Dennis M. Oakley

Since its commercialization in the late 1980's, confocal laser scanning microscopy (CLSM) has since become one of the most prevalent fluorescence microscopy techniques for three-dimensional structural studies of biological cells and tissues. The flexibility of the approach has enabled its application in a diverse array of studies, from the fast imaging of dynamic processes in living cells, to meticulous morphological analyses of tissues, and co-localization of protein expression patterns. In this chapter, we introduce the principles of confocal microscopy and discuss how the approach has become a mainstay in the biological sciences. We describe the components of a CLSM system and assess how modern implementations of the approach have further expanded the use of the technique. Finally, we briefly outline some practical considerations to take into account when acquiring data using a CLSM system. © 2018 by John Wiley & Sons, Inc.

The effect of intravesical Bacillus Calmette-Guérin (BCG; Pasteur strain) treatment on the frequency of implantation and growth rate of the murine transitional cell carcinoma of the bladder, MBT-2, was studied. MBT-2 cells were instilled into the bladder immediately after electrocauterization, and BCG instillations (40, 80, and 160 micrograms/instillation) were initiated 24 hr later and continued on a weekly basis for 4 weeks. BCG treatment significantly (p less than 0.0002) reduced the incidence of tumor implantation in a dose-dependent manner and resulted in significantly (p less than 0.0001) smaller tumors when they appeared in BCG-treated mice. The therapeutic effect of BCG correlated with augmentation of natural killer cell (NK) activity and positive purified protein derivative (PPD) footpad reactions. In experiments in which treatment was initiated with rapidly growing BCG organisms (10(7) colony-forming units/mg), tumor implantation was inhibited, there was a dose-dependent increase in NK activity, and mice had positive footpad reactions in PPD. In experiments in which BCG with reduced viability (10(6) colony-forming units/mg) and slower growth rates was used for treatment, no significant inhibition of tumor implantation was observed, NK activity was depressed, and PPD footpad tests were uniformly negative. The results suggest that the therapeutic effects of BCG therapy in this murine model correlate with augmentation of NK activity and positive footpad reactions to PPD and further suggest that the viability and growth rate of BCG organisms are important factors in determining the efficacy of intravesical BCG therapy.

Four methods of intravesical implantation of the transplantable mouse bladder tumor, MBT-2, and their effects on intravesical therapy with Bacillus Calmette-Guérin (BCG) were compared, and modifications which improved implantation are described. Pretreatment of the bladder with N-methyl-N-nitrosourea (MNU) resulted in tumor implantation in approximately two-thirds of the animals; however, all tumors penetrated the bladder wall. Using the MNU implantation procedure, intravesical BCG therapy was shown to reduce MBT-2 outgrowth by 77%. Tumor cell instillation after electrocautery produced an incidence of tumor implantation similar to that of the MNU procedure. The efficacy of BCG for the electrocautery implantation procedure also was similar to the MNU method. With the electrocautery procedure, the electrode and tumor cells were introduced into the bladder via a catheter prepared from PE 10 polyethylene tubing. The procedure required two catheterizations and produced a 24% incidence of extravesical tumors. Use of a Teflon catheter and a single catheterization for tumor cell instillation resulted in a reproducible method for implanting MBT-2 tumors which were all confined within the bladder. The efficacy of BCG therapy was unchanged from that described for the other implantation techniques.