Improving the axial and lateral resolution of three-dimensional fluorescence microscopy using random speckle illuminations.

We consider a fluorescence microscope in which several three-dimensional images of a sample are recorded for different speckle illuminations. We show, on synthetic data, that by summing the positive deconvolution of each speckle image, one obtains a sample reconstruction with axial and transverse resolutions that compare favorably to that of an ideal confocal microscope.

Bessel-like photonic nanojets from core-shell sub-wavelength spheres.

It is accepted so far that the formation of photonic nanojets requires the use of large dielectric spheres (several wavelengths in diameter). Here we show both numerically and experimentally that similar effects can be obtained with properly engineered sub-wavelength core-shell colloids. The design of the spheres is strongly inspired by a far-field approach for the generation of Bessel beams.

Development of a nonlinear fiber-optic spectrometer for human lung tissue exploration.

Several major lung pathologies are characterized by early modifications of the extracellular matrix (ECM) fibrillar collagen and elastin network. We report here the development of a nonlinear fiber-optic spectrometer, compatible with an endoscopic use, primarily intended for the recording of second-harmonic generation (SHG) signal of collagen and two-photon excited fluorescence (2PEF) of both collagen and elastin. Fiber dispersion is accurately compensated by the use of a specific grism-pair stretcher, allowing laser pulse temporal width around 70 fs and excitation wavelength tunability from 790 to 900 nm. This spectrometer was used to investigate the excitation wavelength dependence (from 800 to 870 nm) of SHG and 2PEF spectra originating from ex vivo human lung tissue samples. The results were compared with spectral responses of collagen gel and elastin powder reference samples and also with data obtained using standard nonlinear microspectroscopy. The excitation-wavelength-tunable nonlinear fiber-optic spectrometer presented in this study allows performing nonlinear spectroscopy of human lung tissue ECM through the elastin 2PEF and the collagen SHG signals. This work opens the way to tunable excitation nonlinear endomicroscopy based on both distal scanning of a single optical fiber and proximal scanning of a fiber-optic bundle.

Refractive effects in coherent anti-Stokes Raman scattering microscopy.

Coherent anti-Stokes Raman scattering (CARS) microscopy with high sensitivity and high three- dimensional resolution has been developed for the vibrational imaging of chemical species. Due to the coherent nature of the CARS emission, it has been reported that the detection of epi-CARS and forward-CARS (F-CARS) signals depends on the size and shape of the sample. We investigate theoretically and experimentally the effects on the CARS signal of refractive index mismatches between the sample and its surroundings. Backward-CARS and F-CARS signals are measured for different polystyrene bead diameters embedded in different refractive index solvents. We show that index mismatches result in a backward-reflected F-CARS signal that generally dominates the experimentally backward-detected signal. Simulations based on geometrical and wave optics comparing forward- and backward-detected signals for polystyrene beads embedded in different index solvents confirm our findings. Furthermore, we demonstrate that the maxima of forward- and backward-detected signals are generated at different positions along the optical axis in the sample if refractive index mismatches are present between the sample and its surroundings.

Increasing the lateral resolution of 4Pi fluorescence microscopes.

The axial resolution of fluorescence microscopes can be considerably improved by superposing two illumination beams and by adding coherently the two wavefronts emitted by the luminescent sample. This solution has been implemented in 4Pi microscopes. Theoretical and experimental results have shown that a considerable improvement of the axial resolution can be obtained with these microscopes. However, the lateral resolution remains limited by diffraction. We propose a configuration of a 4Pi microscope in which the lateral displacement of the source modifies the collection efficiency function (CEF). Numerical calculations based on an approximate scalar theory and on exact vector-wave-optics results of the field distribution of the electromagnetic field in image space show that the lateral extent of the CEF can be reduced by a factor greater than 2 with respect to the diffraction limit. We show that, with this solution, the resolution in the transverse plane of 4Pi type B and 4Pi type C microscopes can be improved significantly.