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Biophys J, October 1998, p. 2119-2120, Vol. 75, No. 4
*Centro de Química-Física Molecular, Complexo I, Instituto Superior Técnico, Lisboa, Portugal; #Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal; and §Department of Chemical and Nuclear Engineering, University of California, Santa Barbara, California 93106 USA
In a recent paper, Carlson et al. (1997) Independently, we observed a similar effect on a phospholipid bilayer
in the absence of protein. The results were first obtained on a control
sample used in our study of the interaction of the polyene antibiotic
filipin with planar bilayers (Santos et al., 1998 Fig. 1 a shows the AFM
image of the respread bilayer. The bilayer contains numerous holes with
a depth of about 2.5 nm, which is consistent with the thickness of a
monolayer. A 5 × 5 µm2 square
region in the central area of the image was generated by
scanning at a force of 20 nN for 2 minutes, then imaged by increasing
the scan area and immediately recording the image at a force of 5 nN.
The scanning process heals the defective bilayer and produces a flat
hole-free area. Fig. 1 b displays the AFM image of another
DPPE/cholesterol sample prepared under the same conditions as the
previous sample with an air exposure of 2 s. Monolayer patches are
clearly seen within holes, which are also one layer thick. We could not
produce a hole-free area like in Fig. 1 a by applying a
higher force, although fusion of patches could be induced. Fig. 1
c shows the same area as in Fig. 1 b after 10 min
of scanning with a force of 25 nN and a scan rate of 10 Hz. Some
patches fused with their nearest neighbors. Applying a higher force did
not accelerate fusion and eventually led to the rupture and removal of
the bilayer.
LETTER
reported
the use of the atomic force microscope (AFM) to initiate bilayer fusion in a controlled manner. In their study of reconstituted high-density lipoproteins (HDL) they obtained discoidal bilayers of dipalmitoyl phosphatidylcholine (DPPC) and apolipoprotein A-I (apoA-I), the primary
protein component of HDL. Under certain experimental conditions the
fusion of the discoidal bilayers into a planar bilayer was induced by
the scanning process in the AFM. In other cases, scanning of the HDL
particles resulted in digging a monolayer-thick hole. The difference
between fusion and digging did not appear to depend on force or scan
rate. It was not clear whether protein played a role in the fusion
process.
). The bilayer of 7:3
(w:w) dipalmitoyl phosphatidylethanolamine (DPPE):cholesterol was
prepared by Langmuir-Blodgett deposition on freshly cleaved mica at a
surface pressure of 25 mN/m. The sample was then imaged by contact-mode
AFM under water. Silicon nitride tips with a nominal spring constant of
0.12 N/m (Digital Instruments, Santa Barbara, CA) were used. The
bilayer was unintentionally exposed to air for a moment during transfer
to the fluid cell of the AFM. The short exposure to air probably led to
the disruption of the bilayer into monolayer and trilayer domains;
subsequent respreading led to a highly defective bilayer.
View larger version (64K):
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FIGURE 1
AFM height images of a DPPE/cholesterol bilayer which
was exposed to air for a short time. All images are taken in a contact
mode under water. (a) Area: 9 × 9 µm2. A square defect-free region in the central part of
the image was reconstituted by the AFM tip during several scannings at
a force of 20 nN. (b) Area: 900 × 900 nm2. Monolayer-thick patches within holes are clearly seen.
(c) Area: 900 × 900 nm2, the same
region as in (b) after scanning at 25 nN for 10 minutes.
Some patches fused with their nearest neighbors.
Exposure to air was not necessary to induce the healing of holes. Fig.
2 a shows a pure DPPE
bilayer deposited at a surface pressure of 25 mN/m. The sample was kept
under water during transfer to the AFM. A flat hole-free square region
in the center of the image was generated by scanning for 2 min with a
force of 100 nN, then imaged by increasing the scan size and recording
the image at a force of 5 nN. Note that such healing could be induced only for samples with monolayer-thick holes. It is not clear why Langmuir-Blodgett deposition sometimes produces monolayer-thick holes
in DPPE bilayers. It is much more common to have bilayer-thick holes in
bilayers (Mou et al., 1995, Shao et al., 1996) although monolayer-thick
holes have also been observed (Hui et al., 1995). In the samples with
bilayer-thick holes we did not observe any healing. Applying a higher
force led to the coalescence of nearby holes and then to removal of the
bilayer.
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The mechanism of the tip-induced reconstitution is not clear although it is logical that energy brought by the AFM tip helps to convert unstable holes or disks into a lower-energy bilayer. One possible explanation is that the AFM tip drags lipid molecules that are loosely attached or extracted from bilayer until they fill in holes in bilayer. These molecules might also adsorb on the AFM tip during this process, forming a layer on it. Fig. 2 b shows an AFM image of a hole in a DPPE/cholesterol bilayer. The hole appears to be one layer thick and to contain another monolayer-thick hole inside. However, it is clearly seen that the lower edges of both holes have exactly the same shape, which is a very unlikely event. Such an image might be generated if the hole were two layers thick and the AFM tip had an adsorbed monolayer on it. As the substrate step is deeper than the step on the AFM tip, the tip cannot reach the bottom of the hole and the hole edge is imaged twice, once with the tip itself and once with monolayer on it (double tip effect). Thus, the image in Fig. 2 b suggests that lipids can adsorb to the AFM tip during the scanning of bilayers in water.
Our results show that patterning similar to that shown by Carlson et
al. (1997) can be obtained on samples without protein. We could not
establish the precise conditions necessary for patterning but have
shown that it is crucial to have a bilayer with monolayer-thick holes.
While additional work will be necessary before the mechanism of
patterning is fully understood, the effect opens new possibilities for
manipulation of phospholipid bilayers and formation of nanoscopic defect-free regions with a desired size and shape.
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FOOTNOTES |
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Received for publication 20 February 1998 and in final form 7 July 1998.
Address reprint requests to Dr. Joseph A. Zasadzinski, Department of Chemical and Nuclear Engineering, University of California, Santa Barbara, CA 93106. Tel.: 805-893-4769; Fax: 805-893-4731; E-mail: gorilla{at}engineering.ucsb.edu.
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REFERENCES |
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Biophys J, October 1998, p. 2119-2120, Vol. 75, No. 4
© 1998 by the Biophysical Society 0006-3495/98/10/2119/02 $2.00
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