Biomedical Engineering Reference
In-Depth Information
feedback loop dampens the cantilever resonance within the drive circuit. This
increases feedback effi ciency and scan speed (Sulchek et al.
2002
) . Using an actively
damped z-scanner, 240 × 240-nm images of Myosin in a buffer solution were
acquired at a frame rate of 21 per second (Kodera et al.
2005
) . Using such devices,
the slowest component in the AFM system is the cantilever. Since the feedback
bandwidth cannot exceed 1/8 of the cantilever resonance frequency (Kodera et al.
2005
), high-resonance-frequency cantilevers are needed to further improve the tem-
poral resolution.
2.3
Determination of Structural Identity
Identifi cation of features observed on biological samples can be diffi cult. Unless
the sample contains well-defi ned and purifi ed biological materials, such as for
instance gap junctions or reconstituted ion channel proteins and receptors (Lal
et al.
1993
; Lin et al.
2001
; Quist et al.
2005a
), it is necessary to use complemen-
tary techniques for proper identifi cation. Using the AFM itself, force recognition
imaging (Kienberger et al.
2005
) can be performed by linking specifi c antibodies
to the tip via a spacer that recognize the presence of its antigen on the surface and
result in images that do not only refl ect topography but also interaction strength
between the AFM tip and the sample. For further direct identifi cation of identity,
the open architecture of AFM allows for integration of optical techniques for
fl uorescence.
2.4
Integration of Optical and Assay Tools
Besides the comparison with images obtained with electron microscopy or crystal-
lographic techniques, the design of AFM allows for easy access with optical
techniques. For example, simultaneous AFM and fl uorescence microscopy, i.e., epi-
fl uorescence, confocal fl uorescence, and total internal refl ection fl uorescence (TIRF)
microscopy, can be performed using fl uorescent markers for biochemical/immuno-
logical identifi cation of imaged features (Fig.
2
).
As an example of the power of the multimodal imaging, simultaneous fl uores-
cence and AFM imaging were performed on neuroblastoma cells and other cell
lines (Quist et al.
2000
), linking changes in cell mechanical properties (using AFM)
to the functionality of hemichannels (using fl uorescence). Recently, a TIRF add-on
for AFM systems was developed that allows for simultaneous AFM and TIRF imag-
ing using light-emitting diode illuminators without the requirement of specialized
TIRF objectives (Ramachandran et al.
2008
). Besides integration of optical tools,
AFM sample supports can be designed to function as in situ lab-on-chips. For
instance, using porous substrates to support biological (or other) specimens, and a
conducting AFM tip, imaging can be combined with ionic conductance measure-
ments (Ionescu-Zanetti et al.
2004
; Quist et al.
2007
) .
