Abstract
Background: Intravital microscopy is an emerging technique in life science with applications in kidney research. Longitudinal observation of (patho-)physiological processes in living mice is possible in the smallest functional unit of the kidney, a single nephron (sn). In particular, effects on glomerular filtration rate (GFR) - a key parameter of renal function - can be assessed. Methods: After intravenous injection of a freely filtered, non-resorbable, fluorescent dye in C57BL/6 mice, a time series was captured by multiphoton microsopy. Filtration was observed from the glomerular capillaries to the proximal tubule (PT) and the tubular signal intensity shift was analyzed to calculate the snGFR. Results: Previously described methods for snGFR analysis relied on two manually defined measurement points in the PT and the tubular volume was merely estimated in 2D images. We present an extended image processing workflow by adding continuous measurement of intensity along the PT in every frame of the time series using ImageJ. Automatic modelling of actual PT volume in a 3D dataset replaced 2D volume estimation. Subsequent data analysis in R, with a calculation of intensity shifts in every frame and normalization against tubular volume, allowed exact assessment of snGFR by linear regression. Repeated analysis of image data obtained in healthy mice showed a striking increase of reproducibility by reduction of user interaction. Conclusions: These improvements in image processing and data analysis maximize the reliability of a sophisticated intravital microscopy technique for the precise assessment of snGFR, a highly relevant predictor of kidney function.
Highlights
Intravital microscopy is an emerging technique in life science with applications in kidney research
In the time series acquired after application of LY, a line region of interest (ROI) was set to manually define the position and direction of the measurement
For the automatic 3D modelling of proximal tubule (PT) volume the z-stack of the same field of view was acquired
Summary
Intravital microscopy is an emerging technique in life science with applications in kidney research. Filtration was observed from the glomerular capillaries to the proximal tubule (PT) and the tubular signal intensity shift was analyzed to calculate the snGFR. Results: Previously described methods for snGFR analysis relied on two manually defined measurement points in the PT and the tubular volume was merely estimated in 2D images. We present an extended image processing workflow by adding continuous measurement of intensity along the PT in every frame of the time series using ImageJ. Subsequent data analysis in R, with a calculation of intensity shifts in every frame and normalization against tubular volume, allowed exact assessment of snGFR by linear regression. Conclusions: These improvements in image processing and data analysis maximize the reliability of a sophisticated intravital microscopy technique for the precise assessment of snGFR, a highly relevant predictor of kidney function
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