The QDs used in this study are referred to as GSH-QD650 and were made of CdSe/CdZnS core with a GSH coating, and they were about 8 nm in total diameter, emitting red (around 650 nm in wavelength ) fluorescent signals with ultraviolet (UV) light (Fig. 42.1c). In cortical tissues of mouse kidneys at 2.5 and 5 s after injection of QDs, red QD signals were clearly detected. There were three ways to observe the renal tissue structures. First, pure morphology was obtained with hematoxylin-eosin (HE) staining of the serial sections, enabling us to compare the fluorescence image. Second, detection of autofluorescence images with UV excitation was useful on the same section, as shown by the blue color in Fig. 42.1d, e, while the fluorescence intensity of QD signals was very strong. Third, diffraction interference contrast (DIC) images were useful to visualize morphological contours of blood vessel s due to the appearance of erythrocyte shapes on the exactly same section.

In the renal cortex, at 2.5 s after the QD injection (Fig. 42.1d), QDs were detected only in renal glomerular capillaries as well as afferent arterioles , but not in blood capillaries around renal tubules. The QDs were seen to be distributed in restricted areas in glomerular capillary loop s . This finding is consistent with the concept of blood flow in the renal cortex, showing that the flow enters from afferent arterioles into glomerular capillary loops and exits through efferent arterioles and into peritubular blood capillaries. At 5 s after the QD injection (Fig. 42.1e), they were detected in almost all peritubular blood capillaries, as well as those in all renal glomeruli. Thus, the in vivo blood flow, reflecting their living state s , was clearly visualized by the QD distribution in tissue sections captured by IVCT.

With the confocal laser scanning microscope (CLSM), tissue sections were exposed to UV laser light (405 nm in wavelength ), and the best GSH-QD650-fluorescence images were detected at a 630–730 nm wavelength. After FS, the tissue specimens became very hard, and it was possible to trim them directly with a razor blade, and the cut tissue surface was exposed to laser beams by CLSM. Both afferent and efferent arterioles were visualized at a vascular pole, where the QD distribution was three-dimensionally detected in the glomerulus as a whole, as obtained at any levels by optionally cut sections after the image reconstruction. Thus, vascular structures of glomerular capillary loop s were clearly demonstrated by the reconstruction of QD distribution with CLSM.

The HRP has been a well-known soluble protein often used as a tracer by visualization with DAB-enzyme reaction to examine diffusive states in the interstitium in various animal organs after artificial injection into blood vessel s [15]. The HRP distribution was evaluated by the simultaneous injection of HRP and QDs into the mouse left ventricle s , and IVCT was performed at 30 s after the injection. Because glutaraldehyde treatment usually reduced the QD intensity, the IVCT-frozen tissues containing both HRP and QDs were divided into two pieces and processed to each detection protocol. At 30 s after the simultaneous injection of HRP and QDs, only DAB-reaction products were detected in the interstitium, as well as in blood vessels of the renal cortex, showing HRP localization. In addition, DAB-reaction products were detected at basal striations of some renal tubules. On the other hand, QDs were detected only inside blood vessels, but not in the interstitium of the renal cortex. Thus, dynamic aspects of blood supply into organs were clearly visualized by detecting different soluble extrinsic components with the IVCT, which probably reflect living animal functions.