We demonstrate room temperature detection of single 20 nm superparamagnetic nanoparticles (SPNs) with a wide-field optical microscope platform suitable for biological integration. The particles are made of magnetite (Fe 3 O 4 ) and are thus non-toxic and biocompatible. Detection is accomplished via optically detected magnetic resonance imaging using nitrogen-vacancy defect centers in diamond, resulting in a DC magnetic field detection limit of 2.3 µT. This marks a large step forward in the detection of SPNs, and we expect that it will allow for the development of magneticfield-based biosensors capable of detecting a single molecular binding event.Keywords: Nitrogen-vacancy center, super-paramagnetic nanoparticles, optically detected magnetic resonance, magnetometry, microscopy.Super-paramagnetic nanoparticles (SPNs) are particles made of ferromagnetic material which, due to their small size, exhibit paramagnetic behavior with magnetic susceptibilities orders of magnitude larger than typical paramagnetic materials. greatly from this capability include highly sensitive assays for cancer, 11,12 HIV, 13 and nonacute-coronary-syndrome cardiac conditions. 14 In particular, digital immuno-assays rely on the detection of single bio-molecules to achieve ultra-high detection sensitivities. 15,16 In this work, we experimentally realize a room temperature platform capable of detecting single 20 nm diameter magnetite SPNs using wide-field optical imaging. NV centers can be viewed as a localized 2-electron system with the energy-level diagram shown in Figure 1(a). The ground state of the system is a spin-triplet, for which the m s = ±1 spin states are degenerate under zero applied magnetic field, and split from the m s = 0 spin state by an energy E ss due to spin-spin interactions. In this work, we perform imaging ODMR measurements on a 200 nm-thick, highdensity sheet of NV centers near the {111} surface of a diamond chip. As depicted in Using this setup, the spatial distribution of the component of the magnetic field normal to the chip surface is imaged. We use a simple difference scheme utilizing only NVs aligned with the applied DC field. Images taken with an RF magnetic field applied to the sensing surface are subtracted from images with no applied RF. The frequency of the RF field is set just below the steepest part of the ODMR curve (f 1 in Figure 1(b)), 5 maintaining a high small-field sensitivity while increasing the signal from the high field in proximity to SPNs.In order to characterize the system's SPN detection capability, magnetite SPNs were deposited on the sensing surface in isolated single particles and small groups. The SPNs were prepared according to a previously reported method. [39][40][41][42] Particle distributions were obtained by drying colloidal suspensions on a lithographically defined pattern on the sensor surface, resulting in a grid pattern with small groups of particles at each grid point. Figures 2(a-b) show scanning electron microscope (SEM) images of a resulting pattern of particle g...