28 In Situ Detection of Viral Nucleic Acids by Chemiluminescence
349
erly identify the morphological structure of the sample. A series of corrections should be performed after the acquisition of the images, such
as background, gamma, geometric, flat-field, defect corrections. A series of mathematical functions should also be available to improve the
quality of the chemiluminescent and live images regarding image processing and analysis. Pseudocolor and overlay functions are necessary
to superimpose chemiluminescent image to light-transmitted image in
order to appropriately localize the light emission from the target. A
quantitative analysis of the light emitted in a given area of the sample
(pixel) should be possible. It should also be possible to standardize the
above mentioned parameters to permit a precise, accurate and reliable
comparison of specimens analyzed in different sets of experiments.
High quality color prints of the images with appropriate comments,
according to the guidelines of good laboratory practice and method
standardization should be carried out.
In our studies, the luminescent signal from the hybrid formation was detected and analyzed using a high-performance, low light-level imaging apparatus (Luminograph LB 980, EG&G Berthold, Bad Wilbad, Germany),
which permits emitted light measurement at the single-photon level.
The video system consists of a l"Saticon, high dynamic range pick-up
tube (which is a Vidicon-type tube with Se-As-Tllight target photoconductor) linked to an image intensifier, by high transmission lenses, and
also to a videoamplifier. This system is connected to a model BH-2 optical
microscope (Olympus Optical, Tokyo, Japan), and to a PC provided with
software for quantitative image analysis. The microscope is enclosed in a
dark box to prevent contact with the external light.
Alternatively, when a higher degree of detectability was required, a Luminograph LB 981 (EG&G Berthold, Bad Wilbad, Germany), based on a
slow-scan, back-illuminated, cooled CCD was used. The instrument set-up
and CL imaging processing are quite similar to those of the LB 980 luminograph.
Both instrument systems for the detection of chemiluminescence in in situ
hybridization operate in three steps:
1. firstly, tissue structures and cells are recorded in transmitted light;
2. the luminescent signal is then measured with an optimized photon accumulation lasting 1 min;
3. after a computer elaboration of the luminescent signal with pseudocolors corresponding to the light intensity, an overlay of the two
Detection of
luminescence
349
erly identify the morphological structure of the sample. A series of corrections should be performed after the acquisition of the images, such
as background, gamma, geometric, flat-field, defect corrections. A series of mathematical functions should also be available to improve the
quality of the chemiluminescent and live images regarding image processing and analysis. Pseudocolor and overlay functions are necessary
to superimpose chemiluminescent image to light-transmitted image in
order to appropriately localize the light emission from the target. A
quantitative analysis of the light emitted in a given area of the sample
(pixel) should be possible. It should also be possible to standardize the
above mentioned parameters to permit a precise, accurate and reliable
comparison of specimens analyzed in different sets of experiments.
High quality color prints of the images with appropriate comments,
according to the guidelines of good laboratory practice and method
standardization should be carried out.
In our studies, the luminescent signal from the hybrid formation was detected and analyzed using a high-performance, low light-level imaging apparatus (Luminograph LB 980, EG&G Berthold, Bad Wilbad, Germany),
which permits emitted light measurement at the single-photon level.
The video system consists of a l"Saticon, high dynamic range pick-up
tube (which is a Vidicon-type tube with Se-As-Tllight target photoconductor) linked to an image intensifier, by high transmission lenses, and
also to a videoamplifier. This system is connected to a model BH-2 optical
microscope (Olympus Optical, Tokyo, Japan), and to a PC provided with
software for quantitative image analysis. The microscope is enclosed in a
dark box to prevent contact with the external light.
Alternatively, when a higher degree of detectability was required, a Luminograph LB 981 (EG&G Berthold, Bad Wilbad, Germany), based on a
slow-scan, back-illuminated, cooled CCD was used. The instrument set-up
and CL imaging processing are quite similar to those of the LB 980 luminograph.
Both instrument systems for the detection of chemiluminescence in in situ
hybridization operate in three steps:
1. firstly, tissue structures and cells are recorded in transmitted light;
2. the luminescent signal is then measured with an optimized photon accumulation lasting 1 min;
3. after a computer elaboration of the luminescent signal with pseudocolors corresponding to the light intensity, an overlay of the two
Detection of
luminescence