Francesco S. Pavone

Calibration of optical tweezers with positional detection in the back focal plane

We explain and demonstrate a new method of force and position calibrations for optical tweezers with back-focal-plane photodetection. The method combines power spectral measurements of thermal motion and the response to a sinusoidal motion of a translation stage. It consequently does not use the drag coefficient of the trapped object as an input. Thus, neither the viscosity, nor the size of the trapped object, nor its distance to nearby surfaces needs to be known. The method requires only a low level of instrumentation and can be applied in situ in all spatial dimensions. It is both accurate and precise: true values are returned, with small error bars. We tested this experimentally, near and far from surfaces in the lateral directions. Both position and force calibrations were accurate to within 3%. To calibrate, we moved the sample with a piezoelectric translation stage, but the laser beam could be moved instead, e.g., by acousto-optic deflectors. Near surfaces, this precision requires an improved formula for the hydrodynamical interaction between an infinite plane and a microsphere in nonconstant motion parallel to it. We give such a formula.

Scoring of collagen organization in healthy and diseased human dermis by multiphoton microscopy

We have used nonlinear imaging to evaluate collagen organization in connective tissue ex-vivo samples. Image analysis methods were tested on healthy dermis, normal scars, and keloids. The evaluation of the second harmonic to autofluorescence aging index of dermis (SAAID) has allowed a first characterization of tissues by scoring the collagen/elastin content. Further analyses on collagen morphology in healthy dermis and keloids were performed by image-pattern analysis of SHG images. The gray-level co-occurrence matrix (GLCM) analysis method has allowed classification of different tissues based on the evaluation of geometrical arrangement of collagen fibrillar bundles, whereas a pattern analysis of the FFT images has allowed the discrimination of different tissues based on the anisotropy of collagen fibers distribution. This multiple scoring method represents a promising tool to be extended to other collagen disorders, as well as to be used in in-vivo skin-imaging applications.

Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain

Elucidating the neural pathways that underlie brain function is one of the greatest challenges in neuroscience. Light sheet based microscopy is a cutting edge method to map cerebral circuitry through optical sectioning of cleared mouse brains. However, the image contrast provided by this method is not sufficient to resolve and reconstruct the entire neuronal network. Here we combined the advantages of light sheet illumination and confocal slit detection to increase the image contrast in real time, with a frame rate of 10 Hz. In fact, in confocal light sheet microscopy (CLSM), the out-of-focus and scattered light is filtered out before detection, without multiple acquisitions or any post-processing of the acquired data. The background rejection capabilities of CLSM were validated in cleared mouse brains by comparison with a structured illumination approach. We show that CLSM allows reconstructing macroscopic brain volumes with sub-cellular resolution. We obtained a comprehensive map of Purkinje cells in the cerebellum of L7-GFP transgenic mice. Further, we were able to trace neuronal projections across brain of thy1-GFP-M transgenic mice. The whole-brain high-resolution fluorescence imaging assured by CLSM may represent a powerful tool to navigate the brain through neuronal pathways. Although this work is focused on brain imaging, the macro-scale high-resolution tomographies affordable with CLSM are ideally suited to explore, at micron-scale resolution, the anatomy of different specimens like murine organs, embryos or flies.

Photothermally-induced disordered patterns of corneal collagen revealed by SHG imaging.

The loss of organization of the corneal collagen lattice induced by photothermal effects was analyzed by using second-harmonic generation (SHG) imaging. Porcine cornea samples were treated with low-power laser irradiation in order to get localized areas of tissue disorganization. The disorder induced within the irradiated area of corneal stroma was quantified by means of Discrete Fourier Transform, auto-correlation and entropy analyses of the SHG images. Polarization modulated SHG measurements allowed to probe the changes in the structural anisotropy of sub-micron hierarchical levels of the stromal collagen. Our results emphasize the great potential of the SHG imaging to detect subtle modifications in the collagen assembly. The proposed analytical methods may be used to track several genetic, pathologic, accidental or surgical-induced disorder states of biological tissues.

Positioning and Elongation of the Fission Yeast Spindle by Microtubule-Based Pushing

In eukaryotic cells, proper position of the mitotic spindle is necessary for successful cell division and development. We explored the nature of forces governing the positioning and elongation of the mitotic spindle in Schizosaccharomyces pombe. We hypothesized that astral microtubules exert mechanical force on the S. pombe spindle and thus help align the spindle with the major axis of the cell. Microtubules were tagged with green fluorescent protein (GFP) and visualized by two-photon microscopy. Forces were inferred both from time-lapse imaging of mitotic cells and, more directly, from mechanical perturbations induced by laser dissection of the spindle and astral microtubules. We found that astral microtubules push on the spindle poles in S. pombe, in contrast to the pulling forces observed in a number of other cell types. Further, laser dissection of the spindle midzone induced spindle collapse inward. This offers direct evidence in support of the hypothesis that spindle elongation is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Broken spindles recovered and mitosis completed as usual. We propose a model of spindle centering and elongation by microtubule-based pushing forces.

Contrast and depth enhancement in two-photon microscopy of human skin ex vivo by use of optical clearing agents

We investigate the application of hyperosmotic optical clearing agents to improve the image contrast and penetration depth in two-photon microscopy of human dermis ex vivo. We show that the agents glycerol, propylene glycol, and glucose all convey significant improvements and we provide results on their dynamic behaviour and the reversibility of the effect. At suitable concentrations, such agents have the potential to be compatible with living tissue and may possibly enhance in-vivo deep-tissue imaging.

Multiphoton multifocal microscopy exploiting a diffractive optical element

Multiphoton multifocal microscopy (MMM) usually has been achieved through a combination of galvo scanners with microlens arrays, with rotating disks of microlens arrays, and cascaded beam splitters with asynchronous rastering of scanning mirrors. Here we describe the achievement of a neat and compact MMM by use of a high-diffraction-efficiency diffractive-optic element that generates a multiple-spot grid of uniform intensity to achieve higher fidelity in imaging of live cells at adequate speeds.

Combined intracellular three-dimensional imaging and selective nanosurgery by a nonlinear microscope.

We use near-IR femtosecond laser pulses for a combination of microscopy and nanosurgery on fluorescently labeled structures within living cells. Three-dimensional reconstructions of microtubule structures tagged with green fluorescent protein (GFP) are made during different phases of the cell cycle. Further, the microtubules are dissected using the same laser beam but with a higher laser power than for microscopy. We establish the viability of this technique for the cells of a fission yeast, which is a common model to study the mechanics of cell division. We show that nanosurgery can be performed with submicrometer precision and without visible collateral damage to the cell. The energy is primarily absorbed by the GFP molecules, and not by other native structures in the cell. GFP is particularly suitable for multiphoton excitation, as its excitation wavelength near 900 nm is benign for most cellular structures. The ability to use GFP to label structures for destruction by multiphoton excitation may be a valuable tool in cell biology.

Palette of fluorinated voltage-sensitive hemicyanine dyes

Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440–670 nm; the two-photon excitation range is 900–1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.

Calibration of optical tweezers with differential interference contrast signals

A comparison of different calibration methods for optical tweezers with the differential interference contrast (DIC) technique was performed to establish the uses and the advantages of each method. A detailed experimental and theoretical analysis of each method was performed with emphasis on the anisotropy involved in the DIC technique and the noise components in the detection. Finally, a time of flight method that permits the reconstruction of the optical potential well was demonstrated.