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