Researchers at the Beckman Institute for Advanced Science and Technology at the University of Illinois Urbana-Champaign are using microscopic nanodiamonds to calibrate and assess the performance of high-powered microscopes. The work is poised to support a wide range of research and applications, optimizing workflows by saving time in the preparation stages for fluorescence microscopic analysis. Fluorescent nanodiamonds are microscopic particles with small amounts of other chemical elements trapped inside as impurities. For microscopy applications, they are distinctive in the way that they do not bleach, said research leader Mantas Žurauskas, an imaging research scientist for the GlaxoSmithKline Center for Optical Molecular Imaging at the Beckman Institute. According to Žurauskas, each time a fluorescent nanodiamond is observed, it appears the same. “This is very rare in fluorescence microscopy,” Žurauskas said. However, it is a challenge in biomedical microscopy imaging to create reliable calibrations samples, or “phantoms.” “There are changes each time you look at a fluorescent structure. As phantoms, I used fluorescent beads very often; these are like little beads filled with fluorescent dye,” Žurauskas said. “Each time you look at them, they are a bit dimmer. It is really this fluorescence decay that is a big enemy in fluorescence microscopy.” In collaboration with industry partners, researchers at the University of Illinois Urbana-Champaign are using microscopic nanodiamonds (shown here) to calibrate and assess the performance of high-powered microscopes. A stable fluorescent nanodiamond phantom promises wide-reaching applicability for microscopy research and quality control alike. Courtesy of Beckman Institute/University of Illinois. The stability of a calibration sample is fundamental to the ability to assess the optical system’s day-to-day quality. “It is kind of a first-aid kit for a microscope,” Žurauskas said of the phantom-enabled calibration. “Ideally, we want to take the same object each time and see the same image.” The stability and longevity of nanodiamonds allows their continuous reuse as a calibration tool, which eliminates the labor-intensive preparation that is typically required for fluorescence microscopy, the researchers said. The researchers took advantage of the physical properties of nanodiamonds to engineer the imaging phantoms. “The nanodiamonds are distributed randomly, and they are very sparse, so that you can look at individual particles, or on the opposite end of the spectrum you can look at dense distributions of these particles,” Žurauskas said. “A second plane contains a viewfinder grid, which is effectively a laser-machined grid with nanodiamonds embedded in it. This helps to find the same area each time.” A microscopy image of the viewfinder grid with embedded nanodiamonds. The grid simplifies the ability of the user to locate the same area with each view. Courtesy of Beckman Institute/University of Illinois. Two companies are currently evaluating the imaging phantoms for wider use. LiveBx, a University of Illinois Urbana-Champaign spinout company that provides a laser source for life-science research and clinical applications such as label-free histology and real-time tissue biopsy, is looking to see if the phantoms can improve its system, Žurauskas said. The company’s work develops imaging instruments that simplify and speed the observation of living tissue and cells. Industry partner GlaxoSmithKline is assessing the phantom for quality control applications in its own biomedical research labs. The research was published in Photonics Research (www.doi.org/10.1364/PRJ.434236).