Fluorescence correlation spectroscopy is used extensively for quantitative characterization of biomolecules at very low concentration. However, light aberration and scattering from the tissues are two major factors that affect the results strongly. Although adaptive optics arrangement can correct the aberrations of light to some extent, it fails completely to eliminate the light scattering effect. Recently, exploiting the fact that autocorrelation of a speckle pattern is a sharply peaked point spread function and the optical memory effect, non-invasive imaging of fluorescent sample through a scattering medium has been possible. However, it is also very challenging to measure the dynamic properties of the fluorescent molecules or particles through a scattering layer due to poor signal to noise ratio. In this study, we employ a modality based on speckle cross-correlation enabled via optical memory effect to study two dimensional (2D) diffusion of fluorescent particles hidden behind a scattering film. We realized a 2D diffusing model system by confining fluorescent polystyrene beads of 1µm diameter at the water/air interface behind a TiO2 diffuser. The experimental set up was built up in an epifluorescence configuration. The fluorescent beads were excited by an illumination speckle generated by the incident light in a plane-wave geometry while passing through the disordered TiO2 film. Similarly, the emitted fluorescent signal also traversed through the same TiO2 film to generate the detection speckle, which was eventually recorded by a high frame rate CMOS camera. The experimental set up has also been modelled numerically, where speckle pattern has been generated by a spherical wave, transmitted through a scattering object in an optical microscope. Moreover, the dependence of the speckle size on the numerical aperture, magnification, and the distance of the focal plane from the bead plane has also been studied. The numerical results have been compared with the experimental values to estimate the speckle size. Furthermore, we have evaluated the 2D diffusion constant by monitoring the widening of the 2D speckle cross-correlation function versus lag time. This result has been compared with that obtained with the single particle tracking method without the scattering layer. Quantitative agreement between the results obtained by the speckle cross-correlations and the single particle tracking technique without the diffuser establishes the potential application of this technique in correlation spectroscopy. Superimposed multiple beads speckle patterns were also studied and the results will be presented in the conference.
Pathologist examination of tissue slides provides insightful information about a patient’s disease. Traditional analysis of tissue slides is performed under a binocular microscope, which requires staining of the sample and delays the examination. We present a simple cost-effective lensfree imaging method to record 2–4μm resolution wide-field (10 mm2 to 6 cm2) images of unstained tissue slides. The sample processing time is reduced as there is no need for staining. A wide field of view (10 mm2) lensfree hologram is recorded in a single shot and the image is reconstructed in 2s providing a very fast acquisition chain. The acquisition is multispectral, i.e. multiple holograms are recorded simultaneously at three different wavelengths, and a dedicated holographic reconstruction algorithm is used to retrieve both amplitude and phase. Whole tissue slides imaging is obtained by recording 130 holograms with X-Y translation stages and by computing the mosaic of a 25 x 25 mm2 reconstructed image. The reconstructed phase provides a phase-contrast-like image of the unstained specimen, revealing structures of healthy and diseased tissue. Slides from various organs can be reconstructed, e.g. lung, colon, ganglion, etc. To our knowledge, our method is the first technique that enables fast wide-field lensfree imaging of such unlabeled dense samples. This technique is much cheaper and compact than a conventional phase contrast microscope and could be made portable. In sum, we present a new methodology that could quickly provide useful information when a rapid diagnosis is needed, such as tumor margin identification on frozen section biopsies during surgery.
We developed a new imaging tool that can help pathologists in recording wide-field images of tissue slides. We present a simple cost-effective lens-free imaging method to record 2-4μm resolution wide-field (10 mm2 - 6 cm2) images of stained and unstained tissue slides. To our knowledge, our method is the first technique that enables fast (less than 5 minutes) wide-field lens-free imaging of such dense samples. Multiple holograms are recorded with different wavelength illumination, and a multispectral algorithm is used to retrieve both amplitude and phase. Our method can be used to retrieve images of stained tissue slides. For such absorbing object, the useful information is included in the modulus of the reconstructed complex field. Our method can also be applied to retrieve images of unstained tissue slides, where the useful information is in the retrieved phase. This technique is much cheaper and compact than a conventional microscope and could be made portable. Moreover, it enables wide field unstained tissue slides imaging, which could quickly provide useful information, for example on frozen section biopsies, when a rapid diagnosis is needed during surgery.
Fluorescence fluctuation methods, such as fluorescence correlation spectroscopy, are very sensitive to optical
aberrations. That is why it is possible to use a fluctuations-based metric, the molecular brightness, to correct aberrations
using a sensorless modal adaptive optics approach. We have investigated the performance of this method by correcting
known aberrations under various experimental conditions. The signal-to-noise ratio of the brightness measurement was
examined theoretically and experimentally and found to be directly related to the accuracy of aberration correction, so
that the latter can be predicted for a given sample brightness and measurement duration. We have also shown that the
initial measurement conditions play a key role in the correction dynamics and we provide guidelines to optimize the
corrections accuracy and speed. The molecular brightness, used as a metric, has the advantage that it depends on
aberrations as the square of the Strehl ratio, regardless of the nature of the sample. Therefore, it is straightforward to
predict the achievable correction accuracy and the same performance can be obtained in samples with different structure
and contrast, which would not be possible with image-based optimization metrics.
Micro-fabrication and surface functionalization imply to know the equilibrium surface concentration of various kinds of molecules. Paradoxically, this crucial parameter is often poorly controlled and even less quantified. We have used a technique belonging to the family of fluorescence fluctuation microscopy, namely Image Correlation Spectroscopy (ICS), to measure the absolute surface concentration of fibrinogen molecules adsorbed on glass substrates. As these molecules are immobile, the width of the autocorrelation of the confocal image obtained by scanning the sample only reflects that of the confocal Point Spread Function. Conversely, the amplitude of the autocorrelation is directly related to the average number of proteins simultaneously illuminated by the laser beam and therefore to their surface concentration. We have studied the surface concentration of fibrinogen proteins versus the initial concentration of these molecules, solubilized in the solution which has been deposited on the surface. The estimation of this relation can be biased for several reasons: the concentration of fibrinogen molecules in solution is difficult to control; the measurement of the surface concentration of adsorbed molecules can be strongly underestimated if the surface coverage or the molecular brightness is not uniform. We suggest methods to detect these artifacts and estimate the actual surface concentration, together with control parameters. Globally, fluorescence fluctuation microscopy is a powerful set of techniques when one wants to quantify the surface concentration of molecules at the micrometer scale.
Alignment of a laser to a point source detector for confocal microscopy can be a time-consuming task. The problem is
further exacerbated when multiple laser excitation spots are used in conjunction with a multiple pixel single photon detector;
in addition to X, Y and Z positioning, pixels in a 2D array detector can also be misaligned in roll, pitch and yaw
with respect to each other, causing magnification, rotation and focus variation across the array. We present a technique for
automated multiple point laser alignment to overcome these issues using closed-loop feedback between a laser illuminated
computer controlled Liquid Crystal on Silicon Spatial Light Modulator (LCOS-SLM) acting as the excitation source and a
32×32 pixel CMOS Single Photon Avalanche Diode (SPAD) array as the multiple pixel detection element. The alignment
procedure is discussed and simulated to prove its feasibility before being implemented and tested in a practical optical system.
We show that it is possible to align each independent laser point in a sub-second time scale, significantly simplifying
and speeding up experimental set-up times. The approach provides a solution to the difficulties associated with multiple
point confocal laser alignment to multiple point detector arrays, paving the way for further advances in applications such
as Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Imaging Microscopy (FLIM).
Fluorescence Correlation Spectroscopy (FCS) is an attractive method to measure molecular concentration, mobility parameters and chemical kinetics. However its ability to descriminate different diffusing species needs to be improved. Recently, we have proposed a simplified spatial Fluorescence cross Correlation Spectroscopy (sFCCS) method, allowing, with only one focused laser beam to obtain two confocal volumes spatially shifted. Now, we present a new sFCCS optical geometry where the two pinholes, a ring and core, are encapsulated one in the other. In this approach all physical and chemical processes that occur in a single volume, like singlet-triplet dynamics and photobleaching, can be eliminated; moreover, this new optical geometry optimises the collection of fluorescence. The first cross Correlation curves for Rhodamine 6G (Rh6G) in Ethanol are presented, in addition to the effect of the size of fluorescent particules (nano-beads, diameters : 20, 100 and 200 nm). The relative simplicity of the method leads us to propose sFCCS as an appropriate method for the determination of diffusion parameters of fluorophores in solution or cells. Nevertheless, progresses in the ingeniering of the optical Molecular Detection Efficiency volumes are highly desirable, in order to improve the descrimination between the cross correlated volumes.
Fluorescence fluctuation spectroscopy is applied to study molecules, passing through a small observation volume, usually subjected to diffusive or convective motion in liquid phase. We suggest that such a technique could be used to measure the areal absolute concentration of fluorophores deposited on a substrate or imbedded in a thin film, with a resolution of a few micrometers. The principle is to translate the solid substrate in front of a confocal fluorescence microscope objective and to record the subsequent fluctuations of the fluorescence intensity. The validity of this concept is investigated on model substrates (fluorescent microspheres), DNA-chips, and dye-stained histidine molecules anchored on silanized glass surfaces.