Spectral domain phase microscopy (SDPM) is a function extension of spectral domain optical coherence tomography. SDPM achieves exquisite levels of phase stability by employing common-path interferometry. We discuss the theory and limitations of Doppler flow imaging using SDPM, demonstrate monitoring the thermal contraction of a glass sample with nanometer per second velocity sensitivity, and apply this technique to measurement of cytoplasmic streaming in an Amoeba proteus pseudopod. We observe reversal of cytoplasmic flow induced by extracellular CaCl2, and report results that suggest parabolic flow of cytoplasm in the A. proteus pseudopod.
"1(B)-1(C) and 1(F)-1(G), a 20 μm change in bulk path delay δz between the two interferometer arms causes the fringes to shift to a different lateral section of the field of view. The primary consequence of such a finite spectral bandwidth in the context of off-axis interferometry is that the phase sensitivity at the edges of the field of view decreases as the width of the complex envelope decreases . Several groups have investigated the use of diffractive optical elements to alleviate this effect when using a large spectral bandwidth [7,8]. "
[Show abstract][Hide abstract] ABSTRACT: Quantitative phase spectroscopy is presented as a novel method of measuring the wavelength-dependent refractive index of microscopic volumes. Light from a broadband source is filtered to an ~5 nm bandwidth and rapidly tuned across the visible spectrum in 1 nm increments by an acousto-optic tunable filter (AOTF). Quantitative phase images of semitransparent samples are recovered at each wavelength using off-axis interferometry and are processed to recover relative and absolute dispersion measurements. We demonstrate the utility of this approach by (i) spectrally averaging phase images to reduce coherent noise, (ii) measuring absorptive and dispersive features in microspheres, and (iii) quantifying bulk hemoglobin concentrations by absolute refractive index measurements. Considerations of using low coherence illumination and the extension of spectral techniques in quantitative phase measurements are discussed.
"Factors influencing phase sensitivity include mechanical stability , decorrelation , image SNR [32,33], and timing induced errors for the case of swept-source systems . An expression characterizing the lower velocity limit is given by  "
[Show abstract][Hide abstract] ABSTRACT: Recent advances in Doppler techniques have enabled high sensitivity imaging of biological flow to measure blood velocities and vascular perfusion. Here we compare spectrometer-based and wavelength-swept Doppler OCT implementations theoretically and experimentally, characterizing the lower and upper observable velocity limits in each configuration. We specifically characterize the washout limit for Doppler OCT, the velocity at which signal degradation results in loss of flow information, which is valid for both quantitative and qualitative flow imaging techniques. We also clearly differentiate the washout effect from the separate phenomenon of phase wrapping. We demonstrate that the maximum detectable Doppler velocity is determined by the fringe washout limit and not phase wrapping. Both theory and experimental results from phantom flow data and retinal blood flow data demonstrate the superiority of the swept-source technique for imaging vessels with high flow rates.
"To overcome such site-to-site variability, laser Doppler imaging  or laser speckle imaging ,  have been used to visualize heterogeneous blood flow in a relatively large tissue area. Doppler optical coherence tomography ,  has also shown to visualize the functional information of blood flow in a relatively deeper region on a complementary structural image of optical coherence tomography. Instead of the blood flow measurements, multispectral imaging has been recently developed to quantitatively monitor blood volume and oxygen saturation with a large field of view of approximately 250 mm 2 with relatively low resolution . "
[Show abstract][Hide abstract] ABSTRACT: There is emerging evidence that microvascular alterations may occur early, even at early stages of carcinogenesis, as indispensable participants in tumor growth. However, the exact spatial extents of such alterations remain unclear, in part, because detailed microvascular alterations in relatively deep tissue over a relative large area are not easily visualized. Due to the heterogeneous nature of tissue microvasculature, microscopic evaluations with a small field of view often fail to provide a representative assessment. On the other hand, conventional whole-body small-animal optical imaging techniques suffer from unwanted diffuse light, which would otherwise deteriorate image contrast and resolution. To fill such a gap, we take advantage of the high anisotropic property of biological tissue by implementing back-directional gating into an imaging platform to suppress unwanted diffuse light. We further combine a spectral analysis of microvascular hemoglobin (Hb) absorption with back-directional gated imaging to improve image resolution, contrast, and penetration depth that are required for subcutaneous mouse xenograft models. In tissue phantom and pilot animal studies, we demonstrate that our diffuse-light-suppressed spectroscopic imaging platform can be a simple, yet effective, imaging setup to visualize subcutaneous microvascular Hb content over a relatively large area.
IEEE Journal of Selected Topics in Quantum Electronics 09/2010; 16(4-16):815 - 823. DOI:10.1109/JSTQE.2009.2037160 · 2.83 Impact Factor
Elisabete Jorge, Rui Baptista, João Calisto, Henrique Faria, Pedro Monteiro, Manuel Pan, Mariano Pêgo
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