The assessment of materials viscoelastic properties often represents a means of diagnosis or characterization of biological tissues and biomaterials. In this paper, we introduce a new optical method for the evaluation of dynamical properties of viscoelastic media. The approach is based on time-resolved spatial speckle imaging, using a continuous wave CW illumination and a standard CCD detector. We demonstrate that an estimation of viscoelastic properties is possible, by analyzing intensity and contrast profiles of scattering spot images acquired over multiple exposure times. The accuracy of this approach is evaluated using simulated tissue mimicking media having well known optical and dynamical properties.
We developed a fluorescence imaging microscope system intended for the localization within artery slices of a gadolinium-based macromolecular biospecific magnetic resonance (MR) contrast agent used for the visualization of atherothrombosis. As the contrast agent is not initially fluorescent, we substitute some gadolinium ions for terbium ions to make them fluorescent while preserving their chemical characteristics. A long fluorescence emission time constant enables us to have a suitable signal-to-noise ratio, despite a low intensity, using pulsed illumination and time-gated imaging. Images of rat arteries show that the contrast agent is indeed localized on the specific regions of the tissues. We currently have a tool that allows us to understand and optimize the MR contrast agent.
Acousto-optic imaging (AOI) is an emerging technique in the field of biomedical optics which combines the optical
contrast allowed by diffuse optical tomography with the resolution of ultrasound (US) imaging. In this work we report
the implementation, for that purpose, of a CMOS smart-pixels sensor dedicated to the real-time analysis of speckle
patterns. We implemented a highly sensitive lock-in detection in each pixel in order to extract the tagged photons after
an appropriate in-pixel post-processing. With this system we can acquire images in scattering samples with a spatial
resolution in the 2mm range, with an integration time compatible with the dynamic of living biological tissue.
Diffuse light in tissue can be a very interesting tool for medical diagnosis, especially if one considers the fluctuations of
the speckle pattern. Of course, speckle analysis suffers from the low spatial coherence of speckle patterns, and multipixel
detection is required in order to increase the signal to noise ratio. There is therefore a need of a setup with a high
sensitivity, capable of outputting a signal from noise through averaging on a high number of pixels, as the signal can be
lower than the photon level for one image and one frame. Furthermore, such a processing has to be done at a very high
acquisition rate. “Smart-pixels” arrays can represent a major breakthrough in this field.
Diffuse Correlation Spectroscopy (DCS) is based on the temporal correlations of the speckle pattern from the light that
has diffused through a biological media. Measurements must be made on a small coherence area of the size of a speckle
grain. Summing independent measurement increases the SNR as the square root of the number of detectors. We present a
bi-dimensionnal pixel CMOS detector array specially designed for this task, with parallel in-pixel demodulation and
temporal correlations computation. Optical signals can be processed at a rate higher than 10,000 samples per second with
demodulation frequencies in the MHz range.
We have already demonstrated the potentiality of interferometry to perform timeresolved
measurements of the light scattered by a tissue: the fluctuations of the speckle
pattern, linked to a wavelength-modulation of the source, are registered, and the time-resolved
average intensity can be numerically obtained from these data[1]. The competitive results were
obtained with a simple photodiode as detector[2].
Such a method can be cheaper and more accessible for biomedical applications than
direct time-resolved methods, but it is not its unique advantage: this method allows to perform
Diffusing Wave Spectroscopy (DWS) with selected photon pathlengths[3,4]; for instance, we
have shown that we can improve the spatial resolution in transillumination imaging of a
dynamic heterogeneity through the selection of short photon transit times[4]. Therefore such a
method can offer interesting applications, for example in mammography.
A way to improve the signal to noise ratio of this method can consist in multiplying
the number of detectors. That's the reason why we decide to consider the use of a high speed
camera, that can reach a rate of 1000 frames per second. We will present the first results
obtained with this new system. The performance will be discussed, and compared to our
previous setup.
KEYWORDS: Tissue optics, Sensors, Reflectivity, Picosecond phenomena, Streak cameras, Scattering, Optical testing, In vivo imaging, Dielectric filters, Monte Carlo methods
We develop a new sensor for the local in vivo measurement of optical coefficients near the surface of a tissue. To be less sensitive to the heterogeneous surface of the sample, we decided to perform space and time-resolved measurements. The sensor is a bundle of fibres. The
excitation light is generated by a mode-locked Ti-Sa laser at 800nm and filtered by a 1.5nm bandwidth dielectric filter in order to limit group velocity dispersion in the monomode excitation fibre. The reflectance light is collected by gradient index fibres at 250μm and 1.3 mm from the source. The detection is performed with a Hamamatsu M5675 synchroscan streak camera. The whole system allows a time resolution of about 5ps. We made comparisons between time and space resolved Monte-Carlo numerical simulations and in vitro experimental data obtained with unskimmed UHT milk which is a known reference medium. The system does not rely on the absolute value of the reflected light intensity nor depend on the intensity ratio between different fibres since the distance between the medium and the fibres as well as the fibres tip cleanness cannot be guaranteed in vivo. As a consequence we use global characteristic of the time resolved curves such as the FWHM and their evolution with the distance from the source. The good agreement between the simulations and the experimental data lets us envisage to use numerically pre-computed tables for a real time determination of the local scattering mean free path and the anisotropy factor . We soon will be able to perform
measurements with biological tissues, in vitro in a first time and in vivo in a second time.
The aim of this study was to perform a preliminary evaluation of the diagnostic potential of laser induced autofluorescence spectroscopy (LIAFS) for urothelial tumors using fluorescence intensity ratios at different wavelengths. After testing three laser excitation wavelengths (308, 337, and 480 nm) in normal and malignant bladder cell lines, 308 nm appeared to be the most promising wavelength since two fluorescence bands were observed at 360 and 440 nm; these were attributed to tryptophan (Trp) and reduced nicotinamide adenine dinucleotide (NADH) respectively. This study was then performed on freshly removed normal bladder and bladder tumor specimens exclusively using the 308-nm excitation wavelength. The tumor spectra, regardless of stage and grade, were very similar to the malignant cell spectra. However, a marked reduction of overall
intensity was observed for carcinoma in situ (CIS). Normal bladder mucosa exhibited a shift of the first
fluorescence band to 380 nm, indicating an overlap of Trp urothelial cell emission and collagen fluorescence
derived from the lamina propria. The intensity of the NADH emission band was markedly reduced in tumor
tissues compared with normal mucosa, which could indicate different redox conditions in urothelial tumors. A fluorescence intensity ratio at 360 and 440 nm can accurately discriminate normal or inflammatory mucosa from all bladder tumors, including CIS. These findings support the use of LIAFS as a new diagnostic technique for occult urothelial tumors.
We have designed a program using laser induced autofluorescence spectroscopy as a possible way to characterize urothelial tumors of the bladder. The autofluorescence spectra were compared between normal, suspicious and tumor areas of human bladder. Three different pulsed laser wavelengths were used for excitation: 308 nm (excimer), 337 nm (nitrogen) and 480 nm (dye laser). Excitation light was delivered by a specially devised multifiber catheter introduced through the working channel of a regular cystoscope under saline irrigation. The fluorescence light was focused into an optical multichannel analyzer detection system. The data was evaluated in 25 patients immediately before resection of a bladder tumor. Spectroscopic results were compared with histopathology. Upon 337 nm and 480 nm excitations, the overall intensity of the fluorescence spectra from bladder tumors was clearly reduced in comparison with normal urothelium, regardless of the stage and the grade of the tumor. upon 308 nm excitation, the shape of tumor fluorescence spectra, including carcinoma in situ, differed drastically from that of normal tissue. In this case, no absolute intensity measurements are needed and clear diagnosis can be achieved from fluorescence intensity ratio (360/440 nm). This spectroscopic study could be particularly useful for the design of a simplified autofluorescence imaging device for real-time routine detection of occult urothelial neoplastic lesions.
The aim of this paper was to demonstrate the feasibility of routine real time clinical detection of occult urothelial precancerous and cancerous lesions by laser induced autofluorescence (LIAF), that is with no previous instillation of exogenous fluorescent marker. Three different pulsed laser wavelengths were alternately used for excitation: 480 nm (dye laser), 337 nm (nitrogen laser), and 308 nm (XeCl excimer laser). A clinical endoscopic study was performed on 23 patients immediately before transurethral resection of bladder tumor. Spectroscopic results were compared with histological analysis. For 480 nm and 337 nm excitation a single fluorescence broad band was obtained in any case, but for tumors the overall intensity was significantly reduced compared to normal mucosa. For 308 nm excitation, two main broad bands were observed. In the case of neoplasic lesions (including carcinoma in situ), the intensity ratio [I(360 nm)/I(440 nm)] was always greater than 2, but for normal or inflammatory areas this ratio was less than 2. A clear diagnosis could then be achieved for 308 nm excitation without the need of absolute intensity measurements. We get no false positive at this wavelength. XeCl LIAF spectroscopy is therefore a promising technique for the detection of urothelial precancerous lesions and could be used to perform an 'optical biopsy' in a routine mode with real time results.
Laser induced fluorescence has often been used as a diagnostic method. Unfortunately the fluorescence signal is modified during the photons migration towards the detector. The purpose of this study is to determine the alterations of the laser induced fluorescence spectra in white matter of adult brain due to the spectral variations of the optical coefficients (mu) a((lambda) ), (mu) s((lambda) ) and of the mean cosine of the scattering angle g((lambda) ).
Three-hundred-eight nm laser-induced autofluorescence spectra of the normal human brain, astrocytoma grade IV and glioblastoma grade IV specimens, have been recorded in vitro two hours after surgical resection. Typical fluorescence spectra for normal (N) and malignant (M) tissue show 4 maxima at about 352, 362, 383, and 460 nm. These spectra are analyzed in detail. Subtle differences in normalized spectra of N and M tissues appear to be large enough for diagnosis. Several criteria such as maxima and minima absolute intensity and intensity ratios at typical wavelengths are computed and used to classify the tissue. This preliminary study shows that fluorescence spectroscopy with 308 nm UV excitation could be a valid technique for discriminating tumor types. However, it should be noted that these measurements are made in vitro. Living tissues may have different spectral characteristics, therefore future in vivo investigations must be performed.
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