BIOLOGICAL IMAGING: Fluorescent nanocrystals improve upon dyes

By replacing dye markers with semiconductor nanocrystals, scientists at the University of California-Berkeley (UCB) and Lawrence Berkeley National Laboratory (LBNL; Berkeley, CA) have successfully demonstrated a technique for measuring multiple biomarkers simultaneously using a single laser source. In conventional fluorescence microscopy, spectroscopy, flow cytometry, and other methods that use fluorescence in biology to report on different proteins, it takes quite a bit of work to observe different color fluorophores, according to Shimon Weiss, a staff scientist in the LBNL materials sciences division. For example, a paper coauthored by Weiss referred to a conventional flow-cytometry experiment in which an eight-color, three-laser system was required to measure 10 parameters on cellular antigens.¹

"You use a filter wheel. You take data serially. You have to use many filters for excitation, and so on," Weiss said. "Nanocrystals basically eliminate all of that difficulty. They have very narrow emission spectra, and they have broad absorption. Therefore, you can have many different markers, all excited with a single laser."

Earlier work by Paul Alivisatos, a chemistry professor at UCB, senior staff scientist in the LBNL materials sciences division, and coauthor of the Science paper, had already demonstrated that the color of light emitted by a semiconductor nanocrystal depends on its size. Also, unlike dye molecules, which have narrow excitation spectra and emission spectra skewed toward the red wavelengths, nanocrystals can be excited efficiently at any wavelength shorter than the emission peak and have narrow, symmetrical emission spectra that minimize crosstalk between adjacent detection channels.

Typical nanocrystal emission widths in the visible spectrum range from 20 to 30 nm full-width at half maximum with extinction coefficients on the order of 105 M^-1 cm^-1 in the visible and ultraviolet, the researchers wrote. The nanocrystals can also be made to cover a spectral region from 400 nm to 2 µm through variation of nanocrystal material and size. In addition, the emission spectrum is tunable with relatively long fluorescence lifetimes in the hundreds of nanoseconds.

"If you do fluorescence with dye molecules, you have to match your laser to a specific dye," Weiss said. "In this case the restriction is removed because the absorption is more or less semi-continuous. It has an edge, but then it goes into the blue and ultraviolet continuously. Therefore you can position your laser anywhere you want."

Weiss, Alivisatos, and coworkers demonstrated these characteristics in a biological environment by labeling mouse tissue cells (3T3 fibroblasts) with cadmium selenide nanocrystals of two different sizes (2 nm to emit 550-nm green light and 4 nm to emit 630-nm red light). The cadmium selenide core crystals were then enclosed within cadmium sulfide "shells" to boost their performance. "By enclosing a core nanocrystal of one material within a shell of another having a higher bandgap, one can efficiently confine the excitation to the core, eliminating nonradiative relaxation pathways and preventing photochemical degradation," the researchers wrote.

In addition, each core-shell unit was enclosed within a shell of silica for water solubility and biocompatibility with the mouse tissue. Weiss described this process as making the biological and nanocrystal molecules "biologically friendly" so that they could be conjugated into samples for the unconventional immunocytochemistry experiments. The silica shells were also modified to selectively control the site of conjugation between molecules. The 2-nm nanocrystals were coated with trimethoxysilylpropyl urea and acetate groups to bind to the cell nucleus through electrostatic and hydrogen-bonding interactions, while an avidin-biotin interaction attached the 4-nm nanocrystals to actin filaments on the outer cell membrane.

Upon imaging with wide-field microscopy, the green and red labels were visible to the unaided eye and could be photographed with a color Polaroid camera. Confocal microscopy images showed that cell nuclei had penetrated the green probes, while actin fibers had been stained red.

Potential applications for the nanocrystal staining technique include labeling and observation of dynamic events, such as conformation changes in a protein; use in x-ray- and electron-based imaging techniques; and use as a tunable dye for detecting fluorescence in blood samples.

"The development of semiconductor nanocrystals for biological labeling gives biologists an entire new class of fluorescent probes for which no small organic molecule equivalent exists," the researchers wrote. "These nanocrystal probes can be complementary and in some cases may be superior to existing fluorophores."

REFERENCE

1. M. Bruche¥Jr. et al., Science 281, 2013 (Sept. 25, 1998).