Bleed-through can cause inaccuracy in intensity measurements. (A and B) Images of a cell (outlined in white) labeled with DAPI (nuclei) and Bodipy-FL phalloidin (actin). Both images were collected using the same microscope (model 80i; Nikon), a Plan-Apochromat 100x NA 1.4 oil objective lens, the same camera (ORCA R2; Hamamatsu Photonics), and MetaMorph software. The same camera acquisition settings were used for both images, but they were collected using two different filters designed for imaging DAPI. (A) An image collected with a DAPI filter set containing a long-pass emission filter, which allows bleed-through of the Bodipy-FL signal in the cytoplasm. The bleed-through of the actin in the cytoplasm is just barely visible by eye in the image. The average intensity value of the cytoplasm in this image is 205. (B) An image of the same cell as in A, collected with a DAPI filter set containing a band-pass emission filter, which blocks bleed-through of the Bodipy-FL signal in the cytoplasm. The average intensity value of the cytoplasm in this image is 91, over 50% less than the image in A. Bar = 10 µm.