Resolution and sampling. (A–C) Images of the same pair of 150-nm green fluorescent beads collected with a microscope (model TE2000U; Nikon), a Plan-Apochromat 100x 1.4 NA oil objective lens, and MetaMorph software. A camera with 6.45-µm photodiodes (ORCA-AG; Hamamatsu Photonics) was used, and different camera binning settings were used to vary the area of the specimen covered by one pixel. Exposure times were adjusted to reach a maximum intensity value of ∼3,600 for each image. Using the equation for lateral resolution, we can calculate that the diameter of the first minimum of the airy disk, and therefore the diameter of the bead in the optical image, should be equal to ∼465 nm. Bar = 0.5 µm. (A) An image collected with no camera binning, and an exposure time of 200 ms. Each pixel corresponds to ∼65 nm of the specimen, and each bead is sampled with ∼7 pixels. (B) An image collected using 2 × 2 camera binning, and an exposure time of 50 ms. Each pixel corresponds to ∼129 nm of the specimen, and each bead is sampled with about 3.5 pixels. (C) An image collected using 4 × 4 camera binning, and an exposure time of 25 ms. Each pixel corresponds to ∼258 nm of the specimen, and each bead is sampled with less than 2 pixels. The optical image is under-sampled, and the two beads can no longer be distinguished as separate from one another.