Jerome Mertz

Two-photon microscopy in brain tissue: parameters influencing the imaging depth

Light scattering by tissue limits the imaging depth of two-photon microscopy and its use for functional brain imaging in vivo. We investigate the influence of scattering on both fluorescence excitation and collection, and identify tissue and instrument parameters that limit the imaging depth in the brain. (i) In brain slices, we measured that the scattering length at lambda=800 nm is a factor 2 higher in juvenile cortical tissue (P14-P18) than in adult tissue (P90). (ii) In a detection geometry typical for in vivo imaging, we show that the collected fraction of fluorescence drops at large depths, and that it is proportional to the square of the effective angular acceptance of the detection optics. Matching the angular acceptance of the microscope to that of the objective lens can result in a gain of approximately 3 in collection efficiency at large depths (>500 microm). A low-magnification (20x), high-numerical aperture objective (0.95) further increases fluorescence collection by a factor of approximately 10 compared with a standard 60x-63x objective without compromising the resolution. This improvement should allow fluorescence measurements related to neuronal or vascular brain activity at >100 microm deeper than with standard objectives.

Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy

We demonstrate that simultaneous second-harmonic generation (SHG) and two-photon-excited fluorescence (TPEF) can be used to rapidly image biological membranes labeled with a styryl dye. The SHG power is made compatible with the TPEF power by use of near-resonance excitation, in accord with a model based on the theory of phased-array antennas, which shows that the SHG radiation is highly structured. Because of its sensitivity to local asymmetry, SHG microscopy promises to be a powerful tool for the study of membrane dynamics.

Optical sectioning microscopy with planar or structured illumination

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.

Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers

We generalize a simple model describing second-harmonic generation (2HG) by highly focused excitation light to allow the possibility of sample inhomogeneities. Depending on their spatial frequencies these inhomogeneities can significantly modify the 2HG radiation patterns and total powers, and in certain cases provoke backward 2HG propagation. Examples are considered where the distribution of 2HG radiators is axially periodic or spherically localized, illustrating the effects of sample structure or clustering. Our model is specifically applicable to 2HG microscopy geometries.

Radiative absorption, fluorescence, and scattering of a classical dipole near a lossless interface: a unified description

A simple input–output formalism based on the Lorentz reciprocity theorem is presented for the study of a classical radiating dipole near a lossless interface. The problems of dipole absorption, fluorescence, and scattering are considered in a unified description, and the effects of the interface (a simple dielectric here) are shown to be broadly twofold. First, the channeling of radiation into and out of the dipole is modified. Second, the intrinsic dipole polarizability is found to be modified, leading to an effective absorption (or scattering) cross section that depends on the states of both the dipole and the driving field. These results are particularly applicable to studies involving evanescent-wave microscopy.

Nonlinear microscopy: new techniques and applications

Nonlinear microscopy, a general term that embraces any microscopy technique based on nonlinear optics, is further establishing itself as an important tool in neurobiology. Recent advances in labels, labeling techniques, and the use of native or genetically encoded contrast agents have bolstered the capacity of nonlinear microscopes to image the structure and function of not just single cells but of entire networks of cells. Along with novel strategies to image over exceptionally long durations and with increased depth penetration in living brains, these advances are opening new opportunities in neurobiology that were previously unavailable.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection.

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Ultra-deep two-photon fluorescence excitation in turbid media

An important application of two-photon excited fluorescence (TPEF) microscopy is to provide high-resolution images from deep within scattering media. We investigate strategies to further improve TPEF penetration depth by considering the effects of scattering on fluorescence generation and collection separately. In particular, we demonstrate that the redistribution of laser power into higher energy pulses by means of a regenerative amplifier improves the TPEF depth penetration by two to three excitation scattering mean free paths.

Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution

We describe a simple two-photon fluorescence imaging strategy, called targeted path scanning (TPS), to monitor the dynamics of spatially extended neuronal networks with high spatiotemporal resolution. Our strategy combines the advantages of mirror-based scanning, minimized dead time, ease of implementation, and compatibility with high-resolution low-magnification objectives. To demonstrate the performance of TPS, we monitor the calcium dynamics distributed across an entire juvenile rat hippocampus (>1.5mm), at scan rates of 100 Hz, with single cell resolution and single action potential sensitivity. Our strategy for fast, efficient two-photon microscopy over spatially extended regions provides a particularly attractive solution for monitoring neuronal population activity in thick tissue, without sacrificing the signal-to-noise ratio or high spatial resolution associated with standard two-photon microscopy. Finally, we provide the code to make our technique generally available.

Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy

We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. The method involves acquiring two images, one with nonuniform illumination (in our case, speckle) and another with uniform illumination (in our case, randomized speckle). An evaluation of the local contrast in the speckle-illumination image provides an optically sectioned image with low resolution. This is complemented with high-resolution information obtained from the uniform-illumination image. A fusion of both images leads to a full resolution image that is optically sectioned across all spatial frequencies. This hybrid illumination method is fast, robust, and generalizable to a variety of illumination and imaging configurations.