Andor Technology iXon Ultra 897 User Manual page 155

TUTORIAL 3
Performing Basic Low-Light Microscopy Functions Using the
iXon
Additional Requirements
Epifluorescence Microscope and camera adaptor. Optical filter set matched
to fluorescence beads. Fluorescent microspheres, e.g. Fluorescein
containing micron scale polystyrene FluoSpheres® from Molecular Probes.
Darkened ambient conditions.
The object of this experiment is to perform some basic low light Aim
microscopy functions with the iXon, including recording kinetic series and
determination of optimal gain, utilising as a simple example, micron scale
fluorescent microspheres in solution.
Attach the camera to the microscope via the appropriate mounting adaptor. Description
Ensure the camera is stabalized at the chosen temperature (a lower
temperature will essentially negate darkcurrent noise spikes). The
temperature indicator at the bottom left of the Main Window turns blue,
when the system has reached the requested temperature.
Prepare a solution of microspheres - e.g. a 1:100 dilution of the stock
solution. Deposit a small amount onto a microscope slide and place a cover
slip on top. Focus onto the beads using a reasonably high magnification,
e.g. x100 oil immersion objective. While the beads are still in solution, they
can be seen to translate, both laterally and also in and out of the focal plane
in the z-direction.
Obtain an Image on CCD - Switch the microscope from eyepiece to
camera observation. From the Acquisition menu on the Main Window,
select Setup Acquisition. In the Setup CCD tab, ensure gain is set to zero
and set exposure time to approximately 10-20 ms. In the Video Mode tab,
ensure that Use Settings From Standard Setup is selected. In the Setup
Image tab, ensure that 1 x 1 Binning and Full Area is selected. Click the
Video Mode Run button – an image of the beads should appear on screen
at the frame rate accessed through the selected settings.
Section 7. 3
Page 155