Page 213 - Fundamentals of Light Microscopy and Electronic Imaging

196 FLUORESCENCE MICROSCOPY

and AMC. In addition, they contain low-fluorescence glass—a feature that minimizes
background fluorescence and gives high contrast. Since image brightness (photon flux
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4
per unit area and time) is proportional to NA /M , where NA is the numerical aperture
and M is the magnification, a 60 , 1.4 NA planapochromatic objective is among the
brightest objectives and is very well suited for fluorescence imaging.
The spatial resolution d for two noncoherent fluorescent point objects is the same
as in bright-field microscopy with incoherent light, and is given as d 0.61λ/NA,
where λ is the mean wavelength of fluorescent light transmitted by the barrier filter.
Resolution, brightness, and other features defining the optical performance of objective
lenses are described in Chapter 4.

CAUSES OF HIGH-FLUORESCENCE BACKGROUND

For research microscopes with properly selected fluorescence filter sets, the amount of
background fluorescence in the image of a specimen containing a single fluorochrome
is usually 15–30% of maximum specimen brightness—not 0% as might be expected.
An example of background signal in a cell labeled with a single fluorochrome is shown
in Figure 11-10. Because background fluorescence is always present, it is important to
take steps to keep the background signal as low as possible.

• Less than ideal performance of filter sets, where transmission and reflectance by
interference filters and the dichroic mirror are not 100% and where the transition
boundaries between transmission and reflection are not sharply defined, is a major
contributor to background signal. These problems are compounded when poorly
performing filters are used together in a single filter set.
• Specimen preparation must include complete neutralization of unreacted aldehyde
groups and blocking of remaining reactive sites to minimize nonspecific binding of
the fluorescent probe. It is also necessary to completely remove unbound fluo-
rochrome by thorough rinsing. However, even if these precautions are followed,
background fluorescence from unbound antibody can be high for a mounted cover-
slip containing labeled cultured cells. This is because the labeling reaction is an
equilibrium between bound and free states of the antibody. Even with an equilib-
6
rium dissociation constant of 1 nM and a modest concentration of 10 antigen-
binding sites per cell, a significant fraction of antibody would be expected to disso-
ciate and be free in the mounting medium.
• Reflections and scattering in the optical pathway cause rays to enter a filter at an
oblique angle, reducing the filter’s transmission/reflection efficiency. One site of
concern is the back wall of the filter cube, where excitatory rays that are partially
transmitted by the dichroic mirror are reflected and transmitted by the emission fil-
ter because they are not incident at an angle perpendicular to the plane of the emis-
sion filter. In recent Zeiss designs where the back wall of the filter cube is removed,
reflections at this location are removed, and image contrast is improved by
15–20%.
• Dust, fingerprints, and scratches on filters and lens elements scatter significant
amounts of light, resulting in an increase in background signal and reduced con-
trast. Cleaning filters and optics significantly reduces this problem. Interference fil-
ters also deteriorate gradually over time due to handling and the presence of water