Simultaneous Two- and Three-Photon Deep Imaging of Autofluorescence in Bacterial Communities.

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.

Analysis of intensity correlation enhanced plasmonic structured illumination microscopy.

We propose to enhance the performance of localized plasmon structured illumination microscopy (LP-SIM) via intensity correlations. LP-SIM uses sub-wavelength illumination patterns to encode high spatial frequency information. It can enhance the resolution up to three-fold before gaps in the optical transfer function (OTF) support arise. For blinking fluorophores or for quantum antibunching, an intensity correlation analysis induces higher harmonics of the illumination pattern and enlarges the effective OTF. This enables ultrahigh resolutions without gaps in the OTF support, and thus a fully deterministic imaging scheme. We present simulations that include shot and external noise and demonstrate the resolution power under realistic photon budgets. The technique has potential in light microscopy where low-intensity illumination is paramount while aiming for high spatial but moderate temporal resolutions.

Extended focal depth Fourier domain optical coherence microscopy with a Bessel-beam - LP02 mode - from a higher order mode fiber.

We present a robust fiber-based setup for Bessel-like beam extended depth-of-focus Fourier-domain optical coherence microscopy, where the Bessel-like beam is generated in a higher order mode fiber module. In this module a stable guided LP02 core mode is selectively excited by a long period grating written in the higher order mode fiber. Imaging performance of this system in terms of lateral resolution and depth of focus was analyzed using samples of suspended microbeads and compared to the case where illumination is provided by the fundamental LP01 mode of a single mode fiber. Illumination with the LP02 mode allowed for a lateral resolution down to 2.5 µm as compared to 4.5 µm achieved with the LP01 mode of the single mode fiber. A three-fold enhancement of the depth of focus compared to a Gaussian beam with equally tight focus is achieved with the LP02 mode. Analysis of the theoretical lateral point spread functions for the case of LP01 and LP02 illumination agrees well with the experimental data. As the design space of waveguides and long-period gratings allows for further optimization of the beam parameters of the generated Bessel-like beams in an all-fiber module, this approach offers a robust and yet flexible alternative to free-space optics approaches or the use of conical fiber tips.

Analysis of super-resolution via 3D structured illumination intensity correlation microscopy.

Intensity correlation microscopy (ICM), which is prominently known through antibunching microscopy or super-resolution optical fluctuation imaging (SOFI), provides super-resolution through a correlation analysis of antibunching of independent quantum emitters or temporal fluctuations of blinking fluorophores. For correlation order m the PSF in the signal is effectively taken to the mth power, and is thus directly shrunk by the factor m. Combined with deconvolution, a close to linear resolution improvement of factor m can be obtained. Yet, analysis of high correlation orders is challenging, which limits the achievable resolutions. Here we propose to use three dimensional structured illumination along with mth-order correlation analysis to obtain an enhanced scaling of up to m + m = 2m. Including the stokes shift or plasmonic sub-wavelength illumination enhancements beyond 2m can be achieved. Hence, resolutions far below the diffraction limit in full 3D imaging and with already low correlation orders, can potentially be achieved. Since ICM operates in the linear regime our approach may be particularly promising for enhancing the resolution in biological imaging at low illumination levels.

Incoherent Diffractive Imaging via Intensity Correlations of Hard X Rays.

Established x-ray diffraction methods allow for high-resolution structure determination of crystals, crystallized protein structures, or even single molecules. While these techniques rely on coherent scattering, incoherent processes like fluorescence emission-often the predominant scattering mechanism-are generally considered detrimental for imaging applications. Here, we show that intensity correlations of incoherently scattered x-ray radiation can be used to image the full 3D arrangement of the scattering atoms with significantly higher resolution compared to conventional coherent diffraction imaging and crystallography, including additional three-dimensional information in Fourier space for a single sample orientation. We present a number of properties of incoherent diffractive imaging that are conceptually superior to those of coherent methods.

Superresolving Imaging of Arbitrary One-Dimensional Arrays of Thermal Light Sources Using Multiphoton Interference.

We propose to use multiphoton interferences of photons emitted from statistically independent thermal light sources in combination with linear optical detection techniques to reconstruct, i.e., image, arbitrary source geometries in one dimension with subclassical resolution. The scheme is an extension of earlier work [S. Oppel et al., Phys. Rev. Lett. 109, 233603 (2012)], where N regularly spaced sources in one dimension were imaged by use of the Nth-order intensity correlation function. Here, we generalize the scheme to reconstruct any number of independent thermal light sources at arbitrary separations in one dimension, exploiting intensity correlation functions of order m≥3. We present experimental results confirming the imaging protocol and provide a rigorous mathematical proof for the obtained subclassical resolution.