Biomedical Engineering Reference
In-Depth Information
Sample
a
Z stage
b
Lens
UV light source
100 µm
c
20
0
-20
DMD
uM
Designed gray
scale images
100
300
uM
d
100 µm
FIGURE 2.10
Micropatterning. of. gradients. of. photoreactive. proteins. using. the. digital. micromir-
ror.device..(From.Sheng.Wang,.Cheryl.Wong.Po.Foo,.Ajithkumar.Warrier,.Mu-ming.Poo,.Sarah.C..
Heilshorn,.and.Xiang.Zhang,.“Gradient.lithography.of.engineered.proteins.to.fabricate.2D.and.3D.
cell. culture. microenvironments,”.
Biomed. Microdev.
. 11,. 1127,. 2009.. Reprinted. with. permission.
from.Springer.Science+Business.Media.)
University and Paul Cramer presented a general aqueous-based strategy for patterning proteins
on surfaces that exploits the principle that photobleaching creates photogenerated radicals.
hese radicals, he hypothesized, can be used for attaching organic molecules to surfaces. he
patterning process starts by coating the surface with BSA, which acts as a “sticky surface” for the
photobleached molecules. As photobleachable molecules, Cramer's group demonstrated the deposi-
tion of biotin-4-luorescein (0.025 mg/mL, photobleachable with blue light for 30 minutes) and
of Alexa 594-labeled anti-dinitrophenyl IgG (0.25 mg/mL, photobleachable with yellow/green
light for 2 hours). he IgG pattern was visible as deposited (each IgG, which contains three to
four luorescent labels, was only partially photobleached); to visualize the biotin pattern, a solu-
tion of Alexa 488-streptavidin was added. his photochemistry has been recently implemented
by Santiago Costantino's group (University of Montreal, Canada) both in a laser-based setup
and in a wide-ield illumination setup (
Figure 2.11
). By placing a spatial ilter (such as a liquid
crystal display) in the image plane of where a camera's CCD chip would normally be placed
and using the camera port as an illumination port, it is possible to project arbitrary computer-
generated patterns onto the sample with any light source (
Figure 2.11a
) and deposit proteins
with high dynamic range idelity (
Figure 2.11b
). With this setup, Cramer's photobleaching
scheme (
Figure 2.11c
) has achieved submicron resolution (
Figure 2.11d
) and multiprotein pat-
terning capability (using lasers of diferent wavelengths to immobilize proteins tagged with dif-
ferent luorophores).
he high-energy femtosecond pulsed laser of a multiphoton microscope, equipped with a com-
puterized scanner and selectable frequencies, is a powerful tool for biopatterning—for both add-
ing and deleting material. As a notable example, Jason Shear and colleagues from the University
of Texas at Austin have used multiphoton imaging lasers to photo-cross-link albumin, thus con-
structing biocompatible nonadherent microstructures in the presence of the cells (
Figure 2.12
)
that can be used to guide axons and trap bacterial cells (see
Figure 1.16
). Others have used
the laser to desorb proteins selectively, thus deleting patterns in the presence of the cells.
