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In Optical Coherence tomography (OCT), dispersion mismatches cause degradation of the image resolution and are, thus, compensated accordingly. However, dispersion is specific to the material that is causing the effect and can, therefore, carry useful information regarding the composition of the samples. In this summary, we propose a novel technique for estimating tissue dispersion by calculating the cross-correlation of images acquired at different center wavelengths to estimate the shift between their features, also known as walk-off, and use that to calculate the dispersion. Since a distinct reflector is not required, this method is applicable to any sample and can even be implemented in vivo and in situ in human tissues. The proposed technique was verified ex vivo resulting in Group Velocity Dispersion (GVD) values comparable to those obtained from estimating the walk-off from a mirror, as described in the literature. The applicability to cancer diagnosis was evaluated on a small set of gastrointestinal normal and cancer OCT images. Using the statistics of the GVD estimates, tissue classification resulted in 100% sensitivity and 81% specificity (92% correct classification rate). The success of these preliminary results indicates the potential of the proposed method, which should be further investigated to elucidate its advantages and limitations. 10867

Dispersion mismatch between the two arms of an Optical Coherence Tomography (OCT) interferometer causes degradation of the image resolution. However, dispersion in tissue is specific to the chemical constituents of the cells and can therefore carry diagnostically useful information. Unfortunately, dispersion measurement techniques, presented so far in the literature, require the presence of strong distinct reflections in the OCT image which are rarely present when imaging tissues in vivo. The novel method presented here relies on the image speckle to calculate the PSF degradation and is therefore applicable to any tissue and can be implemented in vivo and in situ. The resolution degradation is estimated using a Wiener-type deconvolution and was verified ex vivo resulting in Group Velocity Dispersion (GVD) values comparable to the standard techniques. This technique has also been applied to normal and malignant samples of human colon to evaluate its applicability for cancer diagnosis. Using the statistics of the GVD estimate, the tissue classification resulted in 93% sensitivity and 100% specificity (96% correct classification rate). The success of these preliminary results indicates the potential of the proposed method which should be further.

Dispersion, a result of wavelength-dependent index of refraction variations, causes pulse-width broadening with detrimental effects in many pulsed-laser applications. It is also considered to be one of the major causes of resolution degradation in Optical Coherence Tomography (OCT). However, dispersion is material dependent and, in tissue, Group Velocity Dispersion (GVD) could be used, for example, to detect changes associated with early cancer and result in more accurate disease diagnosis. In this summary we compare different techniques for estimating the GVD from OCT images, in order to evaluate their accuracy and applicability in highly scattering samples such as muscle and adipose tissue. The methods investigated included estimation of the GVD from (i) the point spread function (PSF) degradation, (ii) the shift (walk-off) between images taken at different center wavelengths and (iii) the second derivative of the spectral phase. The measurements were degraded by the presence of strong Mie scattering and speckle noise with the most robust being the PSF degradation and the least robust the phase derivative method. If the GVD is to be used to provide sensitive diagnostic information from highly scattering human tissues, it would be preferable to use the resolution degradation as an estimator of GVD.

Dispersion, resulting from wavelength-dependent index of refraction variations, could be used to detect changes associated with cancer for early and accurate diagnosis. Different techniques for estimating the dispersion from Optical Coherence Tomography (OCT) images were investigated to evaluate their accuracy and applicability to samples such as muscle and adipose tissue. The dispersion was estimated from (i) the point spread function (PSF) degradation, (ii) the shift (walk-off) between images taken at different center wavelengths and (iii) the second derivative of the spectral phase. A novel technique, which uses a Wiener-type deconvolution algorithm to calculate the PSF degradation from the image speckle and is, therefore, applicable in vivo and in situ is also presented. This method was applied to a set of normal and cancer gastrointestinal OCT images resulting in 93% sensitivity and 73% specificity. The success of these preliminary results indicates the potential of using dispersion measurements for disease diagnosis.

In Optical Coherence tomography (OCT), dispersion mismatches cause degradation of the image resolution. However, dispersion is specific to the material that is causing the effect and can therefore carry useful information regarding the composition of the samples. In this summary, we propose a novel technique for estimating the dispersion in tissue which uses the image speckle to calculate the PSF degradation and is therefore applicable to any tissue and can be implemented in vivo and in situ. A Wiener-type deconvolution algorithm was used to estimate the image PSF degradation from the speckle. The proposed method was verified ex vivo resulting in comparable values of the Group Velocity Dispersion (GVD) as obtained by a standard estimation technique described in the literature. The applicability to cancer diagnosis was evaluated on a small set of gastrointestinal normal and cancer OCT images. Using the statistics of the GVD estimate, the tissue classification resulted in 93% sensitivity and 73% specificity (84% correct classification rate). The success of these preliminary results indicates the potential of the proposed method which should be further investigated to elucidate its advantages and limitations.

The index of refraction (n) of materials and/or tissues depends on their physical properties and serves as a source of optical contrast in imaging. The variations of the index of refraction have also been investigated for diagnostic purposes in various fields, such as hematology, oncology, etc., since they can signify disease and cell dynamic changes. Optical coherence tomography (OCT) has been used in the past to measure the index ex vivo. However, most methodologies described in the literature are not appropriate for in vivo imaging since they require either a mirror below the sample or a complicated imaging setup and algorithms. We describe a technique that uses two images, obtained at different angles, to estimate the index of refraction and can, thus, also be applied in vivo. The index of refraction is calculated from the path-length difference observed by the OCT beam at the two different angles. When a reflector is not available, the path-length difference can be estimated using image registration and the cross-correlation of adjacent A-scans. The proposed technique was validated experimentally using both clear and scattering samples. The resulting values of the index of refraction were within ∼1% of the expected. The main limitation of this technique is the effect of misalignment on the results, requiring the precision provided by an angular-resolved OCT system. These very promising results provide evidence that the dual-angle method should be further investigated and validated on human tissues so that it can be developed into a clinically useful diagnostic tool in the future.

The modulations appearing on the backscattering spectrum originating from a scatterer are related to its diameter as described by Mie theory for spherical particles. Many metrics for Spectroscopic Optical Coherence Tomography (SOCT) take advantage of this observation in order to enhance the contrast of Optical Coherence Tomography (OCT) images. However, none of these metrics has achieved high accuracy when calculating the scatterer size. In this work, Mie theory was used to further investigate the relationship between the degree of modulation in the spectrum and the scatterer size. From this study, a new spectroscopic metric, the bandwidth of the Correlation of the Derivative (COD) was developed which is more robust and accurate, compared to previously reported techniques, in the estimation of scatterer size. The self-normalizing nature of the derivative and the robustness of the first minimum of the correlation as a measure of its width, offer significant advantages over other spectral analysis approaches especially for scatterer sizes above 3 μm. The feasibility of this technique was demonstrated using phantom samples containing 6, 10 and 16 μm diameter microspheres as well as images of normal and cancerous human colon. The results are very promising, suggesting that the proposed metric could be implemented in OCT spectral analysis for measuring nuclear size distribution in biological tissues. A technique providing such information would be of great clinical significance since it would allow the detection of nuclear enlargement at the earliest stages of precancerous development.

Infrared fluorescence endoscopy (IRFE), in conjunction with an infrared fluorescent-labelling contrast agent, is a well known technique used for efficient early-stage cancer detection. In this paper we present a cost-effective ( $ $500) screening capsule prototype, which is able to detect infrared (IR) fluorescence emitted by indocyanine green (ICG) fluorophore dye. Rather than image, the capsule works as a high-sensitivity fluorometer that records fluorescence levels throughout the small intestine. The presented mixed-signal system has a small size, consumes very little power ( $\approx$ 6.3 mA) and does not require an external belt and hardware for data collection. By determining fluorescence levels in the intestine, rather than collecting images, we avoid the need for labour intensive video analysis. The whole system is contained within a compact ingestible capsule, that is sized so as to come into close contact with the intestine walls during peristalsis. Ex-vivo experiments, on ICG-impregnated swine intestine, have shown that the prototype system is able to detect low concentrations of ICG in the nanomolar and micromolar region, which is required to detect early cancer in the small intestine.

The modulations appearing on the backscattering spectrum originating from a scatterer are related to its diameter as described by Mie theory for spherical particles. Many metrics for Spectroscopic Optical Coherence Tomography (SOCT) take advantage of this observation in order to enhance the contrast of Optical Coherence Tomography (OCT) images. However, none of these metrics has achieved high accuracy when calculating the scatterer size. In this work, Mie theory was used to further investigate the relationship between the degree of modulation in the spectrum and the scatterer size. From this study, a new spectroscopic metric, the bandwidth of the Correlation of the Derivative (COD) was developed which is more robust and accurate, compared to previously reported techniques, in the estimation of scatterer size. The self-normalizing nature of the derivative and the robustness of the first minimum of the correlation as a measure of its width, offer significant advantages over other spectral analysis approaches especially for scatterer sizes above 3 μm. The feasibility of this technique was demonstrated using phantom samples containing 6, 10 and 16 μm diameter microspheres as well as images of normal and cancerous human colon. The results are very promising, suggesting that the proposed metric could be implemented in OCT spectral analysis for measuring nuclear size distribution in biological tissues. A technique providing such information would be of great clinical significance since it would allow the detection of nuclear enlargement at the earliest stages of precancerous development.

The subtle tissue changes associated with the early stages of malignancies, such as cancer, are not clearly discernible even at the current, improved, resolution of optical coherence tomography (OCT) systems. However, these changes directly affect the spectral content of the OCT image that contains information regarding these unresolvable features. Spectral analysis of OCT signals has recently been shown to provide additional information, resulting in improved contrast, directly related to scatterer size changes. Amplitude modulation–frequency modulation (AM–FM) analysis, a fast and accurate technique for the estimation of the instantaneous frequency, phase, and amplitude of a signal, can also be applied to OCT images to extract scatterer-size information. The proposed technique could make available an extremely valuable tool for the investigation of disease characteristics that now remain below the resolution of OCT and could significantly improve the technology's diagnostic capabilities. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Fourier Domain (FD) Optical Coherence Tomography (OCT) interferograms require a Fourier transformation in order to be converted to A-Scans representing the backscattering intensity from the different depths of the tissue microstructure. Most often, this transformation is performed using a discrete Fourier transform, i.e. the well-known Fast Fourier Transform (FFT). However, there are many alternatives for performing the necessary spectral conversion. Autoregressive (AR) spectral estimation techniques are one such example. The parametric nature of AR techniques offers several advantages, compared to the commonly-used FFT, including better convergence and less susceptibility to noise. They can also be adjusted to represent more or less of the signal detail depending on the order of the autoregression. These features make them uniquely suited for processing the FD OCT data. The advantages of the proposed methodology are illustrated on in vivo skin imaging data and the resolution is verified on single back-reflections from a glass surface. AR spectral estimation can be used to convert the interferograms to A-Scans while at the same time reducing the artifacts caused by high intensity back-reflections (by -20 dB) and diminishing the speckle (by -12 dB) all without the degradation in the resolution associated with other techniques.

The size-dependent spectral variations, predicted by Mie theory, have already been considered as a contrast enhancement mechanism in optical coherence tomography. In this work, a new spectroscopic metric, the bandwidth of the correlation of the derivative, was developed for estimating scatterer size which is more robust and accurate compared to existing methods. Its feasibility was demonstrated using phantoms containing polystyrene microspheres as well as images of normal and cancerous human colon. The results are very promising, suggesting that the proposed metric could be utilized for measuring nuclear size distribution, a diagnostically valuable marker, in human tissues.

A seven-zone pupil filter which can extend the depth of focus (DOF) and provide lateral super resolution in Optical Coherence Tomography is designed. Both amplitude and phase of the transmitted light beam are modified in each zone. The scalar diffraction theory is used to optimize the zone parameters. A broadband source spectrum and a Gaussian beam profile has been taken into account for the design of this filter. Several filters have been reported to extend the depth of focus (DOF) and improve the resolution but they take the assumption that a monochromatic and uniform light beam is used. With the proposed filter the DOF in OCT can be extended by 14 times while the lateral resolution can be improved by a factor of 1.47 and maintained constant within this DOF.

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A new method for lateral resolution improvement of Optical Coherence Tomography (OCT) images is presented. The improvement is independent of the delivery optics and the depth of focus. It is based on the lateral oversampling of the image. In OCT, laterally oversampled signals are backscattered signals from shifted and overlapped resolution volumes. In that way, signals from successive volumes are correlated due to the region shared by adjacent resolution volumes. Resolution can be improved by utilizing the cross correlation of signals from such overlapped volumes. The resolution can be improved by various degrees depending on which pairs of signals are used. In this method signals from all overlapped volumes are combined optimally to improve the resolution using all the available cross correlations. Preliminary results of such an approach on laterally oversampled OCT images have shown that it is possible to achieve a 3.7-fold lateral resolution improvement.

A novel technique for axial resolution improvement in Fourier domain optical coherence tomography (FDOCT) is presented. The technique is based on the deconvolution of modulated optical coherence tomography signals. In FDOCT, the real part of the Fourier transform of the interferogram is modulated by a frequency which depends on the position of the interferogram in k space. A slight numerical k shift results in a different modulation frequency. By adding two shifted signals, beating can appear in the A-scan. When the amount of shifting is appropriately selected, deconvolution of the resulting depth profile, using suitable modulated kernels, yields a narrower resolution width. A resolution improvement by a factor of ∼ 7 can be achieved without the need for a broader bandwidth light source.

A novel method for lateral resolution improvement of Optical Coherence Tomography (OCT) images, which is independent of the focusing of the delivery optics and the depth of field, is presented. This method was inspired by radar range oversampling techniques. It is based on the lateral oversampling of the image and the estimation of the locations of the multiple scatterers which contribute to the signal. The information in the oversampled images is used to estimate the locations of multiple scatterers assuming each contributes a weighted portion to the detected signal, the weight determined by the location of the scatterer and the point spread function (PSF) of the system. A priori knowledge of the PSF is not required since optimization techniques can be employed to achieve the best possible enhancement of the image resolution. Preliminary results of such an approach on laterally oversampled OCT images have shown that it is possible to achieve a two-fold lateral resolution improvement. Moreover by performing deconvolution with the new improved PSF the lateral resolution can be further improved by another factor of two for a total of 4x improvement. Such improvement can be significant, especially in cases where the Numerical Aperture (NA) of the delivery optics is limited, such as, for example, in the case of ophthalmic imaging where the optics of the eye itself limit the lateral resolution.

This manuscript presents a novel technique for axial resolution improvement in Fourier Domain Optical Coherence Tomography (FDOCT). The technique is based on the modulated deconvolution of OCT signals which results in a resolution improvement by a factor of ~ 7 without the need for a broader bandwidth light source. This method relies on a combination of two basic properties: beating, which appears when adding two signals of slightly different frequencies, and the resolution improvement, achieved by deconvolution of an OCT image with the encoded source autocorrelation function. In FDOCT the real part of the FFT of the interferogram is modulated by a frequency which depends on the shift in position of the interferogram. A slightly shifted interferogram will, therefore, result in an A-Scan which will have a different modulation frequency. Beating will appear when two such A-Scans, with an appropriately selected amount of shift, are added. Deconvolution of the resulting signal, using suitable kernels, results in a narrower resolution width.

A novel technique for axial resolution improvement of Optical Coherence Tomography (OCT) systems is proposed. The technique is based on step-frequency encoding, using frequency shifting, of the OCT signal. A resolution improvement by a factor of approximately 7 is achieved without the need for a broader bandwidth light source. This method exploits a combination of two basic principles: the appearance of beating, when adding two signals of slightly different carrier frequencies, and the resolution improvement by deconvolution of the interferogram with an encoded autocorrelation function. In time domain OCT, step-frequency encoding can be implemented by performing two scans, with different carrier frequencies, and subsequently adding them to create the encoded signal. When the frequency steps are properly selected, deconvolution of the resulting interferogram, using appropriate kernels, results in a narrower resolution width.

A novel technique for axial resolution improvement of Optical Coherence Tomography (OCT) systems is presented. The technique is based on step-frequency encoding of the OCT signal, using frequency shifting. A resolution improvement by a factor of ~ 7 is achieved without the need for a broader bandwidth light source. This method exploits a combination of two basic principles: the appearance of beating, when adding two signals of slightly different carrier frequencies, and the resolution improvement of OCT images by deconvolution of the interferogram with the encoded source autocorrelation function. In OCT, step-frequency encoding can be implemented by performing two A-scans, with different carrier frequencies and subsequently adding them to create the encoded signal. Deconvolution of the resulting interferogram, using appropriate kernels, results in a narrower resolution width when the frequency steps are appropriately selected.

A novel spectral analysis technique of OCT images is demonstrated in this paper for classification and scatterer size estimation. It is based on SOCT autoregressive spectral estimation techniques and statistical analysis. Two different statistical analysis methods were applied to OCT images acquired from tissue phantoms, the first method required prior information on the sample for variance analysis of the spectral content. The second method used k-means clustering without prior information for the sample. The results are very encouraging and indicate that the spectral content of OCT signals can be used to estimate scatterer size and to classify dissimilar areas in phantoms and tissues with sensitivity and specificity of more than 90%.

The early stages of malignant diseases, such as cancer, are characterized by cellular and microstructural changes which define both the diagnosis and the prognosis of the disease. Unfortunately, at the current resolution of Optical Coherence Tomography (OCT), such changes associated with early cancer are not clearly discernible. However, spectral analysis of OCT images has recently shown that additional information can be extracted from those signals, resulting in improved contrast which is directly related to scatterer size changes. Amplitude Modulation - Frequency Modulation (AM-FM) analysis is a fast and accurate technique which can also be applied to the OCT images for estimation of spectral information. It is based on the analytic signal of the real data, obtained using a Hilbert Transform, and provides the instantaneous amplitude, phase, and frequency of an OCT signal. The performance of this method is superior to both FFT-based and parametric (e.g. autoregressive) spectral analysis providing better accuracy and faster convergence when estimating scatterer features. Since disease tissues exhibit variations in scatterer size and thus also exhibit marked differences in spectral and phase characteristics, such advanced analysis techniques can provide more insight into the subtle changes observed in OCT images of malignancy. Therefore, they can make available a tool which could prove extremely valuable for the investigation of disease features which now remain below the resolution of OCT and improved the technology's diagnostic capabilities.

The early stages of malignant diseases, such as cancer, are characterized by cellular and microstructural changes which define both the diagnosis and the prognosis of the disease. Unfortunately, at the current resolution of Optical Coherence Tomography (OCT), such changes associated with early cancer are not clearly discernible. However, spectral analysis of OCT images has recently shown that additional information can be extracted from those signals, resulting in improved contrast which is directly related to scatterer size changes. Amplitude Modulation – Frequency Modulation (AM-FM) analysis is a fast and accurate technique which can also be applied to the OCT images for estimation of spectral information. It is based on the analytic signal of the real data, obtained using a Hilbert Transform, and provides the instantaneous amplitude, phase, and frequency of an OCT signal. The performance of this method is superior to both FFT-based and parametric (e.g. autoregressive) spectral analysis providing better accuracy and faster convergence when estimating scatterer features. Since disease tissues exhibit variations in scatterer size and thus also exhibit marked differences in spectral and phase characteristics, such advanced analysis techniques can provide more insight into the subtle changes observed in OCT images of malignancy. Therefore, they can make available a tool which could prove extremely valuable for the investigation of disease features which now remain below the resolution of OCT and improved the technology’s diagnostic capabilities.

Optical Coherence Tomography (OCT) is an emerging medical imaging technology able to detect tissue microstructure in vivo and in situ. However, many changes, associated with diseases such as cancer, result in cellular and sub-cellular variations which are very important for the diagnosis but are below the resolution limit of OCT. Since the spectrum of scattered light is structure-dependent, the spectral content of OCT signals depends on such features and can be used to extract information otherwise unavailable in standard OCT images. Spectral analysis of OCT has resulted in improved contrast which corresponds to scatterer size changes. In this paper, novel spectral analysis techniques, by means of autoregressive spectral estimation, discriminant analysis, clustering, and scatterer size calculations, are presented. These procedures result in quantitative and significantly more accurate measurements compared to previous techniques. They were initially tested on samples of microspheres, successfully identifying each with sensitivity and specificity of Gt90% and yielding diameter estimates within 16% of the actual size. The techniques were also applied to in vivo images of xenopus laevis tadpoles and ex vivo neurological samples and provided increased contrast between different tissues. Such methods could provide a very useful procedure for the identification of subtle changes in OCT images and could make available a tool for differentiating tissues based on scatterer size. Such a tool could prove extremely valuable in the evaluation of disease features which now remain below the resolution limit of OCT.

The diagnostic utility of a conventional transillumination microscope, the most common imaging modality in clinical use today, is limited by the microscope's resolution. It is, however, possible to achieve lateral resolution well beyond the classical limit by using laterally structured illumination in a wide-field, nonconfocal microscope. In this method, the spatially modulated illumination (SMI) makes high-resolution information that is normally inaccessible visible in the observed image. Previously presented SMI microscopy systems operated in epifluorescence mode. We describe the design, construction, and testing of a novel transillumination SMI microscope. As transillumination is necessary for most medical applications, such as histopathologic evaluation of biopsy tissue and chromosomal analysis, such a system should have a significant diagnostic effect.

Human chromosome analysis is an essential task in cytogenetics, especially in prenatal screening, genetic syndrome diagnosis, cancer pathology research and mutagen dosimetry. Chromosomal analysis begins with the creation of a karyotype, which is a layout of chromosome images organized by decreasing size in pairs. Both manual and automatic classification of chromosomes are limited by the resolution of the microscope and imaging system used. One way to improve the results of classification and even detect subtleties now remaining undetected, is to enhance the resolution of the images. It is possible to achieve lateral resolution beyond the classical limit, by using spatially modulated illumination (SMI) in a wide-field, non-confocal microscope. In this case, the sample is illuminated with spatially modulated light, which makes normally inaccessible high-resolution information visible in the observed image by shifting higher frequencies within the OTF limits of the microscope. Although, SMI microscopes have been reported in the past, this manuscript reports the development of a transillumination microscope for opaque, non-fluorescent samples. The illumination path consisted of a light source illuminating a ruled grating which was subsequently imaged on the sample. The grating was mounted on a rotating and translating stage so that the magnification and rotation of the pattern could be adjusted. The imaging lens was a 1.25 NA oil immersion objective. Test samples showed resolution improvement, as judged from a comparison of the experimentally obtained FWHM. Further studies using smaller fringe distance or laser interference pattern illumination will be evaluated to further optimize the SMI results.

Summary form only given. Over the past few years, optical coherence tomography (OCT) has been extensively evaluated as a possible diagnostic tool in a variety of medical fields, demonstrating its feasibility as a high speed, non-invasive, high resolution in vivo and in situ imaging modality with the possibility of being easily implemented into different medical diagnostic instruments; e.g., catheters, microscopes, as well as ophthalmoscopes. We demonstrate an ultrahigh, subcellular level resolution OCT system for in vivo and in vitro imaging. It uses a Kerr-lens mode-locked Ti:sapphire laser that emits pulses as short as 5.5 fs with bandwidths up to 350 nm centred at 800 nm with an average power of up to 150 mW, due to broadband controlled dispersion and high reflectivity enabled by low-dispersion prisms and special designed double chirped mirrors. The output spectrum can be shaped by changing the prisms insertions (P1, P2) or by alteration of the prism separation. Appropriate single mode fibers, special broad bandwidth, wavelength flattened 3 dB fiber coupler, as well as special achromatic and catadioptric objectives are used to maintain this ultrabroad bandwidth to achieve subcellular level resolution.

Optical coherence tomography (OCT) is an optical imaging technique that is capable of performing micron-scale, cross-sectional imaging of internal microstructure in biological systems. OCT is analogous to ultrasound B mode imaging except that it uses light rather than sound and performs imaging by measuring the back-scattered intensity of light from structures in tissue. We describe recent advances in OCT technology including the application of short pulse solid state lasers based on Ti: Al2O3 and Cr: Mg2SiO4 to enable high-resolution, high-speed imaging as well as the development of OCT catheter/endoscope delivery to permit imaging of internal organ systems. OCT enables the nonexcisional, in situ, real-time imaging of tissue microstructure and is thus a powerful and promising technique for optical biopsy.

The effects of dispersion on optical coherence tomography (OCT) images have long been documented. The imbalance of spectral broadening, caused by dispersion mismatches in the two arms of the OCT interferometer, can result in significant resolution degradation. Efforts to correct this phenomenon have resulted in improved image quality using various techniques. However, dispersion is also present and varies in tissues. As a result, group velocity dispersion (GVD) can be used to detect changes in tissues and provide useful information for diagnosis. Several methods can be utilized to measure the GVD from OCT images: (i) the degradation of the point spread function (PSF), (ii) the shift (walk-off) between images taken at different wavelengths, (iii) the changes in the second derivative of the spectral phase, as well as two new methods, which do not require a reflector and are applicable in intact tissues, i.e., using (iv) the speckle degradation, and (v) the speckle cross correlation. A systematic, experimental, evaluation of these methods is presented to elucidate the capabilities, the limitations, and the accuracy of each technique when attempting to estimate the GVD in scattering samples. The most precise values were obtained from the estimation of the PSF degradation, whereas using the phase derivative method was only applicable to minimally scattering samples. Speckle broadening appears to be the most robust method for tissue GVD measurements.