Biomedical Engineering Reference
In-Depth Information
C
core
shell
polymer coating
biomolecule
10 nm
30 nm
A
B
Fig. 10.1.
Biomarkers gallery. (
a
) Confocal maximal projection of fixed HEK293
cells immunostained for actin (
red
) and stained for the nuclei with Hoechst 3342
(
blue
). Only one cell expresses EGFP-labeled uPAR (
green
). (
b
) Confocal maximal
projection of fixed HEK293 stably transfected with EGFP-labeled uPAR (
green
),
and stained with Cy5-uPA ligand (
red
). (
c
)(
Top
) Scheme of a quantum dot and
(
bottom
) an example of application in cell imaging representing the distribution of
GM1 gangliosides found in the plasma membrane in live HeLa cells; nucleus was
stained with Hoechst 3342 (reproduced from [4])
these dyes lack specificity for any particular protein; most applications use
antibodies in fixed and permeabilized cells or specifically labeled ligands
(Fig. 10.1a,b).
Quantum dots (QDs) are inorganic nanocrystals that fluoresce at sharp
and discrete wavelengths depending on their size [4-6]. Their extinction
coe
cients is 10-100 times higher than small fluorophores and fluorescent
proteins (FPs). They also have good quantum yields and exceptional photo-
stability that allow repeated imaging of single molecules [4]. Their absorbance
extends from short wavelengths up to just below the emission wavelength,
so that a single excitation wavelength readily excites QDs of multiple emis-
sion maxima. QDs typically contain a core of CdSe or CdTe and ZnS shell
(Fig. 10.1c). For biological applications, a coating that makes QDs water sol-
uble is necessary to prevent quenching by water, and allow conjugation to
protein targeting molecules such as antibodies and streptavidin. The large
size of QDs conjugated to biomolecules (10-30 nm) prevents e
cient traver-
sal of intact membranes, which restricts their use to permeabilized cells or
extracellular or endocytosed proteins.
10.1.2 Fluorescent Proteins
Rapid advances in live-cell imaging technologies, combined with the use of
genetically encoded fluorescent proteins (FPs), has resulted in a revolution in
cell biology, as it is now possible to track the assembly of protein complexes
within the organized microenvironment of the living cell. In the next sections,
we discuss some of these advances, focusing on fluorescence imaging and spec-
troscopic techniques that are front-edge for the analysis of protein movement
and interactions in living cells.
