Chasing Rainbows

Quantum dot immunofluorescence and nuclear cell staining of macaque dopaminergic neurons in the substantia nigra. The right image is a composite of the three markers.

Courtesy of Pok Man Mendy Chan, Mount Sinai School of Medicine, New York

Gone are the days when "multicolor immuno-fluorescence" meant proteins tagged with dyes of red, green, and blue. Today's fluorescent molecules cover the color spectrum, and researchers are using them to image proteins simultaneously.

Antibodies can be conjugated to two types of fluorescent molecules: organic fluorophores, or dyes, and quantum dots. Dyes, which link to antibodies that directly or indirectly recognize desired proteins, have been a staple in immunfluorescence studies for decades. That means many antibodies are commercially available and protocols are versatile. But dyes have drawbacks for multiplexing. An activated dye emits a wide spectrum of light outside its peak emission level, creating overlap when several are used at once. And absorption spectra are narrow, so a separate laser is needed to excite each dye.

Quantum dots avoid these difficulties. Dots are 20 to 50 times brighter than dyes and emit narrow spectra, and color can be finely tuned by varying the diameter of the core. One color among several is thus easier to resolve. Also, dots are excited by a wide range of wavelengths, so only one excitation source is needed. But dots, too, have drawbacks. They are about 10 times larger than dye molecules, so they don't reliably penetrate cell membranes, limiting their use primarily to labeling cell surface proteins. Also, their size and chemistry may contribute to unnatural conditions in cells.

For multiplexing, cost may not be a big factor in choosing dots versus dyes. Dot-conjugated antibodies generally cost more, but stock solutions keep longer and can be more dilute, so the cost per experiment is similar. Also, dots' narrower emission spectra allow a higher degree of multiplexing, so a study could potentially be conducted with fewer experiments.

The Scientist talked to researchers who have come up with solutions for getting the best multicolor immunofluorescence from dots and dyes.