2 Microscopy and Imaging Systems
65
Fig. 8. a FITC-labeled chromosome paints (24Xcyte mFISH kit from MetaSystems). Plan-Neofluoar lOox/1.3 Oil,
M300 standard CCD, integration time 2.4 s. b Same metaphase captured at very low illumination light level. Integration time was 125 s
creased. However, we obtain a decent, analyzable image, with a background that is still dominated by residual fluorescence of labeled nucleotides that are unspecifically bound to DNA rather than by CCD dark current. This is an important result.
- The dynamic range of typical FISH signals is limited by the sample
itself and does not exceed the dynamic range that an 8-bit digital system can handle.
- In most applications an uncooled standard video CCD is suitable for
long-term exposure of signals down to, and even below the limit of
visibility, if the exposure control is properly done.
To learn about the limit of resolution we will take a closer look at the two
regions marked in Fig. 7. Region A includes two spots of about 0.8 J..Lm
diameter, separated by 0.5 J..Lm. Region B shows two spots of similar
size, separated by 0.4 J..Lm. Spots smaller than 0.8 J..Lm are not found in
the image. The theoretical minimum size, however, was calculated to
be 0.58 J..Lm and 0.54 J..Lm for the 100x/1.3 and the 63x/1.4 objectives, respectively. This theoretical limit is not reached due to several factors: (1) a
65
Fig. 8. a FITC-labeled chromosome paints (24Xcyte mFISH kit from MetaSystems). Plan-Neofluoar lOox/1.3 Oil,
M300 standard CCD, integration time 2.4 s. b Same metaphase captured at very low illumination light level. Integration time was 125 s
creased. However, we obtain a decent, analyzable image, with a background that is still dominated by residual fluorescence of labeled nucleotides that are unspecifically bound to DNA rather than by CCD dark current. This is an important result.
- The dynamic range of typical FISH signals is limited by the sample
itself and does not exceed the dynamic range that an 8-bit digital system can handle.
- In most applications an uncooled standard video CCD is suitable for
long-term exposure of signals down to, and even below the limit of
visibility, if the exposure control is properly done.
To learn about the limit of resolution we will take a closer look at the two
regions marked in Fig. 7. Region A includes two spots of about 0.8 J..Lm
diameter, separated by 0.5 J..Lm. Region B shows two spots of similar
size, separated by 0.4 J..Lm. Spots smaller than 0.8 J..Lm are not found in
the image. The theoretical minimum size, however, was calculated to
be 0.58 J..Lm and 0.54 J..Lm for the 100x/1.3 and the 63x/1.4 objectives, respectively. This theoretical limit is not reached due to several factors: (1) a