Table 1.
Mono- and bi-exponential FLIM benchmarking experiments on standardized dyes.
Figure 1.
Depth-dependent SNR and maximum imaging depth in FLIM.
Three dimensional fluorescence images acquired by means of the GOI and p-TCSPC setup, respectively, at the same region (a) of a fluorescein-isothio-cyanate (FITC) stained skin biopsy (200×200×400 µm3) and (f) of a hippocampal slice of a Thy1 EGFP mouse, in which neuronal subsets express EGFP, (300×300×260 µm3). Corresponding depth dependent signal-to-noise ratio (ddSNR) curves are shown in (b) for the skin biopsy and in (g) for the hippocampal slice. Dependence of the mean fluorescence lifetime on ddSNR is depicted in (c) for the dermal samples and in (h) for the hippocampal slices. Depth dependence of the mean fluorescence lifetime of FITC in the skin biopsy is depicted in (d) and of EGFP in the hippocampal slice is shown in (i). The corresponding widths (Gaussian full-width-at-half-maximum) of the lifetime distributions are shown in (e) and (j), respectively. Setup parameters are listed in Material S1.
Figure 2.
Dynamic FRET-FLIM in living hippocampal slices of CerTN L15 mice.
Projections of three dimensional (3D) fluorescence data sets (a) (221×221×12 µm3, 492×492×5 voxel) acquired by the GOI setup and (c) (200×200×12 µm3, 512×512×5 voxel) acquired by the p-TCSPC setup in a living hippocampal slice of a CerTN L15 mouse. The fluorescence lifetime distributions of the FRET-quenched and unquenched Cerulean corresponding to (a) and (c) are depicted in the graphs (d) and (b). (e) and (f) show corresponding 3D a1·100/(a2+a1) images (FRET-ratio images) before and 7 minutes after perfusion with a 100 mM KCl solution as acquired with the GOI and p-TCSPC setup, respectively. The depolarization of the neurons in the presence of highly concentrated K+ ions leads to an increase of neuronal calcium level, as shown by increased FRET signal. (g) Time-lapse images showing neurons in the demarcated area in (e) depicting the increase of calcium in neuronal somata. The graph (h) shows the increase of calcium level in the soma indicated in (g) by the arrowhead. 3D unit = 28 µm, scale bar = 20 µm. (i) Time-lapse showing neurons in the demarcated area in (f) depicting the increase of calcium in neuronal somata and processes. The magenta arrows in (i) indicate the incomplete depolarization immediately after application, i.e. high calcium levels are still led from the dendrites through the soma to the axon before the neuron is completely flooded by calcium. The graphs in (k) show the neuronal calcium level at the indicated time points along the white line in (i), reaching from the soma to the axon. The magenta arrows in (k) correspond to the Calcium increase also indicated by magenta arrows in the image series (i). The same process of neuronal depolarization as in (h) for the GOI setup can be observed in (j) for the p-TCSPC setup. 3D unit = 30 µm, scale bar = 20 µm. Imaging was performed starting from 15–30 µm depth in tissue, which is beyond the glial scar.
Figure 3.
FLIM-based Calcium calibration of TN L15.
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 Kd and the Hill slope.
Figure 4.
Dynamic intravital FRET-FLIM in the spinal cord of healthy CerTN L15 mice.
(a) 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) 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). (c) 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. (d) 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. (e) 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. (f) 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). (g) 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. (h) Distributions of FRET-ration in the images in (g).
Figure 5.
Dynamic intravital FRET-FLIM in the brain stem of CerTN L15 mice.
(a) Merged 3D a1·100/(a2+a1) image (FRET signal image, 150×150×30 µm3, 256×256×16 voxel in 90 µm depth) showing neuronal calcium and 3D data set of the tdRFP expressing immune cells recorded by the p-TCSPC setup. (b) As shown in the demarcated area in (a), the calcium level in somata and processes interacting with immune cells, directly at the contact site, is significantly higher (FRET signal of up to 58%, 1.66 µM calcium, orange-red area in (b)) than in unaffected somata and processes (FRET signal of approximately 8%, 110 nM calcium). The corresponding fluorescence lifetime distributions of the FRET-quenched and unquenched Cerulean, as shown in the graph (c), are similar to those determined in hippocampal slices. (d) The perfusion with a 100 mM KCl solution led to the depolarization of the neurons followed by a calcium concentration increase. The effect is more prominent in cells with low calcium than in the already affected neurons. (e) Time lapse of 3D a1·100/(a2+a1) images of two neurons in contact with immune cells and then being subject to K+ ion increase. The graph (f) shows the absolute values of the FRET signal in the somata numbered from 1 to 3 in (d). 3D unit = 15 µm, scale bar = 12 µm. Imaging was performed between 80 µm to 100 µm depth in tissue, at a site with high immune cell infiltration grade (Material S1).