Fluorescence microscopy of colour‐tagged nanoparticles that are undergoing thermal motion

Summary To bypass limitations of conventional biochemical analysis, single‐particle biochemical analysis is used. To improve single‐particle biochemical analysis, procedures are needed to keep nanometre‐sized particles in focus while the particles are undergoing thermal motion. A simple, inexpensive procedure is developed here for keeping particles in focus during the continuous observing/discriminating/recording of two different particles, both of which are undergoing thermal motion. This procedure concentrates the particles in a plane of solution that is in focus when the cover glass surface is in focus. An essential component of the procedure is the addition of molten, low‐melt agarose to the specimen. Motionless binding to glass is inhibited by inclusion of anti‐stick additives in the specimen. Both carrier protein (gelatin) and non‐ionic detergent (Triton X‐100) are anti‐stick additives successfully used here. Intact bacteriophages T3 and T7 are used as model particles, in anticipation of the use of the procedures developed here for the analysis of the assembly of bacteriophages. Observing/discriminating/recording of colour‐tagged bacteriophages T3 and T7 is achieved at video frame rate with image splitting to discriminate colours.