrahigh axial resolution surface profiling as well as volumetric optical imaging based on time encoded optical coherence tomography in the frequency domain without any mechanical scanning element is presented. A frequency tuned broad bandwidth titanium sapphire laser is interfaced to an optical microscope (Axioskop 2 MAT, Carl Zeiss Meditec) that is enhanced with an interferometric imaging head. The system is equipped with a 640 x 480 pixel CMOS camera, optimized for the 800 nm wavelength tuning range for transmission and reflection measurements of a microscopic sample. Sample volume information over 1.3 x 1 x 0.2 mm(3) with ~3 mum axial and ~4 mum transverse resolution in tissue is acquired by a single wavelength scan over more than 100 nm optical bandwidth from760 to860 nm with 128-2048 equidistant optical frequency steps with an acquisition time of 1 to 50 ms per step. Topography and tomography with a signal to noise ratio of 83 dB is demonstrated on test surfaces and biological specimen respectively. This novel OCT technique promises to enable high speed, three dimensional imaging by employing high frame rate cameras and state of the art tunable lasers in a mechanically stable environment, due to lack of moving components while reducing the intensity on the sample.

For the first time in vivo retinal imaging has been performed with a new compact, low noise Yb-based ASE source operating in the 1 microm range (NP Photonics, lambdac = 1040 nm, Deltalambda = 50 nm, Pout = 30 mW) at the dispersion minimum of water with ~7 microm axial resolution. OCT tomograms acquired at 800 nm are compared to those achieved at 1040 nm showing about 200 microm deeper penetration into the choroid below the retinal pigment epithelium. Retinal OCT at longer wavelengths significantly improves the visualization of the retinal pigment epithelium/choriocapillaris/choroids interface and superficial choroidal layers as well as reduces the scattering through turbid media and therefore might provide a better diagnosis tool for early stages of retinal pathologies such as age related macular degeneration which is accompanied by choroidal neovascularization, i.e., extensive growth of new blood vessels in the choroid and retina.

Conventionally, cell chemotaxis is studied on two-dimensional (2D) transparent surfaces, due to limitations in optical and image data-collection techniques. However, surfaces that more closely mimic the natural environment of cells are often opaque. Optical coherence tomography (OCT) is a noninvasive label-free imaging technique, which offers the potential to visualize moving cells on opaque surfaces and in three dimensions (3D). Here, we demonstrate that OCT is an effective means of time-lapse videomicroscopy of Dictyostelium cells undergoing 3D (2D+time) cell migration on nitrocellulose substrates and 4D (3D+time) chemotaxis within low-density agarose gels. The generated image sequences are compatible with current computer-based image-analysis software for quantification of cell motility. This demonstrates the utility of OCT for cell tracking and analysis of cell chemotaxis in complex environments. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

We propose an algorithm that effectively cancels complex conjugate mirror terms from single OCT A-scans by utilizing the dispersion mismatch between reference and sample arm to generate full range tomograms. This allows distinguishing between complex conjugate mirror terms and real structures and is therefore called dispersion encoded full range (DEFR). Whereas the computational complexity is higher, acquisition speed is not compromised since no additional A-scans need to be measured which makes this technique also robust against phase fluctuations. The iterative algorithm uses numeric dispersion compensation and exhibits no reduction in resolution compared to standard processing. Residual leakage of mirror terms is reduced by incorporating further knowledge such as the power spectrum of the light source. The suppression ratio of mirror signals is more than 50 dB and thus comparable to other complex FD-OCT techniques which use more than one A-scan.

In this study, depth resolved measurements of absorption pro“les in the wavelength range of 800 nm with abandwidth of 140 nm are demonstrated using high speed spectroscopic frequency domain OCT (SOCT). Withproper calibration, SOCT is able to extract absolute, depth resolved absorption pro“les over the whole wavelengthrange at once without the need of tuning and performing measurements at single wavelengths sequentially. Inaddition, high acquisition speed in excess of 20 kHz allows to measure absorption dynamics with 50 µ s timeresolution, which might be useful for the investigation of pharmacokinetics or pharmacodynamics.SOCT of 600 µ m thick single- and multilayered, weakly scattering phantoms with varying absorption inthe range of 10 60 cm 1 , equivalent to blood absorption in capillaries, is presented. SOCT measurements arecompared with those using a spectrometer in transmission mode. For Indocyanine Green (ICG), a dynamicabsorption measurement is demonstrated.Keywords: optical coherence tomography, spectroscopy, depth-resolved, functional imaging

Using high speed ultrahigh resolution optical coherence tomography (OCT) at 800nm, non-invasive 3D cellular imaging has been accomplished. Cellular resolution imaging on and within these types of substrates is not possible with conventional microscopy techniques such as interference contrast microscopy, and requires the use of fluorescent staining. It is possible to achieve data acquisition rates of 20,000 samples per second with OCT which, in combination with its high axial and transverse resolution (>2-3μm), allows it to be used as a non-invasive technique to analyze cell migration in 3D with time. Comparatively high penetration depth also makes OCT a uniquely suited imaging technique for visualization of cells within a 3D construct. In this paper it is demonstrated that it is possible to resolve ~10μm Dictyostelium discoideum cells, a well established and useful model for investigation of cell motility and chemotaxis, in 3D and follow them in time lapse using an 800nm ultrahigh resolution high speed frequency domain based OCT microscope. Ultimately, these visualization techniques could enable monitoring of cell behavior in regenerative medicine, for example tracking of individual cells within a cell scaffold.

We present in vivo frequency domain optical coherence tomography of the human retina and choroid in the 1060 nm water transmission window with 72 nm optical bandwidth (

Purpose To demonstrate acquisition and analysis methods for depth-resolved observation of slow retinal physiology induced changes in infrared backscatter in vivo. Methods A dark-adapted human was briefly subjected to a localized photobleach. For 20 min before and 30 min after the stimulus, volumetric optical coherence tomograms were collected partially overlapping the bleached region. Tomograms were segmented into retinal layers by a newly described algorithm exploiting information in adjacent B-scans. En face fundus images extracted from major intraretinal layers were laterally registered manually. Time series summarizing the observed backscatter in selected layers for the bleached and unbleached areas are shown with a variety of corrections and normalizations applied: tomograms were corrected for inherent sensitivity roll-off, and the ratio between other layers and an assumed unchanging layer (retinal pigment epithelium), as well as the ratio of the stimulated area to the unstimulated area, were calculated. Results Adjacent B-scan information allows a simpler segmentation algorithm to be used. Sensitivity roll-off correction reduces signal variability due to eye motion. After normalizations, the signal correlated with the stimulus appears strongest at the photoreceptor inner-outer segment junction. Conclusions Demonstrated methods manage data complexity and reduce uncorrelated signal variability. This single trial warrants further investigation of intrinsic optical signals to observe slow physiologic responses.

Biomedical Imaging Group, School of Optometry & Vision Sciences,Cardiff University, Wales, UKAbstract. Ultra-high speed optical coherence tomography employing an ultra-broadband lightsource has been combined with adaptive optics utilizing a single high stroke deformable mirrorandchromaticaberrationcompensation. Thereductionofmotionartefacts,geometricandchro-matic aberrations (pancorrection) permits to achieve an isotropic resolution of 2 3 µminthehuman eye. The performance of this non-invasive imaging modality enables to resolve cellularstructuresincludingconephotoreceptors, nerve“brebundlesandcollagenousplatesofthelam-ina cribrosa, and retinal pigment epithelial (RPE) cells in the human retina in vivo with superiordetail. Alterations of cellular morphology due to cone degeneration in a colour-blind subjectare investigated in ultra-high resolution with selective depth sectioning for the “rst time.Keywords: adaptive optics, achromatizer, pancorrection, photoreceptors, retinal pigment ep-ithelial (RPE) cells, lamina cribrosa, colour-blind, retinal pathology

The amount of light backscattered from neural tissues changes as a function of activation and is the basis of intrinsic optical signal (IOS). Typically IOS is observed by looking at darkfield scatter with a CCD camera. The retina is a calculating portion of the central nervous grey matter with an optical quality window, and is likewise easily stimulated optically. Therefore the retina makes a natural model for observing neural interactions with optical tools. Optical coherence tomography (OCT) is an imaging modality that provides depth resolved maps of the amount of light backscattered from tissue that has wide clinical use for observing structural defects associated with ophthalmic disease. It is therefore also natural to use OCT as means to observe intrinsic optical signal in the retina. Indeed, OCT has recently been used to observe an increase in backscatter at the level of the outer photoreceptor segment after bleaching light stimulation in an excised rabbit retina. We are currently attempting to translate this result towards a diagnostic technique for photoreceptor dysfunction in human patients. Currently patient motion and physiological noise present barriers that must be overcome with increases in technological and experimental sophistication. This proceeding reviews current understanding of retinal intrinsic optical signal and discusses its measurement challenge.

Cellular in vivo visualization of the three dimensional architecture of individual human foveal cone photoreceptors is demonstrated by combining ultrahigh resolution optical coherence tomography and a novel adaptive optics modality. Isotropic resolution in the order of 2-3 microm, estimated from comparison with histology, is accomplished by employing an ultrabroad bandwidth Titanium:sapphire laser with 140 nm bandwidth and previous correction of chromatic and monochromatic ocular aberrations. The latter, referred to as pancorrection, is enabled by the simultaneous use of a specially designed lens and an electromagnetically driven deformable mirror with unprecedented stroke for correcting chromatic and monochromatic aberrations, respectively. The increase in imaging resolution allows for resolving structural details of distal elements of individual foveal cones: inner segment zones--myoids and ellipsoids--are differentiated from outer segments protruding into pigment epithelial processes in the retina. The presented technique has the potential to unveil photoreceptor development and pathogenesis as well as improved therapy monitoring of numerous retinal diseases.

Spectrometer based optical coherence tomography at 1050nm with >70nm of spectral bandwidth, acquiring 48k depth scans per second for deeper penetration and isotropic visualisation of the full three dimensional choroidal vasculature is demonstrated in vivo.

The integration of adaptive optics and an achromatizing lens into an ultra-high resolution OCT system enables three-dimensional imaging of photoreceptor morphology. Optophysiology might lead to the extraction of depth-resolved information about retinal function.

Optical coherence tomography (OCT) with unprecedented submicrometer axial resolution achieved by use of a photonic crystal fiber in combination with a compact sub-10-fs Ti:sapphire laser (Femtolasers Produktions) is demonstrated for what the authors believe is the first time. The emission spectrum ranges from 550 to 950 nm (lambda(c)=725 nm , P(out)=27 mW) , resulting in a free-space axial OCT resolution of ~0.75 mum , corresponding to ~0.5 mum in biological tissue. Submicrometer-resolution OCT is demonstrated in vitro on human colorectal adenocarcinoma cells HT-29. This novel light source has great potential for development of spectroscopic OCT because its spectrum covers the absorption bands of several biological chromophores.

Purpose: Optical coherence tomography is a well established imaging modality for the retina at 800 nm, but is limited in its capability to visualize through scattering tissue, like through cataract or into the choroid. Methods: Utilizing the lower scattering at longer wavelengths (1050 nm) high speed (>17 kHz), high axial resolution (~7 μm) optical coherence tomography (OCT) demonstrates its abil-ity to exploit the three dimensional anatomic microstructure of the choroid. Volumetric images, obtained with spectrometer based frequency domain OCT system are presented and compared with their counterparts acquired at the commonly used 800 nm range. Results: Tomograms of normals and patients with pathologic morphological changes of the retinal structure as well as with cataract depict improved signal strength and image contrast at the longer wavelength. The deeper penetration allows reconstruction of the choroidal vascular structure despite strong scattering of superficial tissues at the retinal pigment epithelium, the choriocapillaris also including pathologic retinal thickening. Visualisation of modifications in the choroidal vascular structure associated with wet age related macu-lar degeneration or diabetic retinopathy indicate the diagnostic potential of this technique. Furthermore, being well outside the spectral sensitivity of the eye the wavelength band at 1050 nm also allows for depth resolved profiling of reflectivity changes to directly moni-tor light stimulus related retinal activity, without interference due to the sampling OCT beam. Conclusions: Imaging at 1050 nm enables not only to track morphological but also functional changes of the retina.

Objective imaging of the optic nerve structure has become central to the management of patients with glaucoma. There is an urgent need in diagnosis and staging for reliable objective precursors and markers. Three-dimensional ultrahigh-resolution frequency domain optical coherence tomography (3D UHR OCT) holds particular promise in this respect since it enables volumetric assessment of intraretinal layers including tomographic data for the retinal nerve fiber layer (RNFL) and optic nerve head. The integrated analysis of this information and the resolution advantage has enabled the development of more informative indices of axonal damage in glaucoma compared with measurements of RNFL thickness and cup-to-disc ratio provided by commercial OCT devices. The potential for UHR OCT in enabling the combined analysis of tomographic and volumetric data on retinal structure is explored. A novel parameter was developed; the three-dimensional minimal distance as the optical correlate of true retinal nerve fiber layer thickness around the optic nerve head region. For the purposes of this pilot study, we present data from a normal subject and from two patients with characteristic optic nerve and retinal nerve fiber layer changes secondary to glaucoma.

Current signal post processing in spectrally encoded frequency domain (FD) optical coherence microscopy (OCM) and optical coherence tomography (OCT) uses Fourier transforms in combination with non-uniform resampling strategies to map the k-space data acquired by the spectrometer to spatial domain signals which are necessary for tomogram generation. We propose to use a filter bank (FB) framework for the remapping process. With our new approach, the spectrometer is modeled as a critically sampled analysis FB, whose outputs are quantized subband signals that constitute the k-space spectroscopic data. The optimal procedure to map this data to the spatial domain is via a suitably designed synthesis FB which has low complexity. FB theory additionally states that 1) it is possible to find a synthesis FB such that the overall system has the perfect reconstruction (PR) property; 2) any processing on critically sampled subband signals (as done in current schemes) results in aliasing artifacts. These perspectives are evaluated both theoretically and experimentally. We determine the analysis FB corresponding to our FD-OCM system by using a tunable laser and show that for our grating-based spectrometer - employing a CCD-line camera - the non-uniform resampling together with FFT indeed causes aliasing terms and depth dependent signal attenuation. Furthermore, we compute a finite impulse response based synthesis FB and assess the desired PR property by means of layered samples. The resulting images exhibit higher resolution and improved SNR compared to the common FFT-based approach. The potential of the proposed FB approach opens a new perspective also for other spectroscopic applications.

Endoscopic ultrahigh-resolution optical coherence tomog- raphy OCT enables collection of minimally invasive cross-sectional images in vivo, which may be used to facilitate rapid development of reliable mouse models of colon disease as well as assess chemopre- ventive and therapeutic agents. The small physical scale of mouse colon makes light penetration less problematic than in other tissues and high resolution acutely necessary. In our 2-mm diameter endo- scopic time domain OCT system, isotropic ultrahigh-resolution is sup- ported by a center wavelength of 800 nm and full-width-at-half- maximum bandwidth of 150 nm mode-locked titanium:sapphire laser combined with 1:1 conjugate imaging of a small core fiber. A pair of KZFSN5/SFPL53 doublets provides excellent color correction to support wide bandwidth throughout the imaging depth. A slight deviation from normal beam exit angle suppresses collection of the strong back reflection at the exit window surface. Our system achieves axial resolution of 3.2 m in air and 4.4-m lateral spot diameter with 101-dB sensitivity. Microscopic features too small to see in mouse tissue with conventional resolution systems, including colonic crypts, are clearly resolved. Resolution near the cellular level is potentially capable of identifying abnormal crypt formation and dysplastic cellular organization. © 2006 Society of Photo-Optical Instrumentation

Noninvasive label-free imaging of biological systems raises demand not only for high-speed three-dimensional prescreening of morphology over a wide-field of view but also it seeks to extract the microscopic functional and molecular details within. Capitalizing on the unique advantages brought out by different nonlinear optical effects, a multimodal nonlinear optical microscope can be a powerful tool for bioimaging. Bringing together the intensity-dependent contrast mechanisms via second harmonic generation, third harmonic generation and four-wave mixing for structural-sensitive imaging, and single-beam/single-pulse coherent anti-Stokes Raman scattering technique for chemical sensitive imaging in the finger-print region, we have developed a simple and nearly alignment-free multimodal nonlinear optical microscope that is based on a single wide-band Ti:Sapphire femtosecond pulse laser source. Successful imaging tests have been realized on two exemplary biological samples, a canine femur bone and collagen fibrils harvested from a rat tail. Since the ultra-broad band-width femtosecond laser is a suitable source for performing high-resolution optical coherence tomography, a wide-field optical coherence tomography arm can be easily incorporated into the presented multimodal microscope making it a versatile optical imaging tool for noninvasive label-free bioimaging.

Three-dimensional ultrahigh resolution optical coherence tomography (UHR OCT) and adaptive optics (AO) are combined using a liquid crystal programmable phase modulator (PPM) as a correcting device for the first time. AO is required for correcting ocular aberrations in moderate and large pupils in order to achieve high resolution retinal images. The capabilities of the PPM are studied using polychromatic light. Volumetric UHR OCT images of the living retina with AO, obtained with up 25000 A scans/s and high resolution (~5x5x3 µm; transverse (x) x transverse (y) x axial) are recorded, enabling visualization of interesting intraretinal morphological structures. Cellular retinal features, which might correspond to groups of terminal bars of photoreceptors at the level of the external limiting membrane, are resolved. Benefits and limitations of the presented technique are finally discussed.

A functional extension of ultrahigh resolution OCT (UHR OCT) has been developed, that has the potential to establish this technique as an optical analogue to electrophysiology, by detecting depth resolved variations in optical backscattering caused by physiological tissue changes. This technique has been used to perform in vitro studies on excised, but physiologically intact, rabbit retinas and in vivo experiments on human retinas. UHR OCT has been synchronized with the white light stimulus to properly detected spatially resolved alterations in optical backscattering over time caused by lightinduced intraretinal, physiological changes and has been correlated with simultaneous ERG recordings. Preliminary results demonstrate the potential of this novel extension of UHR OCT to detect time-dependent optical backscattering changes after application of a white light stimulus in specific retinal layers, especially in the inner and outer segments of the photoreceptor layer. Control experiments, including no light stimulus or application of drugs (in in vitro studies only) that inhibit the physiological responses of certain type of retinal cells confirm the physiological origin of the detected backscattering changes. Detection of cell activity and cell physiology by UHR OCT would enable a better understanding of basic physiological phenomena and may also contribute to better understanding of retinal pathogenesis.

The prospects to combine AO and OCT both theoretically and experimentally are presented. The possibility to significantly improve the performance of ophthalmic UHR OCT systems by means of AO is proved. Moreover, the expected transversal resolution when using larger pupil sizes in the eye in combination with AO should enable the study for the first time of many retinal structures never detected before in the living retina

An ultra-broadband, high spectral resolution acousto-optically tunable light-source for the near infrared has been developed for high acquisition speed, ultrahigh-resolution frequency domain optical coherence tomography. Performance and imaging results are compared with different tuning methods

purpose. To demonstrate a new generation of three-dimensional (3-D) ultrahigh-resolution optical coherence tomography (UHR OCT) technology for visualization of macular diseases. methods. One hundred forty eyes with a distinct disease in each of the posterior pole compartments were examined with 3-D UHR OCT. 3-D imaging was performed with a high axial resolution of 3 ?m with a compact, commercially available, ultra–broad-bandwidth (160 nm) titanium:sapphire laser at a video rate of up to 25 B-scans/s. Each tomogram consisted of 1024 × 1024 pixels, resulting in 25 megavoxels/s. results. 3-D UHR OCT offers high-precision 3-D visualization of macular diseases at all structural levels. The UHR modality allows identification of the contour of the hyaloid membrane, tractive forces of epiretinal membranes, and changes within the inner limiting membrane. The system provides quality 3-D images of the topographic dynamics of traction lines from the retinal surface down to the level of the photoreceptor segments. Intraretinal diseases are identified by their specific location in different layers of the neurosensory ultrastructure. Photoreceptor inner and outer segments are clearly delineated in configuration and size, with a characteristic peak in the subfoveal area. The microarchitecture of choroidal neovascularization is distinctly imaged, related leakage can be identified, and the volume can be quantified. conclusions. High-speed UHR OCT offers unprecedented, realistic, 3-D imaging of ocular diseases at all epi-, intra- and subretinal levels. A complete 3-D data set of the macular layers allows a comprehensive analysis of focal and diffuse diseases, as well as identification of dynamic pathomechanisms.

A liquid crystal programmable phase modulator (PPM) is used as correcting device in an adaptive optics system for three-dimensional ultrahigh-resolution optical coherence tomography (UHR OCT). The feasibility of the PPM to correct high order aberrations even when using polychromatic light is studied, showing potential for future clinical use. Volumetric UHR OCT of the living retina, obtained with up 25,000A-scans/s and high resolution enables visualization of retinal features that might correspond to groups of terminal bars of photoreceptors at the external limiting membrane.

Fourier Domain Optical Coherence Tomography (FD OCT) recently gained large reputation as a high speed imaging modality with increased sensitivity. Since the spectrum of the backscattered light is directly available, the method is a natural candidate for performing spectroscopic tissue analysis. Spectroscopic data is extracted by applying a windowed Fourier transform. The precision of the method is analyzed by measuring the absorbance of ICG samples sandwiched between microscope glass plates. A Ti:Sapph laser serves as light source with a spectral bandwidth of 160nm. For spectral analysis of tissue two distinct spectral regions are selected by multiplying the FD OCT signal spectrum with appropriate Gaussian window functions. One obtains two tomograms that enhance slightly different structures. Finally light sources of different spectral widths are synthesized and the resulting OCT tomograms that exhibit different axial resolutions are compared with respect to image quality and structure recognition.

Mouse models are increasingly important for studying human GI pathology. OCT provides minimally invasive, cross-sectional images that indicate the thickness and scattering density of underlying tissue. We have developed endoscopic ultrahigh resolution OCT (UHR-OCT) for the purpose of in vivo imaging in mouse colon. The reduced scale of the mouse colon makes tissue light penetration much less problematic, and high resolution acutely necessary. Higher lateral resolution requires a departure from the traditional cemented GRIN lens design. We support the need for better chromatic aberration than can be achieved by a GRIN lens using commercial raytracing software. We have designed and built a 2mm diameter endoscopic UHR-OCT system achromatized for 770-1020nm for use with a Titanium:sapphire laser with 260 nm bandwidth at full-width-half-maximum centered at 800 nm while achieving a 4.4um lateral spot dimension at focus. A pair of KZFSN5/SFPL53 doublets provides excellent primary and secondary color correction to maintain wide bandwidth through the imaging depth. A slight deviation from normal beam exit angle suppresses collection of the strong back reflection at the exit window surface. The novel design endoscope was built and characterized for through focus bandwidth, axial resolution, signal to noise, and lateral spot dimension. Performance is demonstrated on a variety of ex vivo tissues and in situ mouse colon. Ultrahigh-resolution images of mouse tissue enable the visualization of microscopic features, including crypts that have previously been observed with standard resolution OCT in humans but were too small to see in mouse tissue. Resolution near the cellular level is potentially capable of identifying abnormal crypt formation and dysplastic cellular organization.

In vivo ultrahigh resolution ophthalmic OCT has been performed in more than 300 eyes of 200 patients with several retinal pathologies, demonstrating unprecedented visualization of all major intraretinal layers, in particular the photoreceptor layer. Visualization as well as quantification of the inner and outer segment of the photoreceptor layer especially in the foveal region has been acvhieved. In normal subjects the photoreceptor layer thickness in the center of the fovea is about of 90 μm, approximately equally distributed to the inner and the outer photoreceptor segment. In the parafoveal region this thickness is reduced to ~50 μm (~30 μm for the inner and ~20 μm for the outer segment). This is in good agreement with well known increase of cone outer segments in the central foveal region. Photoreceptor layer impairment in different macular pathologies like macular hole, central serous chorioretinopathy, age related macular degeneration, foveomacular dystrophies, Stargardt dystrophy as well as retinitis pigmentosa has been investigated. Photoreceptor layer loss significantly correlated with visual acuity (R2 = 0.6, p < 0.001) and microperimetry findings for the first time in 22 eyes with Stargardt dystrophy. Visualization and quantification of photoreceptor inner and outer segment using ultrahigh resolution OCT has the potential to improve early ophthalmic diagnosis, contributes to a better understanding of pathogenesis of retinal diseases as well as might have impact in the development and monitoring of novel therapy approaches.

The feasibility of ultrahigh resolution optical coherence tomography to image both healthy and pathological brain tissue morphology as well as the morphology and functional response of neuron cells is investigated.

The morphology of healthy and pathological human brain tissue, as well as the brain structural organization of various animal models has been imaged in-vitro using ultrahigh resolution optical coherence tomography (UHR OCT). Micrometer-scale OCT resolution (< 2 µm axial resolution) was achieved at different central wavelengths by interfacing three state-of-the-art broad bandwidth light sources (Ti:Al2O3, λc = 790 nm, ∆λ = 260 nm and Pout = 50 mW; PCF based laser, λc = 1150 nm, ∆λ = 350 nm and Pout = 2 W; Fiber laser based light source, λc = 1350 nm, ∆λ = 470 nm and Pout = 4 mW) to a modular free-space OCT system, utilizing a dynamic focusing and designed for optimal performance in the appropriate wavelength regions. Images acquired from a fixed honeybee brain demonstrated the ability of UHR OCT to image the globular structure of the brain, some fine morphological details such as the nerve fiber bundles connecting the medulla (visual center) to the honeybee eyes, and the interfaces between different tissue layers in the medulla. Tomograms of various human neuropathologies demonstrated the feasibility of UHR OCT to visualize morphological details such as small (~20 μm) calcifications typical for fibrous meningioma, and enlarged nuclei of cancer cells (~10-15 µm) characteristic for many other neuropathologies. In addition UHR OCT was used to image cellular morphology in living ganglion cells.

Ultrahigh axial resolution OCT is demonstrated in human cells and other human biopsies for two fiber broadened femtosecond light sources, achieving 0.5μm axial resolution in the visible and 1.4μm in the in the 1300nm wavelength region.

Summary from only given. It is expected that this visible light OCT system will provide sub-micrometer axial resolution and spectroscopic OCT imaging in biological tissue, since its emission bandwidth of the PCF light source overlaps with the so called therapeutic window, covering absorption features of several biological chromophores, like melanin, oxy- and deoxyhemoglobin as well as water. Furthermore high resolution ophthalmologic OCT imaging with enhanced penetration and spectroscopic OCT for differential water absorption measurements in the 900-1100 nm wavelength region should also be enabled with this novel OCT system.

Ophthalmic diagnosis represents one of the most clinically developed optical coherence tomography (OCT) applications1,2. Although standard ophthalmic OCT with 10-15 µm axial resolution provides more detailed structural information than any other ophthalmic diagnostic technique, many of the early pathologic changes associated with disease are still below the resolution limit of standard OCT. Ultrahigh resolution OCT imaging employing a laboratory prototype femtosecond Titanium-sapphire laser light source has been demonstrated to achieve an axial resolution of 1-3 µm, thus enabling unprecedented in vivo sub-cellular3 as well as intraretinal4 visualization. Recently, a compact, clinically viable ultrahigh resolution (~3 µm) ophthalmic OCT system has been developed and used in clinical imaging for the first time5.

We demonstrate for the first time optical coherence tomography (OCT) in the 900-1100 nm wavelength range. A photonic crystal fiber (PCF) in combination with a sub-15fs Ti:sapphire laser is used to produce an emission spectrum with an optical bandwidth of 35 nm centered at ~1070 nm. Coupling the light from the PCF based source to an optimized free space OCT system results in ~15 micrometers axial resolution in air, corresponding to ~10 micrometers in biological tissue. The near infrared wavelength range around 1100 nm is very attractive for high resolution ophthalmologic OCT imaging of the anterior and posterior eye segment with enhanced penetration. The emission spectrum of the PCF based light source can also be reshaped and tuned to cover the wavelength region around 950-970 nm, where water absorption has a local peak. Therefore, the OCT system described in this paper can also be used for spatially resolved water absorption measurements in non-transparent biological tissue. A preliminary qualitative spectroscopic Oct measurement in D2O and H2 O phantoms is described in this paper.

We demonstrate for the first time optical coherence tomography (OCT) in the visible wavelength range with unprecedented sub-micrometer axial resolution, achieved by employing a photonic crystal fiber in combination with a sub-15fs Ti:sapphire laser (FEMTOLASERS). The shaped emission spectrum produced by the photonic crystal fiber ranges from 535 nm to 700 nm (centered at ~600 nm) resulting in ~0.9 micrometers axial OCT resolution in air corresponding to ~0.6 micrometers in biological tissue. Preliminary demonstration of the sub-micrometer resolution achieved with this visible light OCT setup is demonstrated on a 2.2 micrometers thick nitrocellulose membrane. The visible wavelength range not only enables extremely high axial resolution for OCT imaging, but also offers an attractive region for spectroscopic OCT.

This study aims to investigate the sensitivity of spectroscopic optical coherence tomography (OCT) to small changes in the absorption properties of the imaged object as well as to evaluate its ability to resolve spatial variations in the object's absorption coefficient. Spectroscopic OCT would have the advantage to provide spatially resolved spectroscopic information at multiple wavelengths across the available bandwidth of the light source in a single measurement. An ultrahigh resolution OCT system based on a Ti:sapphire source emitting in the range of 700 nm to 900 nm with an optical bandwidth of up to 165 nm was used to measure optical absorption of specially designed, non-scattering phantoms. High speed and high resolution digitization in combination with a Morlet wavelet transform was utilized to derive spectroscopic information from the full interference OCT data. Using a non scattering phantom, the preliminary results of the present work reveal the challenges that have to be overcome in order to extract spatially resolved quantitative spectroscopic information by OCT.