Critical issues related to real-time fluorescence imaging of renal tissues using confocal microscopy

To the Editor

The recent article by Horbelt et al.¹ reported a very important methodological development in the area of real-time visualization of renal dynamics. The technique involving time-resolved two-photon microscopy in vivo is surely a step ahead of the more popular LSCM (laser scanning confocal microscopy) technique using in situ examination of different mechanisms in renal glomerular, vascular, and tubular tissues and cells. The authors have presented their findings in a very detailed way, making it easy for others to follow the procedure in an organized way. Our experience while establishing a technique involving real-time [Ca²⁺]_i imaging of ‘intact’ renal glomeruli and arterioles using isolated glomeruli and whole-kidney slices in LSCM has been quite challenging. One thing that we came to know was that research in this area is a lot more demanding than many of the other airway and vascular smooth muscle tissues and cells, which we have been regularly using for [Ca²⁺]_i imaging. We would like the readers, especially researchers new to this area of research, to have know-how about some of the problems they might experience in designing protocols for real-time imaging of Ca²⁺ signaling in intact renal glomeruli and resistance arterioles. Table 1 enumerates some key elements related to dissection of animals and experimental conditions, which can be selected after going through the relevant literature and optimized to get the best results. There are usually two thoughts related to acquisition of ‘intact’ renal tissues. Some groups perform microdissection² to obtain the required vascular tissues or whole glomeruli from the kidneys with minimally disturbing their integrity, whereas others use chemical³ (dissociation enzymes) or physical means⁴ (sieving) for isolation. We have experienced that the microdissection method of minimal manipulations yields better results. The conclusions given here are a result of repeated trials and we hope that these would be helpful to the researchers new in this area.

Table 1.

Factors that might prove critical in the eventual success of real-time Ca²⁺ imaging experiments of intact renal tissues using confocal microscopy

Dissection	Experimentation
Its better to do dissection under anesthesia	Be sure about the species to be used (mice, rats, rabbits and so on)
Some prefer injectable, whereas others prefer gaseous anesthesia	Perfusion system should allow accurate transport of drugs to the tissue to avoid tachyphylaxis seen with some commonly used agonists such as angiotensin II
Time taken to dissect should be as minimum as possible	Experiments should be performed at 37 °C with carbogenaerated perfusion solution at pH 7.4
Solutions used for flushing the kidneys should be at 37 °C	Choice of perfusion solution and Ca2+ fluorescent dyes depends on protocol followed from the literature
Dissection is better done at 4 °C to avoid metabolic damage It is better to dissect out whole glomeruli and arterioles than to isolate them using chemical or physical means to avoid any damage	Minimize photobleaching while recording Use up the tissues within 3 h of dissection