This dataset was created for the online microscopy course given by the Live Cell Imaging core facility, Karolinska Institutet, Sweden. Images in this dataset were acquired with the goal of demonstrating the effect bleedthrough, saturation, underexposure or soft focus have on image analysis. Images of a section of Convallaria labelled with Acridine Orange (Boston Electronics) mounted between a coverslip and a slide were acquired using a Nikon A1 point confocal on a Ti2 microscope with a 10x/0.45 air objective. The pixel size was set to Nyquist sampling. Apart from the seq_saturated and se_underexposed images, the laser power and exposure time were adjusted to have no saturation and no underexposure in the images. The excitation and emission wavelengths were as follows: Channel 1 Excitation: 488 nm laser; Emission filter: 525/50 nm. Channel 2 Excitation: 561 nm; Em595/50 nm. Dichroic Mirror: 405/488/561/640. For the sequential.nd2 file, the 2 channels were acquired sequentially. For the simultaneous.nd2 file, the 2 channels were acquired simultaneously. For the seq_underexposed.nd2 and respectively seq_saturated.nd2 files, the 2 channels were acquired sequentially but the illumination power and gain, respectively the detector offset, were adjusted to result in image saturation, respectively underexposure. The soft-focus.nd2 file was acquired in the same way as the sequential.nd2 file but the image was set slightly out of focus by moving the sample some microns away from the focal plane.