Deciphering Brain Function by Miniaturized Fluorescence Microscopy in Freely Behaving Animals.

Animal behavior is regulated by environmental stimuli and is shaped by the activity of neural networks, underscoring the importance of assessing the morpho-functional properties of different populations of cells in freely behaving animals. In recent years, a number of optical tools have been developed to monitor and modulate neuronal and glial activity at the protein, cellular, or network level and have opened up new avenues for studying brain function in freely behaving animals. Tools such as genetically encoded sensors and actuators are now commonly used for studying brain activity and function through their expression in different neuronal ensembles. In parallel, microscopy has also made major progress over the last decades. The advent of miniature microscopes (mini-microscopes also called mini-endoscopes) has become a method of choice for studying brain activity at the cellular and network levels in different brain regions of freely behaving mice. This technique also allows for longitudinal investigations while animals carrying the microscope on their head are performing behavioral tasks. In this review, we will discuss mini-endoscopic imaging and the advantages that these devices offer to research. We will also discuss current limitations of and potential future improvements in mini-endoscopic imaging.

Resolution and contrast enhancement in coherent anti-Stokes Raman-scattering microscopy.

We implement switching laser mode coherent anti-Stokes Raman-scattering (SLAM-CARS) microscopy to enhance the spatial resolution and contrast in label-free vibrational microscopy. The method, based on the intensity difference between two images obtained with Gaussian and doughnut-shaped modes, does not depend on the specimen and relies on minimal modifications of the typical CARS setup. We demonstrate subdiffraction resolution imaging of myelin sheaths in a mouse brainstem. A lateral resolution of 0.36λ(p) is achieved.

Resolution and contrast enhancement in laser scanning microscopy using dark beam imaging.

Laser scanning microscopy allows for three-dimensional imaging of cells with molecular specific labeling. However the spatial resolution of optical microscopy is fundamentally limited by the diffraction of light. In the last two decades many techniques have been introduced to enhance the resolution of laser scanning microscopes. However most of these techniques impose strong constraints on the specimen or rely on complex optical systems. These constraints limit the applicability of resolution improvement to various imaging modalities and sample types. To overcome these limitations, we introduce here a novel approach, which we called Switching LAser Mode (SLAM) microscopy, to enhance resolution and contrast in laser scanning microscopy. SLAM microscopy relies on subtracting images obtained with dark and bright modes, and exploits the smaller dimensions of the dark spot of the azimuthally polarized TE 01 mode. With this approach, resolution is improved by a factor of two in confocal microscopy. The technique is not based on complex nonlinear processes and thus requires laser power similar to that used in conventional imaging, minimizing photo-damage. The flexibility of the approach enables retrofitting in commercial confocal and two-photon microscopes and opens avenues for resolution enhancement in fluorescence-independent microscopy.

Needles of longitudinally polarized light: guidelines for minimum spot size and tunable axial extent.

Optical beams exhibiting a long depth of focus and a minimum spot size can be obtained with the tight focusing of a narrow annulus of radially polarized light, leading to a needle of longitudinally polarized light. Such beams are of increasing interest for their applications, for example in optical data storage, particle acceleration, and biomedical imaging. Hence one needs to characterize the needles of longitudinally polarized light obtained with different focusing optics and incident beams. In this paper, we present analytical expressions for the electric field of such a nearly nondiffracting, subwavelength beam obtained with a parabolic mirror or an aplanatic lens. Based on these results, we give expressions of the transverse and longitudinal full widths at half maximum of the focal lines as a function of the width of the incident annular beam and we compare the performances of the two focusing systems. Then, we propose a practical solution to produce a needle of longitudinally polarized light with a tunable axial extent and a transverse width reaching the theoretical limit of 0.36λ.

Enhanced resolution in two-photon imaging using a TM(01) laser beam at a dielectric interface.

We have experimentally demonstrated that the resolution of a commercial two-photon microscope is improved using a TM(01) laser beam. With a water immersion objective having a 1.2 NA, the measured point- spread function has an area of 0.15lambda(2). We used a plane interface between dielectrics instead of an annular aperture to increase the relative contribution of the longitudinal field of the TM(01) laser beam. The results are in agreement with the vectorial diffraction theory established by Richards and Wolf [Proc. R. Soc. Lond. A253, 358 (1959)]. The TM(01) laser beam was produced with a quadrant of half-wave plates. We have also used the same mode converter to generate a TE(01) laser beam with a zero at the beam center.