In order to confirm that our approach is suitable to monitor changes in the cellular environment, we analyzed rat kidney biopsies after a 24-h incubation step at 37 °C in the tissue culture incubator (24 h 37 °C) or following permeabilization of the cell membrane by digitonin treatment, which results in the collapse of cellular homeostasis and cell death. The pictures shown in Fig. 2 focus on the corticular area of the kidney and comprise several tubuli. Double staining with either Syto 16 (green) or PI (red; Fig. 2a–c) or TMRM (red) and WGA (green; Fig. 2b–d) documents the cell viability. Despite immediate processing of the samples (i.a.b) a heterogeneity in the staining pattern can be observed which documents the presence of viable and nonviable regions in the tubular area. After the 24-h incubation step at 37 °C, the number of PI-positive (dead) cells increases substantially, while only a fraction of Syto 16 positive/PI negative cells is still present at the end of the incubation period. After the digitonin addition all nuclei are PI/Syto 16 positive resulting in a yellow overlay color. A similar picture emerges when looking at the mitochondrial membrane potential. At the earliest time point after the biopsy areas with decreased mitochondrial function are already detectable (Fig. 2b). Finally, the mitochondrial transmembrane potential has completely collapsed after the permeabilization of the cell membrane by digitonin (Fig. 2f).

The staining pattern obtained with WGA showed the same heterogeneity as did the staining with TMRM. After cultivation for 24 h at 37 °C, the structural integrity of the biopsy was lost and increased cell death was apparent as documented by Syto16/PI staining. Permeabilization with digitonin finally resulted in massive cell death (Fig. 2c and d) but has no influence on the staining pattern of WGA (Fig. 2d).

In order to demonstrate the suitability of our method to monitor the status of a graft prior to transplantation, murine kidneys were exposed to 24 h of cold ischemia. Following this, longitudinal biopsies were taken and incubated with Syto 16 (green), propidium iodide (PI; red), and WGA. Figure 3 shows the result of such a staining documenting the viability in a tubular area before (left image) and after 24 h of cold ischemia (right image).