Ratio imaging has been adapted to light sheet–based fluorescence microscopy, which is a preferential 3-D method for long-term observation at a minimum light dose applied to living cells. Wavelength-dependent light scattering, which might create some shadows and, therefore, artifacts, in ratio imaging was not considered, so far, and obviously did not play a major role in the present images. Those artifacts, however, might be more pronounced in larger spheroids and could in principle be reduced or avoided by improved conditions of illumination, e.g., by variation of the illumination angle in a wobbling mode19 or by use of a self-reconstructing Bessel beam instead of a Gaussian laser beam.11 A further method for reduction of artifacts due to light scattering could be light sheet microscopy with laser scanning excitation and confocal slit detection, as previously described in the literature.20,21 In addition, the method of ratio imaging can be easily modified for other wide-field techniques, e.g., structured illumination microscopy.5,6 When using those techniques, nonphototoxic light doses may be similar to light sheet–based microscopy, if only one cell layer is examined. For measurements of several cell layers, however, the light doses sum up, since in contrast to light sheet microscopy, the whole sample is illuminated upon measurement of each layer, thus reducing the possible number of exposures. Coupling to a microfluidic system is advantageous for observation of rapid redox changes and for minimizing the quantity of agents (e.g., antioxidizing or pharmaceutical agents) used for cancer treatment.