348
MONICA MUSIANI et al.
Materials
Equipment The basic instrumentation for chemiluminescence in situ hybridization
includes:
- a conventional light microscope whose optics must be selected in order
to obtain a magnification factor of at least 360 with a 40 x objective and
a minimum spatial resolution of 1 /.lm. The lens coupling system
should be as simple as possible and objectives should have the highest
numerical aperture compatible with focal aberration and depth of
field, thus reducing the potential loss of light derived from the chemiluminescent reaction and achieving the maximum analytical detectability. The microscope should also be provided with a micrometric
system to automatically adjust the position of the sample, allowing exactly the same field of view to be found when the slide has been replaced after washing and handling procedures. The microscope has
to be enclosed in a dark box to prevent contact with the external light
during the measurements;
- an ultra-sensitive video camera specifically designed to pick up the static chemiluminescent signal. The video camera must be characterized
by high sensitivity, very low instrument noise, wide dynamic range,
and suitable spatial resolution. In the last few years, high-performance
CCD cameras have been proposed as an alternative to older devices
based on vidicon tubes. Cooled, back-illuminated CCD cameras provide very high detection sensitivity, due to their high quantum efficiency and low background noise, allowing a quantification of emitted
light at a single photon level. In addition, often these devices do not
require an image intensification step, which could negatively affect the
image quality and the signal-to-noise ratio. The camera should be calibrated with a light standard such as a liquid scintillation radioactive
isotope (l4C, 3H) or an electronically controlled light emitting diode
(LED);
- a personal computer (PC) provided with software for the camera management and the image processing and analysis. Concerning the camera management, the mode of integration of light should be chosen to
have a wide dynamic range without saturation of the light sensitive
elements. It should be possible to acquire the light signal for different
intervals of time with the possibility of having a multi-frame time-dependent image. Besides chemiluminescent images, the camera has to
be able to acquire and store light-transmitted images in order to prop-
MONICA MUSIANI et al.
Materials
Equipment The basic instrumentation for chemiluminescence in situ hybridization
includes:
- a conventional light microscope whose optics must be selected in order
to obtain a magnification factor of at least 360 with a 40 x objective and
a minimum spatial resolution of 1 /.lm. The lens coupling system
should be as simple as possible and objectives should have the highest
numerical aperture compatible with focal aberration and depth of
field, thus reducing the potential loss of light derived from the chemiluminescent reaction and achieving the maximum analytical detectability. The microscope should also be provided with a micrometric
system to automatically adjust the position of the sample, allowing exactly the same field of view to be found when the slide has been replaced after washing and handling procedures. The microscope has
to be enclosed in a dark box to prevent contact with the external light
during the measurements;
- an ultra-sensitive video camera specifically designed to pick up the static chemiluminescent signal. The video camera must be characterized
by high sensitivity, very low instrument noise, wide dynamic range,
and suitable spatial resolution. In the last few years, high-performance
CCD cameras have been proposed as an alternative to older devices
based on vidicon tubes. Cooled, back-illuminated CCD cameras provide very high detection sensitivity, due to their high quantum efficiency and low background noise, allowing a quantification of emitted
light at a single photon level. In addition, often these devices do not
require an image intensification step, which could negatively affect the
image quality and the signal-to-noise ratio. The camera should be calibrated with a light standard such as a liquid scintillation radioactive
isotope (l4C, 3H) or an electronically controlled light emitting diode
(LED);
- a personal computer (PC) provided with software for the camera management and the image processing and analysis. Concerning the camera management, the mode of integration of light should be chosen to
have a wide dynamic range without saturation of the light sensitive
elements. It should be possible to acquire the light signal for different
intervals of time with the possibility of having a multi-frame time-dependent image. Besides chemiluminescent images, the camera has to
be able to acquire and store light-transmitted images in order to prop-