Carl Zeiss has developed
the DirectFRAP imaging system for all microscope users in cell biology and molecular
genetics who examine processes in living cells on the basis of fluorescence-labeled
proteins. Thanks to its operating principle and the photomanipulation in the
wide field, DirectFRAP opens up the possibility of also studying highly dynamic
processes in the cell and receiving detailed image information at the same time.
It can be used on the Axio Observer microscope system from Carl Zeiss and allows
the photomanipulation of a region of interest (ROI) in the specimen with laser
light and the direct observation of the subsequent processes.
All versions of photomanipulation are possible: bleaching of GFP, photoactivation
of PA-GFP, conversion of Dendra, reversible on/off switching of Dronpa, and
others. Laser pulse control and data acquisition are performed by the ZEISS
The extremely short pulse times of DirectFRAP and the benefits of widefield
microscopy such as short exposure times and larger observation field for fast
processes offer optimal conditions for highly dynamic experiments like the examination
of the interaction of proteins with macromolecules in the cell. The time sequence
of the processes is clearly resolved.
The diaphragm options enable a high level of flexibility during experiments.
This permits the use of DirectFRAP for answering dynamics-related questions
or for the manipulation of fine cell structures. For this purpose, a broad spectrum
of geometric ROI shapes is available on a diaphragm wheel in the slider for
DirectFRAP. In addition to constant laser intensity when the diaphragm size
is changed, the main benefit of the principle is the simultaneous manipulation
of all points of the ROI.
DirectFRAP can be optionally used with other Carl Zeiss imaging systems such
as Laser TIRF 3 or Cell Observer SD (Spinning Disc). System combinations allow
the observation of processes in only one Z plane and are ideal, for example,
for the isolated examination of small cell structures. The same lasers can be
used simultaneously for DirectFRAP and Laser TIRF 3 or Cell Observer SD.
DirectFRAP helps to record dynamic processes in the cell. Changes in these
processes over time can also be recognized in detail. Reactions of the cell
can be examined in detail, for example. Fast, cellular processes become transparent.