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Fluorescence Lifetime Imaging of pH along the Secretory Pathway.

ABSTRACT: Many cellular processes are dependent on correct pH levels, and this is especially important for the secretory pathway. Defects in pH homeostasis in distinct organelles cause a wide range of diseases, including disorders of glycosylation and lysosomal storage diseases. Ratiometric imaging of the pH-sensitive mutant of green fluorescent protein, pHLuorin, has allowed for targeted pH measurements in various organelles, but the required sequential image acquisition is intrinsically slow and therefore the temporal resolution is unsuitable to follow the rapid transit of cargo between organelles. Therefore, we applied fluorescence lifetime imaging microscopy (FLIM) to measure intraorganellar pH with just a single excitation wavelength. We first validated this method by confirming the pH in multiple compartments along the secretory pathway and compared the pH values obtained by the FLIM-based measurements with those obtained by conventional ratiometric imaging. Then, we analyzed the dynamic pH changes within cells treated with Bafilomycin A1, to block the vesicular ATPase, and Brefeldin A, to block endoplasmic reticulum (ER)-Golgi trafficking. Finally, we followed the pH changes of newly synthesized molecules of the inflammatory cytokine tumor necrosis factor-α while they were in transit from the ER via the Golgi to the plasma membrane. The toolbox we present here can be applied to measure intracellular pH with high spatial and temporal resolution and can be used to assess organellar pH in disease models.

SUBMITTER: Linders PTA

PROVIDER: S-EPMC8787756 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a novel technique that measures in vivo autofluorescence intensity decay over time of endogenous fluorophores in the retina. The Heidelberg Engineering FLIO system was used to obtain two 30 degree scans centered on the fovea of both eyes. The FLIO system uses a 473nm blue scanning laser light source and the emitted fluorescence is detected in two wavelengths channels, short and long spectral channels (SSC, LSC). Since the mydriatic status influence the FLIO result, the impact of mydriasis on FLIO need to be clarified. In this prospective, observational study, the impact of mydriasis on measurements from fluorescence lifetime imaging ophthalmoscope (FLIO) images in normal subjects were evaluated. 12 healthy participants (24 eyes) were volunteered and all subjects were scanned twice and the mean fluorescence lifetime (?m) values were computed with dilation and without dilation on different days. Intraclass correlation coefficients (ICC) and coefficients of variation (CV) were calculated from the measured ?m in dilated, nondilated and between the dilated and non-dilated setting. Test duration was also compared and correlated with lifetimes in both settings. Repeatability was excellent for both the dilation and non-dilation settings (ICC; 0.967-0.996; 0.926-0.986, respectively). The agreement between the dilation and non-dilation settings, however, were lower (ICC; 0.688-0.970). The ?m in the non-dilation setting was significantly longer than in the dilation setting for the SSC (P<0.05). The FLIO test duration in the non-dilation setting was significantly longer than with dilation for the SSC (P <0.05). Although good repeatability in ?m measurements between imaging sessions were observed both with and without dilation, the agreement was not as good when comparing dilated with non-dilated measurements. Since FLIO without mydriasis results in longer ?m in the SSC and takes a longer time for image acquisition, maximal dilation is recommended for FLIO testing.

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Fluorescence Lifetime Imaging of pH along the Secretory Pathway.

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