56
ANDREAS PLESCH
The imaging system
To get an idea how these fundamental concepts are related to real imaging
systems, we have to take a closer look at the main functional units that
imaging systems are composed of. Each component will potentially degrade the quality of the image:
- illuminating system of the microscope - including fluorescence lamp,
fluorescence filter set, and optical path
- magnifying system of the microscope - the objective lens
- camera - including the digitizer
- image processing system
The illuminating system
The fluorescence
lamp
Fluorescence filters
Usually, high-pressure mercury lamps are used in FISH applications, as
their spectral characteristics provide efficient excitation of many dyes -
from ultraviolet excitation (DAPI) to deep red (Cyanine 5 and Cyanine
5.5). Mercury lamps of 100 W as well as of 50 Ware available for microscopy. The difference in light intensity reaching the object (slide) is less
than a factor of 2. Some microscopists prefer the 50-W lamp as photo
bleaching is less of a problem during lengthy manual adjustments. The
use of infrared dyes like Cyanine 7 requires a Xenon lamp with its
high intensity in the far-red and infrared spectral range. Perfect stabilization of the lamp power supply is normally not an issue as the integration
times required will effectively smooth out residual ripples of the supply
current. While the fluorescence lamp does not influence the optical resolution, incorrect lamp alignment will severely reduce the illumination intensity, lead to an inhomogeneous illumination within the image and reduce image contrast.
Fluorescent dyes are characterized by their excitation and emission spectra. The emission spectrum of a fluorochrome is shifted towards longer
wavelengths as compared to the excitation spectrum. The spectral distance
between excitation and emission maxima is called Stokes shift. Fluorescence filter sets are used to separate the illuminating light (excitation) and
fluorescence light (emission). Figure 6 shows the principle. The excitation
filter selects the spectral band to illuminate the dye. The dichroic mirror
reflects the excitation light down to the specimen. Dichroic mirrors are
ANDREAS PLESCH
The imaging system
To get an idea how these fundamental concepts are related to real imaging
systems, we have to take a closer look at the main functional units that
imaging systems are composed of. Each component will potentially degrade the quality of the image:
- illuminating system of the microscope - including fluorescence lamp,
fluorescence filter set, and optical path
- magnifying system of the microscope - the objective lens
- camera - including the digitizer
- image processing system
The illuminating system
The fluorescence
lamp
Fluorescence filters
Usually, high-pressure mercury lamps are used in FISH applications, as
their spectral characteristics provide efficient excitation of many dyes -
from ultraviolet excitation (DAPI) to deep red (Cyanine 5 and Cyanine
5.5). Mercury lamps of 100 W as well as of 50 Ware available for microscopy. The difference in light intensity reaching the object (slide) is less
than a factor of 2. Some microscopists prefer the 50-W lamp as photo
bleaching is less of a problem during lengthy manual adjustments. The
use of infrared dyes like Cyanine 7 requires a Xenon lamp with its
high intensity in the far-red and infrared spectral range. Perfect stabilization of the lamp power supply is normally not an issue as the integration
times required will effectively smooth out residual ripples of the supply
current. While the fluorescence lamp does not influence the optical resolution, incorrect lamp alignment will severely reduce the illumination intensity, lead to an inhomogeneous illumination within the image and reduce image contrast.
Fluorescent dyes are characterized by their excitation and emission spectra. The emission spectrum of a fluorochrome is shifted towards longer
wavelengths as compared to the excitation spectrum. The spectral distance
between excitation and emission maxima is called Stokes shift. Fluorescence filter sets are used to separate the illuminating light (excitation) and
fluorescence light (emission). Figure 6 shows the principle. The excitation
filter selects the spectral band to illuminate the dye. The dichroic mirror
reflects the excitation light down to the specimen. Dichroic mirrors are