Israël Veilleux

Cell Lineage Reconstruction of Early Zebrafish Embryos Using Label-Free Nonlinear Microscopy

Zebrafish Development in 3D Vertebrate development has classically been characterized qualitatively, but—by combining expertise in physics, mathematics, and biology—Olivier et al. (p. 967) used label-free conformal nonlinear time-lapse microscopy and image analysis to calculate the spatiotemporal cell lineage of zebrafish embryos throughout their first 10 division cycles. The work reconstructs complete lineage trees, annotated with cell-shape measurements, and allows for visualization with interactive tools. Time-lapse recording characterizes the rhythm and cleavage pattern of the embryo during early stages of development. Quantifying cell behaviors in animal early embryogenesis remains a challenging issue requiring in toto imaging and automated image analysis. We designed a framework for imaging and reconstructing unstained whole zebrafish embryos for their first 10 cell division cycles and report measurements along the cell lineage with micrometer spatial resolution and minute temporal accuracy. Point-scanning multiphoton excitation optimized to preferentially probe the innermost regions of the embryo provided intrinsic signals highlighting all mitotic spindles and cell boundaries. Automated image analysis revealed the phenomenology of cell proliferation. Blastomeres continuously drift out of synchrony. After the 32-cell stage, the cell cycle lengthens according to cell radial position, leading to apparent division waves. Progressive amplification of this process is the rule, contrasting with classical descriptions of abrupt changes in the system dynamics.

Reference optical phantoms for diffuse optical spectroscopy. Part 1 – Error analysis of a time resolved transmittance characterization method

Development, production quality control and calibration of optical tissue-mimicking phantoms require a convenient and robust characterization method with known absolute accuracy. We present a solid phantom characterization technique based on time resolved transmittance measurement of light through a relatively small phantom sample. The small size of the sample enables characterization of every material batch produced in a routine phantoms production. Time resolved transmittance data are pre-processed to correct for dark noise, sample thickness and instrument response function. Pre-processed data are then compared to a forward model based on the radiative transfer equation solved through Monte Carlo simulations accurately taking into account the finite geometry of the sample. The computational burden of the Monte-Carlo technique was alleviated by building a lookup table of pre-computed results and using interpolation to obtain modeled transmittance traces at intermediate values of the optical properties. Near perfect fit residuals are obtained with a fit window using all data above 1% of the maximum value of the time resolved transmittance trace. Absolute accuracy of the method is estimated through a thorough error analysis which takes into account the following contributions: measurement noise, system repeatability, instrument response function stability, sample thickness variation refractive index inaccuracy, time correlated single photon counting system time based inaccuracy and forward model inaccuracy. Two sigma absolute error estimates of 0.01 cm(-1) (11.3%) and 0.67 cm(-1) (6.8%) are obtained for the absorption coefficient and reduced scattering coefficient respectively.

Multiplexed two-photon microscopy of dynamic biological samples with shaped broadband pulses

Coherent control can be used to selectively enhance or cancel concurrent multiphoton processes, and has been suggested as a means to achieve nonlinear microscopy of multiple signals. Here we report multiplexed two-photon imaging in vivo with fast pixel rates and micrometer resolution. We control broadband laser pulses with a shaping scheme combining diffraction on an optically-addressed spatial light modulator and a scanning mirror allowing to switch between programmable shapes at kiloHertz rates. Using coherent control of the two-photon excited fluorescence, it was possible to perform selective microscopy of GFP and endogenous fluorescence in developing Drosophila embryos. This study establishes that broadband pulse shaping is a viable means for achieving multiplexed nonlinear imaging of biological tissues.

Accurately characterized optical tissue phantoms: how, why and when?

Optical tissue phantoms are very important tools for the development of biomedical imaging applications. Optical phantoms are often used as ground truth against which instruments results can be compared. It is therefore important that the optical properties of reference phantoms be measured in a manner that is traceable to the international system of units. SI traceability insures long term consistency of results and will therefore improve the effectiveness of diffuse optics research effort more effective by reducing unwanted variability in the data produced and shared by the community. The ultimate benefit of rigorous SI traceability is the reduction of variability in the data produced by novel diagnostic devices, which will in turn increase the statistical power of clinical trials aiming at validating their clinical usefulness. SI traceability, and therefore uncertainty analysis, is also relevant to traceability aspects mandated by FDA regulations. SI traceability is achieved through a thorough analysis of the measurement principle and its potential error sources. The uncertainty analysis should be ultimately validated by inter-laboratory comparison until a consensus is attained on the best practices for measuring the optical properties of tissue phantoms.

The role of optical tissue phantom in verification and validation of medical imaging devices

Optical tissue phantoms are very important tools for the development of biomedical imaging applications. They play an important role from proof of concept to clinical trials. Optical phantoms are often used as reference materials against which instruments results can be compared. It is therefore important that the optical properties of reference phantoms be measured in a manner that is traceable to the international system of units. SI traceability insures long term consistency of results and will therefore improve the effectiveness of diffuse optics research effort more effective by reducing unwanted variability in the data produced and shared by the community. The ultimate benefit of SI traceability is the reduction of variability in the data produced by novel diagnostic devices, which will in turn increase the statistical power of clinical trials aiming at validating their clinical usefulness. SI traceability, which implies uncertainty analysis, is also relevant to traceability aspects mandated by FDAs Good Manufacturing Practices.

Monitoring changes in tissue optical properties following interstitial photothermal therapy of ex vivo human prostate tissue

We are developing a method of monitoring treatment progression of interstitial photothermal therapy of focal prostate cancer using transrectal diffuse optical tomography (TRDOT) combined with transrectal 3D ultrasound (3D-TRUS). Measurements of prostate tissue optical properties were made on ex vivo human prostate samples prior to and post coagulation. Interstitial photothermal treatments were delivered to the ex vivo samples and monitored using an interstitial probe near the treatment fiber. After treatment, bulk optical properties were measured on native and coagulated zones of tissue. Changes in optical properties across the boundary between native and coagulated tissues were spatially mapped using a small diffuse reflectance probe. The optical property estimates and spatial information obtained using each method was compared.