RNA in cells - Single Molecule Dynamics in Life Science - page 175

The concept behind the MCP system led to the creation of an alternative system
based on the U1A splicing factor and its recognition sequences [25 - 27]. This
approach works well in yeast but is not exploitable in mammalian cells, which
contain the endogenous U1A protein.

8.2.2
Advancements in Imaging Technologies

A critical aspect in single-molecule imaging is the development of the best optical
system tomatch the requirements for imaging singlemoving particles. This includes
acquiring images at rates as fast as, or faster than, the particle movements projected
optically onto the capture chip; applying a minimal amount of light to avoid
phototoxicity and bleaching of the sample, and all of this while maximizing the
signal-to-noise ratio and tracking the particles in three dimensions (3D) and in time
(4D imaging). In the past, the classical confocal microscope was preferred for
imaging fluorescently-labeled cells, but now alternative options are available. The
confocal platform itself has been recently modi ed to increase speed and enable fast
imaging in the z-plane, in time and with different wavelengths simultaneously. More
advances have also been made in the wide- eld epi uorescent microscope. The
improvements in the Charge-Coupled Device (CCD) camera, that converts incident
photons at the detector into electronic information, and the technology of electron-
multiplying CCDs (EMCCDs) which detect very weak signals, are providing higher
levels of sensitivity than the photomultiplying tubes (PMT) used in the confocal
microscope. In this latter instrument, photons are first converted into electrons
which then converge in an electronmultiplier where a systemof electrodes ampli es
the signal by a secondary emission process. However, the major difference between
confocal andwide- eld imaging is that the confocal microscope discards photons that
are not within the image plane and this loss of information reduces the sensitivity
required to detect single molecules. Instead, with the wide- eld microscope all the

information from out of focus photons collected during the acquisition process
becomes important and useful. In fact applying the images deconvolution
algorithms, a point spread function will allow reconstructing them in three
dimensions. In this way the light is reassigned to its point of origin to recreate
an image with a signal-to-noise ratio much higher than that in a confocal micro-
scope [28]. In conclusion, the developments in biophotonics, imaging technologies,
bioinformatics and computational analysis are continuously increasing their
relevance and indispensable roles in the discovery of new principles of cellular and
molecular biology in living cells.

8.3
RNA Dynamics in the Nucleus

The birth site of RNAs, namely the transcription site or RNA factory, is located in
the nucleus. Transcription by RNA polymerase I (pol I) occurs inside the nucleoli,