approach to quantitative
Fluorescence microscopy can detect concentrations of important biomarkers that
are barely visible. Learn the three factors that influence the quality of quantitative
fluorescence imaging, and what you can do to improve the signal.
Enter any microbiol- ogy or bioimaging lab, and you’ll find shelves and drawers
full of fluorescent dyes and fluorescence filter sets. In the cabinets, you may find mercury arc or
metal halide lamps, light-emitting
diodes (LEDs), or lasers. And on
the workbench, of course, is the
microscope with its camera and
set of objective lenses, the workhorse that brings everything into
focus. All one needs to do is pick
a dye to stain the specimen of
the day, match it with the recommended filter set from the manufacturer, plug in the lamp on the
microscope, and in no time dazzling fluorescent images of micro-tubules and golgi apparati appear.
But what if instead of a beautiful image, one needs a quantitative result? What if the images are
of simple beads, micro capillaries,
or gels; and the point of the image
is to detect the concentration of a particular biomolecules trapped within them?
The concentration of fluorescent
markers can be a very important result,
as known biomarkers can alert doctors
to impending heart failure, early onset
of cancer, or the early stages of a deadly
infection. 1, 2 Even when these biomolecules
are present at concentrations that would
worry a physician, they might barely light
up under a fluorescence microscope. For
these applications, a quantitative image
is more important than a qualitative one,
and the basic recommendations for filters,
sources, and dyes might not be enough.
Dragon Green beads imaged with a 10x/0.3NA Nikon CFI Plan Fluor objective (left column), and the same
beads imaged with a 10/x0.5NA Nikon Super Fluor objective (right column).
BRIAN MCCALL is an optical engineer with Edmund Optics; www.edmundoptics.com.