On the fundamental imaging-depth limit in two-photon microscopy

One of the principle advantages of two-photon microscopy over one-photon techniques is that it can provide high-resolution images from very deep within living tissue. While imaging depths of 500 micron in brain tissue have become standard performance, larger depths have been inaccessible mainly due to the power limitation of current femto-second laser sources. Here we investigate strategies to improve the imaging depth in two-photon microscopy. In particular, we show that the two-photon imaging depth can be significantly improved using optically amplified femto-second laser pulses. Using a regenerative amplifier as the excitation source we obtained images of stained vasculature and GFP-labeled neurons down to a depth of about 1000 micron below the brain surface in the cortex of mice in vivo. The maximum imaging depth was now limited by out-of-focus background fluorescence and not by the available excitation power. In order to provide a quantitative description of this behavior, we have investigated the effects of scattering on fluo-rescence excitation and detection. The most prominent parameters that influence the maximum two-photon imaging depth are the excitation numerical aperture and the sample staining charac-teristics. The largest depths can be achieved with the largest excitation numerical aperture and the lowest out-of-focus volume staining

Methanol immersion reduces spherical aberration of water dipping lenses at long wavelengths used in multi-photon laser scanning microscopy

Dipping objectives were tested for multi-photon laser scanning microscopy, since their large working distances are advantageous for thick specimens and the absence of a coverslip facilitates examination of living material. Images of fluorescent bead specimens, particularly at wavelengths greater than 850 nm showed defects consistent with spherical aberration. Substituting methanol for water as the immersion medium surrounding the beads corrected these defects and produced an increase in fluorescence signal intensity. The same immersion method was applied to two representative biological samples of fixed tissue: mouse brain labeled with FITC for tubulin and mouse gut in which the Peyer’s patches were labeled with Texas Red bilosomes. Tissue morphology was well preserved by methanol immersion of both tissues; the two-photon-excited fluorescence signal was six times higher than in water and the depth of penetration of useful imaging was doubled. No modification of the microscope was needed except the provision of a ring to retain a sufficient depth of methanol for imaging

High-speed focal modulation microscopy using acousto-optical modulators

Focal Modulation Microscopy (FMM) is a single-photon excitation fluorescence microscopy technique which effectively rejects the out-of-focus fluorescence background that arises when imaging deep inside biological tissues. Here, we report on the implementation of FMM in which laser intensity modulation at the focal plane is achieved using acousto-optic modulators (AOM). The modulation speed is greatly enhanced to the MHz range and thus enables real-time image acquisition. The capability of FMM is demonstrated by imaging fluorescence labeled vasculatures in mouse brain as well as self-made tissue phantom

Electric field Monte Carlo simulations of focal field distributions produced by tightly focused laser beams in tissues

The focal field distribution of tightly focused laser beams in turbid media is sensitive to optical scattering and therefore of direct relevance to image quality in confocal and nonlinear microscopy. A model that considers both the influence of scattering and diffraction on the amplitude and phase of the electric field in focused beam geometries is required to describe these distorted focal fields. We combine an electric field Monte Carlo approach that simulates the electric field propagation in turbid media with an angular-spectrum representation of diffraction theory to analyze the effect of tissue scattering properties on the focal field. In particular, we examine the impact of variations in the scattering coefficient (µs), single-scattering anisotropy (g), of the turbid medium and the numerical aperture of the focusing lens on the focal volume at various depths. The model predicts a scattering-induced broadening, amplitude loss, and depolarization of the focal field that corroborates experimental results. We find that both the width and the amplitude of the focal field are dictated primarily by µs with little influence from g. In addition, our model confirms that the depolarization rate is small compared to the amplitude loss of the tightly focused field

Multimode fibre:Light-sheet microscopy at the tip of a needle

We also thank the UK Engineering and Physics Sciences Research Council for funding under grant EP/J01771X/1. Finally, we would like to thank EXCELLENT TEAMS (CZ.1.07/2.3.00/30.0005) from European Social Fund and CEITEC - Central European Institute of Technology (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund for support.Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre - an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.Publisher PDFPeer reviewe

A CANDLE for a deeper in-vivo insight

A new Collaborative Approach for eNhanced Denoising under Low-light Excitation (CANDLE) is introduced for the processing of 3D laser scanning multiphoton microscopy images. CANDLE is designed to be robust for low signal-to-noise ratio (SNR) conditions typically encountered when imaging deep in scattering biological specimens. Based on an optimized non-local means filter involving the comparison of filtered patches, CANDLE locally adapts the amount of smoothing in order to deal with the noise inhomogeneity inherent to laser scanning fluorescence microscopy images. An extensive validation on synthetic data, images acquired on microspheres and in vivo images is presented. These experiments show that the CANDLE filter obtained competitive results compared to a state-of-the-art method and a locally adaptive optimized non-local means filter, especially under low SNR conditions (PSNR < 8 dB). Finally, the deeper imaging capabilities enabled by the proposed filter are demonstrated on deep tissue in vivo images of neurons and fine axonal processes in the Xenopus tadpole brain.We want to thank Florian Luisier for providing free plugin of his PureDenoise filter. We also want to thank Markku Makitalo for providing the code of their OVST. This study was supported by the Canadian Institutes of Health Research (CIHR, MOP-84360 to DLC and MOP-77567 to ESR) and Cda (CECR)-Gevas-OE016. MM holds a fellowship from the Deutscher Akademischer Austasch Dienst (DAAD) and a McGill Principal's Award. ESR is a tier 2 Canada Research Chair. This work has been partially supported by the Spanish Health Institute Carlos III through the RETICS Combiomed, RD07/0067/2001. This work benefited from the use of ImageJ.Coupé, P.; Munz, M.; Manjón Herrera, JV.; Ruthazer, ES.; Collins, DL. (2012). A CANDLE for a deeper in-vivo insight. Medical Image Analysis. 16(4):849-864. https://doi.org/10.1016/j.media.2012.01.002S84986416

Acousto-optical Scanning-Based High-Speed 3D Two-Photon Imaging In Vivo.

Recording of the concerted activity of neuronal assemblies and the dendritic and axonal signal integration of downstream neurons pose different challenges, preferably a single recording system should perform both operations. We present a three-dimensional (3D), high-resolution, fast, acousto-optic two-photon microscope with random-access and continuous trajectory scanning modes reaching a cubic millimeter scan range (now over 950 × 950 × 3000 μm3) which can be adapted to imaging different spatial scales. The resolution of the system allows simultaneous functional measurements in many fine neuronal processes, even in dendritic spines within a central core (>290 × 290 × 200 μm3) of the total scanned volume. Furthermore, the PSF size remained sufficiently low (PSFx < 1.9 μm, PSFz < 7.9 μm) to target individual neuronal somata in the whole scanning volume for simultaneous measurement of activity from hundreds of cells. The system contains new design concepts: it allows the acoustic frequency chirps in the deflectors to be adjusted dynamically to compensate for astigmatism and optical errors; it physically separates the z-dimension focusing and lateral scanning functions to optimize the lateral AO scanning range; it involves a custom angular compensation unit to diminish off-axis angular dispersion introduced by the AO deflectors, and it uses a high-NA, wide-field objective and high-bandwidth custom AO deflectors with large apertures. We demonstrate the use of the microscope at different spatial scales by first showing 3D optical recordings of action potential back propagation and dendritic Ca2+ spike forward propagation in long dendritic segments in vitro, at near-microsecond temporal resolution. Second, using the same microscope we show volumetric random-access Ca2+ imaging of spontaneous and visual stimulation-evoked activity from hundreds of cortical neurons in the visual cortex in vivo. The selection of active neurons in a volume that respond to a given stimulus was aided by the real-time data analysis and the 3D interactive visualization accelerated selection of regions of interest

A Compact Multiphoton 3D Imaging System for Recording Fast Neuronal Activity

We constructed a simple and compact imaging system designed specifically for the recording of fast neuronal activity in a 3D volume. The system uses an Yb:KYW femtosecond laser we designed for use with acousto-optic deflection. An integrated two-axis acousto-optic deflector, driven by digitally synthesized signals, can target locations in three dimensions. Data acquisition and the control of scanning are performed by a LeCroy digital oscilloscope. The total cost of construction was one order of magnitude lower than that of a typical Ti:sapphire system. The entire imaging apparatus, including the laser, fits comfortably onto a small rig for electrophysiology. Despite the low cost and simplicity, the convergence of several new technologies allowed us to achieve the following capabilities: i) full-frame acquisition at video rates suitable for patch clamping; ii) random access in under ten microseconds with dwelling ability in the nominal focal plane; iii) three-dimensional random access with the ability to perform fast volume sweeps at kilohertz rates; and iv) fluorescence lifetime imaging. We demonstrate the ability to record action potentials with high temporal resolution using intracellularly loaded potentiometric dye di-2-ANEPEQ. Our design proffers easy integration with electrophysiology and promises a more widespread adoption of functional two-photon imaging as a tool for the study of neuronal activity. The software and firmware we developed is available for download at http://neurospy.org/ under an open source license

Line-Scanning Particle Image Velocimetry: An Optical Approach for Quantifying a Wide Range of Blood Flow Speeds in Live Animals

The ability to measure blood velocities is critical for studying vascular development, physiology, and pathology. A key challenge is to quantify a wide range of blood velocities in vessels deep within living specimens with concurrent diffraction-limited resolution imaging of vascular cells. Two-photon laser scanning microscopy (TPLSM) has shown tremendous promise in analyzing blood velocities hundreds of micrometers deep in animals with cellular resolution. However, current analysis of TPLSM-based data is limited to the lower range of blood velocities and is not adequate to study faster velocities in many normal or disease conditions.We developed line-scanning particle image velocimetry (LS-PIV), which used TPLSM data to quantify peak blood velocities up to 84 mm/s in live mice harboring brain arteriovenous malformation, a disease characterized by high flow. With this method, we were able to accurately detect the elevated blood velocities and exaggerated pulsatility along the abnormal vascular network in these animals. LS-PIV robustly analyzed noisy data from vessels as deep as 850 µm below the brain surface. In addition to analyzing in vivo data, we validated the accuracy of LS-PIV up to 800 mm/s using simulations with known velocity and noise parameters.To our knowledge, these blood velocity measurements are the fastest recorded with TPLSM. Partnered with transgenic mice carrying cell-specific fluorescent reporters, LS-PIV will also enable the direct in vivo correlation of cellular, biochemical, and hemodynamic parameters in high flow vascular development and diseases such as atherogenesis, arteriogenesis, and vascular anomalies