Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy

California Institute of Technology, Bioengineering, Pasadena, California 91125, USA.
Journal of Biomedical Optics (Impact Factor: 2.86). 11/2011; 16(11):116009. DOI: 10.1117/1.3647570
Source: PubMed


Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.

Download full-text


Available from: Jiun-Yann Yu, Sep 11, 2015
  • Source
    • "Recent studies have shown that temporal focusing MPE microscopy (TFMPEM) can generate widefield and axially-resolved excitation on a plane-by-plane basis [3–12]. Different diffraction components such as a grating [3–9], an optical diffuser [10], and a combination of two prisms and a grating [11] can be utilized to diffract illuminating light frequencies for temporal focusing. The most common temporal focusing configuration uses a diffraction grating to separate frequencies into monochromatic waves at different spatial angles; then recombines them at the front focal plane of an objective lens. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.
    Full-text · Article · Aug 2014 · Biomedical Optics Express
    • "Instead of allowing laser pulse traveling through the optical system with constant pulse duration, temporal focusing broadens the pulse duration along with the propagation path, and reaches the shortest pulse duration only at the focal plane of the objective. This approach, also called plane-projection multiphoton microscopy, enables video-rate wide-field optical sectioning of live tissues.[34] Rapid 3D imaging of living cells has been achieved using novel Bessel beam plane illumination microscopy.[35] "
    [Show abstract] [Hide abstract]
    ABSTRACT: Pathology informatics encompasses digital imaging and related applications. Several specialized microscopy techniques have emerged which permit the acquisition of digital images ("optical biopsies") at high resolution. Coupled with fiber-optic and micro-optic components, some of these imaging techniques (e.g., optical coherence tomography) are now integrated with a wide range of imaging devices such as endoscopes, laparoscopes, catheters, and needles that enable imaging inside the body. These advanced imaging modalities have exciting diagnostic potential and introduce new opportunities in pathology. Therefore, it is important that pathology informaticists understand these advanced imaging techniques and the impact they have on pathology. This paper reviews several recently developed microscopic techniques, including diffraction-limited methods (e.g., confocal microscopy, 2-photon microscopy, 4Pi microscopy, and spatially modulated illumination microscopy) and subdiffraction techniques (e.g., photoactivated localization microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy). This article serves as a primer for pathology informaticists, highlighting the fundamentals and applications of advanced optical imaging techniques.
    No preview · Article · May 2012
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A simple technique for remote scanning of the focal plane in temporal focusing multiphoton microscopy is demonstrated both theoretically and experimentally. A new on-axis light propagation optical setup design enables this scanning, which was considered not feasible in previous studies. The focal plane is axially displaced by the movement of a remote optical device, consisting of a double prism grating, and optionally a cylindrical lens. The displacement is linear, and its slope is inversely proportional to the square of the optical system's magnification.
    Full-text · Article · Jul 2012 · Optics Letters
Show more