2.4. Confocal microscopy and image analysis
For determination of PI image stacks with 0.2 mm intervals
from whole cells were acquired using Olympus confocal
IX81microscope, 60 objective, 405 and 488 laser, and DIC.
The image stacks for colocalization analysis were obtained
using Nikon A1R confocal microscope with resonant scanning,
60 objective, and 488 and 561 lasers. In both experiments
altogether 40 and 30 cells, respectively, from three
independent experiments, were randomly selected and optically
sectioned. The brightness and contrast settings of all
images, as well as quantification of B. burgdorferi engulfment
by macrophages from animated multidimensional z-stacks,
were applied using open source Fiji software (NIH, USA). The
noise from green and red fluorescent images was suppressed