Single-molecule microscopy and SPIM of single nanoparticles and mRNA molecules in vivo

We analyzed the intranuclear dynamics of fluorescence labelled single, native mRNA molecules in living cell nuclei. High speed and high sensitivity laser microscopy was employed to analyse the intranuclear movement of the mRNA molecules from the site of transcription towards the nuclear envelope, and thus to characterize their mode of motion within the nucleosol [1-3]. For reference, we also extensively analyzed the intranuclear mobility of inert fluorescent nanoparticles and quantum dots. These studies were performed using a special cell system, namely the salivary gland cells in the larvae of Chironomus tentans. They provide an elegant model system for the analysis of specific messenger ribonucleoprotein particles, the Balbiani Ring (BR) mRNPs [4]. BR mRNPs contain highly repetitive RNA transcripts of roughly 35-40 kb in length, which are packed into huge, granular BR mRNPs with a diameter of almost 50 nm. Transcription and splicing of the BR mRNA, and the formation of the BR mRNPs has been genetically and biochemically thoroughly investigated. Also, in recent studies their intranuclear localisation in fixed glands was visualized by electron microscopy [5]. Labelling of the BR RNPs was achieved in situ by nuclear microinjection of fluorescence labelled oligonucleotides, which were complementary to the highly repetitive sequence on the BR mRNA. This approach generated native, fluorescent mRNPs in vivo. Combining single-molecule tracking with selective-planeillumination microscopy (SPIM) we assessed the movement of the BR particles and fluorescent nanoparticles in the complex 3D architecture of the living gland tissue with high contrast at a millisecond time resolution [6].