Quantitative analysis of hidden particles diffusing behind a scattering layer using speckle correlation.

Speckle-correlation imaging is a family of methods that makes use of the "memory effect" to image objects hidden behind visually opaque layers. Here, we show that a correlation analysis can be applied to quantitative imaging of an ensemble of dynamic fluorescent beads diffusing on a 2D surface. We use an epi-fluorescence microscope where both the illumination and detection light patterns are speckled, due to light scattering by a thin disordered layer. The spatio-temporal cross-correlation of the detection speckle pattern is calculated as a function of lag time and spatial shift and is used to determine the diffusion constant and number of fluorescent particles in the sample without requiring any phase retrieval procedure. It is worth to note that the "memory effect" range is not required to extend beyond a distance of few speckle grains, thus making our method potentially useful for nearly arbitrary values of the thickness of the scattering layer.

Confocal fluorescence correlation spectroscopy through a sparse layer of scattering objects.

In the presence of strong light scattering, as often encountered in biological tissue, optical microscopy becomes challenging and technical demanding. Beside image quality, the quantitative determination of molecular properties is also strongly affected by scattering. We have carried out fluorescence correlation spectroscopy (FCS) experiments, in a solution of fluorophores, through a sparse scattering layer made of dielectric beads. We observe that the fluorescence signal steadily decreases as the focus is moved away from the scattering layer. By contrast, the estimated number of molecules recovers its normal value beyond a characteristic distance of about twice the bead diameters, below which it is strongly biased. Accompanying theoretical modeling demonstrates how diffraction and refraction by the scattering layer and their impact on FCS measurements depend on size and refractive index of the beads.

Cellular response to heat shock studied by multiconfocal fluorescence correlation spectroscopy.

Heat shock triggers a transient and ubiquitous response, the function of which is to protect cells against stress-induced damage. The heat-shock response is controlled by a key transcription factor known as heat shock factor 1 (HSF1). We have developed a multiconfocal fluorescence correlation spectroscopy setup to measure the dynamics of HSF1 during the course of the heat-shock response. The system combines a spatial light modulator, to address several points of interest, and an electron-multiplying charge-coupled camera for fast multiconfocal recording of the photon streams. Autocorrelation curves with a temporal resolution of 14 µs were analyzed before and after heat shock on eGFP and HSF1-eGFP-expressing cells. Evaluation of the dynamic parameters of a diffusion-and-binding model showed a slower HSF1 diffusion after heat shock. It is also observed that the dissociation rate decreases after heat shock, whereas the association rate is not affected. In addition, thanks to the multiconfocal fluorescence correlation spectroscopy system, up to five spots could be simultaneously located in each cell nucleus. This made it possible to quantify the intracellular variability of the diffusion constant of HSF1, which is higher than that of inert eGFP molecules and increases after heat shock. This finding is consistent with the fact that heat-shock response is associated with an increase of HSF1 interactions with DNA and cannot be explained even partially by heat-induced modifications of nuclear organization.

Adaptive optics for fluorescence correlation spectroscopy.

Fluorescence Correlation Spectroscopy (FCS) yields measurement parameters (number of molecules, diffusion time) that characterize the concentration and kinetics of fluorescent molecules within a supposedly known observation volume. Absolute derivation of concentrations and diffusion constants therefore requires preliminary calibrations of the confocal Point Spread Function with phantom solutions under perfectly controlled environmental conditions. In this paper, we quantify the influence of optical aberrations on single photon FCS and demonstrate a simple Adaptive Optics system for aberration correction. Optical aberrations are gradually introduced by focussing the excitation laser beam at increasing depths in fluorescent solutions with various refractive indices, which leads to drastic depth-dependent bias in the estimated FCS parameters. Aberration correction with a Deformable Mirror stabilizes these parameters within a range of several tens of µm into the solution. We also demonstrate, both theoretically and experimentally, that the molecular brightness scales as the Strehl ratio squared.

Chromophore decomposition in multispectral time-resolved diffuse optical tomography.

Multicomponent phantom measurements are carried out to evaluate the ability of multispectral time domain diffuse optical tomography in reflectance geometry to quantify the position and the composition of small heterogeneities at depths of 1-1.5 cm in turbid media. Time-resolved data were analyzed with the Mellin-Laplace transform. Results show good localization and correct composition gradation of objects but still a lack of absolute material composition accuracy when no a priori geometry information is known.

Measuring hemoglobin oxygen saturation during graded hypoxic hypoxia in rat striatum.

We evaluated in vivo reflectance spectroscopy of visible light as a method to assess brain tissue hemoglobin oxygen saturation in rat striatum (SstrO2). Seven anesthetized and mechanically ventilated rats were subjected to incremental reduction in the fraction of inspired oxygen (Fio2): 0.35, 0.25, 0.15, 0.12, and 0.10, followed by a reoxygenation period (Group 1). At each episode, local changes in SstrO2 and in cerebral blood flow (LCBF) were simultaneously determined in the two striatal regions, using reflectance spectroscopy and laser Doppler flowmetry, respectively. Another group of rats (Group 2, n = 6) was also studied to measure sagittal sinus blood hemoglobin saturation (SssO2) during graded hypoxic hypoxia. Corpus striatum exhibited a significant graded decrease in SstrO2, from 38% +/- 17% at Fio2 of 0.35 (control) to 16% +/- 10% at Fio2 of 0.12 and to 13% +/- 7% at Fio2 of 0.10 (P < 0.05), with no difference between the two hemispheres. These local changes in SstrO2 were associated with a significant graded increase in LCBF: 161% +/- 26% of control values and 197% +/- 34% during these 2 hypoxic episodes, respectively (P < 0.05). All local changes were fully reversed during the reoxygenation period. In Group 2, SssO2 decreased from 38% +/- 8% at Fio2 of 0.35 (control) to 10% +/- 3% at Fio2 of 0.10, closely related to SstrO2 decreasing in hypoxia. This study shows that reflectance spectroscopy of the visible light in rat striatum could be a possible measure of continuous changes in SstrO2. SssO2 and LCBF measurements during graded hypoxic hypoxia indicate that changes in SstrO2 reflect primarily those in brain venous oxygenation.

Time-domain diffuse optical tomography using silicon photomultipliers: feasibility study.

Silicon photomultipliers (SiPMs) have been very recently introduced as the most promising detectors in the field of diffuse optics, in particular due to the inherent low cost and large active area. We also demonstrate the suitability of SiPMs for time-domain diffuse optical tomography (DOT). The study is based on both simulations and experimental measurements. Results clearly show excellent performances in terms of spatial localization of an absorbing perturbation, thus opening the way to the use of SiPMs for DOT, with the possibility to conceive a new generation of low-cost and reliable multichannel tomographic systems.

Quantification in time-domain diffuse optical tomography using Mellin-Laplace transforms.

Simulations and phantom measurements are used to evaluate the ability of time-domain diffuse optical tomography using Mellin-Laplace transforms to quantify the absorption perturbation of centimetric objects immersed at depth 1-2 cm in turbid media. We find that the estimated absorption coefficient varies almost linearly with the absorption change in the range of 0-0.15 cm-1 but is underestimated by a factor that depends on the inclusion depth (~2, 3 and 6 for depths of 1.0, 1.5 and 2.0 cm respectively). For larger absorption changes, the variation is sublinear with ~20% decrease for 뫵a = 0.37 cm-1. By contrast, constraining the absorption change to the actual volume of the inclusion may considerably improve the accuracy and linearity of the reconstructed absorption.

Time-domain reflectance diffuse optical tomography with Mellin-Laplace transform for experimental detection and depth localization of a single absorbing inclusion.

We show how to apply the Mellin-Laplace transform to process time-resolved reflectance measurements for diffuse optical tomography. We illustrate this method on simulated signals incorporating the main sources of experimental noise and suggest how to fine-tune the method in order to detect the deepest absorbing inclusions and optimize their localization in depth, depending on the dynamic range of the measurement. To finish, we apply this method to measurements acquired with a setup including a femtosecond laser, photomultipliers and a time-correlated single photon counting board. Simulations and experiments are illustrated for a probe featuring the interfiber distance of 1.5 cm and show the potential of time-resolved techniques for imaging absorption contrast in depth with this geometry.

Multi-confocal fluorescence correlation spectroscopy.

We report a multi-confocal Fluorescence Correlation Spectroscopy (mFCS) technique that combines a Spatial Light Modulator (SLM), with an Electron Multiplying-CCD camera (EM-CCD). The SLM is used to produce a series of laser spots, while the pixels of the EM-CCD play the roles of virtual pinholes. The phase map addressed to the SLM, calculated by using the spherical wave approximation, makes it possible to produce several diffraction limited laser spots. The fastest acquisition mode leads to a time resolution of 100 microseconds. By using solutions of sulforhodamine G we demonstrated that the observation volumes are similar to that of a standard confocal set-up. mFCS experiments have also been conducted on two stable cell lines: mouse embryonic fibroblasts expressing eGFP-actin and H1299 cells expressing the heat shock factor fusion protein HSF1-eGFP. In the first case we could recover the diffusion constant of G-actin within the cytoplasm, although we were also sensitive to interactions with F-actin. Concerning HSF1, we could clearly observe the modifications of the number of molecules and of the HSF1 dynamics during heat shock.

Measuring, in solution, multiple-fluorophore labeling by combining fluorescence correlation spectroscopy and photobleaching.

Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process but also to quantify interactions, for instance within molecular complexes. We combined fluorescence correlation spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutions of cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here, photobleaching is used as a control parameter to vary the experimental outputs (brightness and number of molecules). Assuming a Poissonian distribution of the number of fluorescent labels per cDNA, the FCS-photobleaching data could be easily fit to yield the mean number of fluorescent labels per cDNA strand (approximately = 2). This number could not be determined solely on the basis of the cDNA brightness, because of both the statistical distribution of the number of fluorescent labels and their unknown brightness when incorporated in cDNA. The statistical distribution of the number of fluorophores labeling cDNA was confirmed by analyzing the photon count distribution (with the cumulant method), which showed clearly that the brightness of cDNA strands varies from one molecule to the other. We also performed complementary continuous photobleaching experiments and found that the photobleaching decay rate of Alexa Fluor 647 in the excited state decreases by about 30% when incorporated into cDNA, while its nonradiative decay rate is increased such that the brightness of individual Alexa labels is decreased by 25% compared to free Alexa dyes.

Spatial fluorescence cross-correlation spectroscopy by means of a spatial light modulator.

Spatial fluorescence cross-correlation spectroscopy is a rarely investigated version of fluorescence correlation spectroscopy, in which the fluorescence signals from different observation volumes are cross-correlated. In the reported experiments, two observation volumes, typically shifted by a few microm, are produced, with a spatial light modulator and two adjustable pinholes. We illustrated the feasibility and potentiality of this technique by: i) measuring molecular flows, in the range 0.2-1.5 microm/ms, of solutions seeded with fluorescent nanobeads or rhodamine molecules (simulating active transport phenomenons); ii) investigating the permeability of the phospholipidic membrane of giant unilamellar vesicles versus hydrophilic or hydrophobic molecules (in that case the laser spots were set on both sides of the membrane). Theoretical descriptions are proposed together with a discussion about fluorescence-correlation-spectroscopy-based, alternative methods.

Spatial fluorescence cross-correlation spectroscopy.

We present an alternative method for diffusion measurements of fluorescent species in solution by use of confocal microscopy and fluorescence correlation spectroscopy techniques. It consists of making a time and spatial dual correlation in which one detects the fluorescence signals from two nearby separate confocal volumes and cross correlates them. To improve the spatial discrimination between the two confocal volumes we propose filtering of fluorescence photocounts by rejecting the fluorescence background, which corresponds to particles located far from the center of the detection volumes.