64
ANDREAS PLESCH
Fig. 7. Small hybridization
spots on interphase nuclei.
Only Texas Red fluorescence is
shown. Taken with Plan-Neofluoar IOOx/1.3 Oil and a
standard CCO (M300) with an
integration time of 3.8 s. Regions A and B show spots close
to the resolution limit
The image shows bright hybridization spots along with some unspecific
signals and an elevated fluorescence background within the nuclei. The
signal to background ratio is approximately 4.
The situation for whole chromosome painting signals (FITC-Iabeled
chromosomes of a metaphase hybridized with the "24XCyte" mFISH
kit from MetaSystems) is shown in Fig. 8a. The intensity ratio of the
painted and unpainted chromosomes is 4 again. Is the dynamic range possibly limited by the dark current of the uncooled CCD sensor (M300) used
for capturing? This is not the case, as the background intensity between the
chromosomes is significantly lower than the fluorescence intensity of the
unlabeled chromosomes. And even this inter-chromosomal background is
not only due to dark current, but is caused to a great extent by auto fluorescence within the cytoplasm, by the very small fraction of reflected excitation light that passes the emission filter and by ambient light. Painting
probes labeled with Texas Red usually yield a higher dynamic range at
comparable exposure times. The difference in the dynamic range is probably due to different behavior of the fluorochromes leading to increased
non-specific FITC fluorescence and to increased auto fluorescence caused
by the shorter wavelength of the FITC excitation. Figure 8b illustrates the
influence of dark current on the image quality. The fluorescence lamp was
dimmed and the same metaphase was captured again. Focusing had to be
performed before reducing the light level, as the signals were much too
dark to be visible in the microscope or on the screen. The required exposure time was 125 s. Of course, the dynamic range fell to 2.5 and the intrachromosomal background as well as the overall noise level has been in-
ANDREAS PLESCH
Fig. 7. Small hybridization
spots on interphase nuclei.
Only Texas Red fluorescence is
shown. Taken with Plan-Neofluoar IOOx/1.3 Oil and a
standard CCO (M300) with an
integration time of 3.8 s. Regions A and B show spots close
to the resolution limit
The image shows bright hybridization spots along with some unspecific
signals and an elevated fluorescence background within the nuclei. The
signal to background ratio is approximately 4.
The situation for whole chromosome painting signals (FITC-Iabeled
chromosomes of a metaphase hybridized with the "24XCyte" mFISH
kit from MetaSystems) is shown in Fig. 8a. The intensity ratio of the
painted and unpainted chromosomes is 4 again. Is the dynamic range possibly limited by the dark current of the uncooled CCD sensor (M300) used
for capturing? This is not the case, as the background intensity between the
chromosomes is significantly lower than the fluorescence intensity of the
unlabeled chromosomes. And even this inter-chromosomal background is
not only due to dark current, but is caused to a great extent by auto fluorescence within the cytoplasm, by the very small fraction of reflected excitation light that passes the emission filter and by ambient light. Painting
probes labeled with Texas Red usually yield a higher dynamic range at
comparable exposure times. The difference in the dynamic range is probably due to different behavior of the fluorochromes leading to increased
non-specific FITC fluorescence and to increased auto fluorescence caused
by the shorter wavelength of the FITC excitation. Figure 8b illustrates the
influence of dark current on the image quality. The fluorescence lamp was
dimmed and the same metaphase was captured again. Focusing had to be
performed before reducing the light level, as the signals were much too
dark to be visible in the microscope or on the screen. The required exposure time was 125 s. Of course, the dynamic range fell to 2.5 and the intrachromosomal background as well as the overall noise level has been in-