Design, Labeling, and Application of Probes for RNA smFISH.

Visualization of single mRNA molecules in fixed cells can be achieved using single molecule fluorescent in situ hybridization (smFISH). This approach enables accurate quantification of mRNA numbers and localization at a single-cell level. To ensure reliable results using smFISH, it is critical to use fluorescent probes that are highly specific to their RNA target. To facilitate probe design, we have created anglerFISH, a user-friendly command-line based pipeline. In this chapter, we present how to perform a smFISH experiment using user-designed and labeled probes.

deepBlink: threshold-independent detection and localization of diffraction-limited spots.

Detection of diffraction-limited spots in single-molecule microscopy images is traditionally performed with mathematical operators designed for idealized spots. This process requires manual tuning of parameters that is time-consuming and not always reliable. We have developed deepBlink, a neural network-based method to detect and localize spots automatically. We demonstrate that deepBlink outperforms other state-of-the-art methods across six publicly available datasets containing synthetic and experimental data.

Some optimisation studies relevant to the production of high-purity 124I and 120gI at a small-sized cyclotron.

Optimisation experiments on the production of the positron emitting radionuclides 124I(T(1/2) = 4.18d) and (120g)I (T(1/2) = 1.35 h) were carried out. The TeO(2)-target technology and dry distillation method of radioiodine separation were used. The removal of radioiodine was studied as a function of time and the loss of TeO(2) from the target as a function of oven temperature and time of distillation. A distillation time of 15 min at 750 degrees C was found to be ideal. Using a very pure source and comparing the intensities of the annihilation and X-ray radiation, a value of 22.0 +/- 0.5% for the beta(+) branching in 124I was obtained. Production of 124I was done using 200 mg/cm(2) targets of 99.8% enriched 124TeO(2) on Pt-backing, 16 MeV proton beam intensities of 10 microA, and irradiation times of about 8 h. The average yield of 124I at EOB was 470 MBq(12.7 mCi). At the time of application (about 70 h after EOB) the radionuclidic impurity 123I (T(1/2) = 13.2 h) was <1%. The levels of other impurities were negligible (126I < 0.0001%;125I = 0.01%). Special care was taken to determine the 125I impurity. For the production of (120g)I only a thin 30 mg target (on 0.5 cm(2) area) of 99.9% enriched 120TeO(2) was available. Irradiations were done with 16 MeV protons for 80 min at beam currents of 7 microA. The 120gI yield achieved at EOB was 700 MBq(19 mCi), and the only impurity detected was the isomeric state 120 mI(T(1/2) = 53 min) at a level of 4.0%. The radiochemical purity of both 124I and 120gI was checked via HPLC and TLC. The radioiodine collected in 0.02 M NaOH solution existed >98% as iodide. The amount of inactive Te found in radioiodine was <1 microg. High purity 124I and 120gI can thus be advantageously produced on a medium scale using the low-energy (p,n) reaction at a small-sized cyclotron.