Living Image 3.1

F. Fluorescent Imaging
Specifying Signal
Levels and f/stop
Settings
F.4 Image Data Display
214
Figure F.6 Illustration of the in vivo fluorescence process.
At 600-900 nm, light transmission through tissue is highest and the generation of
autofluorescence is lower. Therefore it is important to select fluorophores that are active
in the 600-900 nm range. Fluorophores such as GFP that are active in the 450-600 nm
range will still work, but the depth of detection may be limited to within several
millimeters of the surface.
Fluorescent signals are usually brighter than bioluminescent signals, so imaging times are
shorter, typically from one to 30 seconds. The bright signal enables a lower binning level
that produces better spatial resolution. Further, the f/stop can often be set to higher values;
f/2 or f/4 is recommended for fluorescence imaging. A higher f/stop improves the depth
of field, yielding a sharper image. For more details on the f/stop, see Lens Aperture,
page 191.
Fluorescent image data can be displayed in units of counts or photons (absolute,
calibrated), or in terms of efficiency (calibrated, normalized). For more details, see
Quantifying Image Data, page 199.
If the image is displayed in photons, you can compare images with different exposure
times, f/stop setting, or binning level. When an image is displayed in terms of efficiency,
the fluorescent image is normalized against a stored reference image of the excitation light
intensity. Efficiency image data is without units and represents the ratio of emitted light
to incident light. For more details on efficiency, see page 201.
Fluorescent Efficiency The detected fluorescent signal depends on the amount of fluorophore present in the
sample and the intensity of the incident excitation light. At the sample stage, the incident
excitation light is not uniform over the FOV. It peaks at the center of the FOV and drops
of slowly toward the edges (Figure F.7). To eliminate the excitation light as a variable from
the measurement, the data can be displayed in terms of efficiency (Figure F.8).