Optical Coherence Tomography.

<p>Differences in retinal optical coherence tomography measurements between HNPP patients (in red) and healthy controls (HC, in grey). A) Peripapillary retinal nerve fiber layer thickness (pRNFL), B) pRNFL in the papulomacular bundle (PMB), C) pRNFL in the nasal hemisphere, D) total macular volume (TMV), E) ganglion cell and inner plexiform layer (GCIP), F) inner nuclear layer (INL). P values are derived from generalized estimating equation models.</p

MRS measurements.

<p>MRS measurements.</p

Dynamic intravital FRET-FLIM in the spinal cord of healthy <i>CerTN L15</i> mice.

<p><b>(a)</b> Intensity of Cerulean and FRET-ratio (a₁·100/(a₁ + a₂) ratio) maps of axons in the spinal cord of a CerTN L15 mouse. 300×300 µm² (256×256 pixel) FRET-FLIM images are acquired every 468 ms using the p-TCSPC device. <b>(b)</b> The accuracy of the FRET-ratio in these data is quantified by the width of its distribution over an image (e.g. 9.04±1.11% corresponding to 242±76 nM calcium, t = 0 ms). This value corresponds to the expected calcium concentration in healthy neurons (44). The distributions of the FRET-ratio do not change over time (after 50 illumination steps). <b>(c)</b> Intensity of Cerulean and FRET-ratio maps (300×300 µm², 256×256 pixel) of axons in the spinal cord of a CerTN L15 mouse must be acquired every 20 s using the high-performance single-channel TCSPC to avoid pile up effects (maximally 10⁶ photons/s) which artificially reduce the fluorescence lifetime of Cerulean and increase the FRET-ratio, i.e. the apparent calcium concentration. Thus, a much lower excitation power (1.8 mW instead of 18 mW as used in the p-TCSPC setup at 850 nm excitation wavelength) was applied. The full potential of the hybrid detector could not be exploited due to the limited average counting rate of the electronics. <b>(d)</b> The distributions of the FRET-ratio corresponding to the images in (c) are similar to the distributions measured using the p-TCSPC setup. Thus, the accuracy of the FRET-ratio measured by p-TCSPC (a) and single-channel TCSPC (c) is also similar. <b>(e)</b> 75×75 µm² (131×131 pixel) FRET-ratio maps in the spinal cord of the same mouse line could be acquired every 82 ms using the p-TCSPC device. <b>(f)</b> The accuracy of the images in (e) is restored by the distribution of the FRET-ratio (9.09±1.58% corresponding to 244±10.6 nM calcium, t = 0 ms), which remains stable over time (50 illumination steps). <b>(g)</b> FRET-ratio maps of the same dimensions (75×75 µm², 131×131 pixel) must be acquired every 10 s in order to simultaneously avoid pile-up effects and to achieve the same accuracy as by p-TCSPC-FLIM. <b>(h)</b> Distributions of FRET-ration in the images in (g).</p

OCT measurements.

<p>OCT measurements.</p

Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation

<div><p>Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET <i>in vivo</i>. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm²) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm²) can be acquired every 82 ms in 115 µm depth in the spinal cord of <i>CerTN L15</i> mice. The <i>CerTN L15</i> mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of <i>CerTN L15</i> mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation.</p></div

FLIM-based Calcium calibration of TN L15.

<p>The FRET signal of TN L15 (Troponin C bound to the FRET pair Cerulean and Citrine) was measured by FLIM in buffered solutions of different free Calcium concentrations in the range 0 µM to 39 µM (Ca Calibration Buffer Kit, Invitrogen, Germany). The fluorescence decays were biexponentially approximated. In all cases, the fluorescence lifetime of the FRET-quenched Cerulean amounts to 808 ps and that of the unquenched Cerulean to 2491 ps. These values well agree according to the Strickler-Berg dependence on refractive index to the values measured in brain slices and in the brain stem of live mice. The ratio a1/(a1+a2) of FRET-quenched to unquenched Cerulean represents the FRET signal and is depicted on the ordinate. The inset shows the values for K_d and the Hill slope.</p

Vision-related quality of life.

<p>Vision-related quality of life.</p

Depth-dependent SNR and maximum imaging depth in FLIM.

<p>Three dimensional fluorescence images acquired by means of the GOI and p-TCSPC setup, respectively, at the same region <b>(a)</b> of a fluorescein-isothio-cyanate (FITC) stained skin biopsy (200×200×400 µm³) and <b>(f)</b> of a hippocampal slice of a <i>Thy1 EGFP</i> mouse, in which neuronal subsets express EGFP, (300×300×260 µm³). Corresponding depth dependent signal-to-noise ratio (ddSNR) curves are shown in <b>(b)</b> for the skin biopsy and in <b>(g)</b> for the hippocampal slice<b>.</b> Dependence of the mean fluorescence lifetime on ddSNR is depicted in <b>(c)</b> for the dermal samples and in <b>(h)</b> for the hippocampal slices. Depth dependence of the mean fluorescence lifetime of FITC in the skin biopsy is depicted in <b>(d)</b> and of EGFP in the hippocampal slice is shown in <b>(i)</b>. The corresponding widths (Gaussian full-width-at-half-maximum) of the lifetime distributions are shown in <b>(e)</b> and <b>(j)</b>, respectively. Setup parameters are listed in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060100#pone.0060100.s009" target="_blank">Material S1</a></i>.</p

Mono- and bi-exponential FLIM benchmarking experiments on standardized dyes.

<p>The values in brackets represent FWHM (full width at half maximum) of the corresponding distribution of the parameters τ₁, τ₂, a₁/(a₁+a₂), respectively, in the (mono- or bi-) exponential approximations. The experimental parameters, i.e. frame dimension, photon flux, acquisition time and count rate, are given for each FLIM detector. λ_exc  = 800 nm, λ_emission  = 593±20 nm.</p

Brain atrophy.

<p>Brain atrophy.</p