|
By Meghana Honnatti and Gareth Hughes, Zyvex Instruments
and Rocky Draper, The University of Texas at Dallas
Download a printable copy here.
Introduction
Part of the Zyvex NanoWorks® product line, the L200 Nanomanipulator
System is a highly versatile nano-manipulation platform adaptable
to both scanning electron and optical microscopes. The L200
possesses a much smaller form factor than existing manipulation
systems [1]. Its small footprint easily accommodates four
independently controllable nanopositioners [2] and is upgradeable
to six nanopositioners with fine positioning (5 nm) and eight
nanopositioners with standard (100 nm) positioning. Each nanopositioner
is capable of holding and manipulating end-effectors, such
as glass capillaries for micro- or nanoinjection, mechanical
probes functionalized with pH-sensitive chemistry for intracellular
pH measurement, and electrodes for accurate, precise electrical
characterization.
With standard 100 nm precision for positioning of glass capillaries
(down to 5 nm precision for positioning of mechanical probes),
the L200 enables the user to carefully maneuver end-effectors
near cell membranes and within the intracellular space. For
non-cellular applications, such as biomaterials characterization,
the L200 enables the fine manipulation of micron and
sub-micron scale materials.
The L200 is a piezo-motor actuated nanomanipulation tool
with four degrees of freedom – one in each orthogonal
axis (X, Y, and Z) and a fourth for tilting capability.
The rail-mounted positioners can be easily re-positioned
for arbitrary placement. The integrated electrical interfaces
(up to 5 per positioner) make it possible to electrically
stimulate samples and provide a path for electrical power
to attached electromechanical devices (such as microgrippers).
Inserting electrodes into electrical interfaces enables precise
electrical measurements. In addition, the L200 allows for
intuitive control of positioners using a single joystick or
integrated software.
This application note briefly describes the following applications
enabled by the L200:
· Biomaterials mechanics
· Intracellular pH sensing
· Nanoinjection
· Intra- and extracellular probing
· Patch clamping
· Optical manipulation
· Cellular nanosurgery
· Nanowriting and nanoetching
Figure 1: Compact L200 control cabinet, 27"
H x 21" W x 26" D (left).
L200 installed on a Nikon TE2000 inverted confocal microscope
(right).
Figure 2: L200 with capillary adaptors and
NanoEffector® Probes.
Biomaterials Mechanics
The Zyvex L200 is an enabling instrument for quantitative
characterization of a wide range of mechanical characteristics
of biomaterials. Some potential applications include studying
the morphology, structure, and nanomechanical properties of
cells. This has applications in the study of different cell
types, comparison of normal cells to cancer or other pathological
cells, etc. Cellular mechanics, functions of cytoskeletal
and membrane proteins, and nanomechanics of actin-myosin systems
can be quantitatively characterized to yield information at
the molecular level.
The Zyvex L200 also has the potential to study the elastic,
viscous, and plastic properties of connective tissue (such
as ligament, tendon, cartilage and bone). Techniques that
can be used to estimate these properties include nano-indentation,
and micro-tensile and micro-fatigue testing, thus revolutionizing
research in tissue regeneration, bone re-growth, and impact
biomechanics. The L200 could also enable mechanical characterization
of other biomaterials such as nanofibers, proteins, DNA molecules,
etc.
The biomechanical characterization of collagen fibers used
as scaffolding material in tissue engineering has been recently
demonstrated. Current techniques for measuring the mechanical
properties of collagen (involving custom built or proprietary
tensile loading systems) can only handle macro-scale fibers
and fibrils; they are not suitable for micron to sub-micron
fibers such as collagen. The L200 provides a solution to problems
that limit the science of biological material characterization.
The L100 (an earlier version of the L200) has been used to
grasp, pull, and twist collagen fibers for mechanical characterization
[3].
Figure 3: Manipulation of a collagen fiber
with the L100. (a) A MEMS gripper grasping onto a collagen
fiber. (b) A MEMS gripper holding a free collagen fiber. (c)
The collagen fiber formed into a loop.
Intracellular pH Sensing
Intracellular pH, and the pH within subcellular organelles,
varies widely depending on conditions within the cell and
the functions of subcellular organelles. The pH within cells
and certain subcellular organelles has been measured using
fluorescent dyes, such as fluorescein, whose fluorescence
emission is a strong function of pH. However, localizing the
dye at precise locations in cells, or within organelles, can
be problematic. An alternative approach to measuring pH is
to coat a nanoprobe with a pH-sensitive fluorescent dye and
position the probe tip at the desired location within a cell
using the Zyvex L200, followed by monitoring changes in the
pH-sensitive fluorophore by fluorescence microscopy.
This method depends on coating probes with fluorescent dyes.
A gold-coated tungsten probe was incubated with an eleven-carbon
alkane thiol terminated at one end with biotin. Via binding
of the thiol to the gold-coated probe, a self-assembled monolayer
displaying biotin was generated. The probe was then incubated
with fluorescent streptavidin, which binds tightly to the
biotin, labeling the probe with the fluorescent dye on the
streptavidin. This same technique can be used to attach a
pH-sensitive fluorophore to the probe and, upon inserting
the probe into cells using the nanomanipulator, measurement
of pH in the immediate environment of the probe becomes possible.
Figure [4] demonstrates the insertion of a fluorescent probe
into a normal rat kidney (NRK) cell.
Figure 4: Intracellular component probing
of NRK cells with a mechnical probe functionalized with quantum
dots. Scale bar equals 5 mm.
Nanoinjection
Cell microinjection techniques are generally of two basic
types, depending on whether the target cells adhere to the
bottom of a culture dish or whether the cells are non-adherent
and float freely in the medium. With adherent cells, the objective
is usually to inject DNA or protein into either the nucleus
or the cytoplasm while the cell is spread out on the bottom
of the dish. The injection procedure is tedious for large
numbers of cells and the procedure frequently damages and
kills the cells, depending on the skill of the operator. One
factor contributing to cell death is that most microinjection
instruments do not have very fine control over needle movement
and the cell membrane is torn during injection. The Zyvex
L200 has very fine positional control, which increases the
efficiency of the injection process by reducing cell death.
Microinjection with non-adherent cells involves a holder
pipette that applies gentle suction to the cell, holding it
in place, so that the injection needle does not move the cell.
This situation is typical in veterinary cloning laboratories
where material is injected into non-adherent oocytes or embryos
during cloning procedures; it also occurs in certain types
of human in vitro fertilization where a sperm is directly
injected into the oocyte. Fine control over needle movement,
possible with the Zyvex L200, reduces the probability of cell
damage. Nanoinjection into intracellular organelles is depicted
in Figure [5].
Figure 5: Microinjection Process Illustration.
Intra and Extracellular Probing
The 5 nm positioning accuracy of the Zyvex L200 opens up
exciting new opportunities in intracellular stimulation and
probing; just a few are discussed in this section. One application
is to probe subcellular domains of the plasma membrane. For
example, one could compare the membrane potentials (in different
plasma membrane domains) of live neurons during the firing
process. One could also measure the potentials across the
membrane of subcellular organelles. With probe tips of less
than 1 micron diameter, it is possible to insert a probe into
an organelle of a living cell while keeping another probe
in the cytoplasm. Thus, the real-time membrane potential across
various subcellular membranes under varying conditions can
also be recorded. One example of intracellular probing is
demonstrated in Figure [6] where two probes were inserted
into an NRK cell.
By inserting electrodes into a cell, and measuring the impedance
between the electrodes at various locations in the cell, one
could construct an impedance map of the intracellular space
– a technique known as “electrical impedance tomography.”
Since different biomolecules have different electrical properties,
a map of the intracellular space would provide information
about the distribution of these molecules inside the cell.
This knowledge provides new insights into biological processes
at the single-cell level. One could insert a functionalized
probe into a cell and extract proteins from specific subcellular
compartments for further detection or even detect certain
proteins within the cell by optical or electrochemical methods.
With this new advance in single-cell proteomics, one will
now be able to study biological responses evident only at
the single-cell level, but which have, for decades –
for lack of better technology – been studied at the
bulk level.
Figure 6: Intracellular component probing
of an NRK cell, with 2 mechanical probes and 2 micropipettes
using a 4 manipulator L200 setup.
Patch Clamping
The L200’s ability for sub-micron precision in manipulation
using multiple probes (while still having a small foot-print)
opens up new vistas in electrophysiology. Not the least among
them is the possibility of simultaneously patch clamping multiple
locations on neuronal processes such as dendrites and axons.
This will enable the study of electrical and biophysical properties,
such as the local distribution and modulation of ion channels
in dendrites [7]. With its multifunctional capability, the
L200 makes it possible to monitor the propagation of synaptic
and action potentials down the axon within the dendritic tree.
Combining patch clamping with either atomic force microscopy
(AFM), scanning probe microscopy (SPM) or scanning ion conductance
microscopy (SICM) brings up the possibility of simultaneously
obtaining both topographical and electrophysiological information
from the same cell. This information greatly aids the study
of the distribution and effects of various ion channels in
dendritic processes. Imaging and controlled application of
reagents and biomolecules and controlled drug delivery are
now feasible.
In addition, the Zyvex L200 could also enable the investigation
of mechano-sensitive ion-channels in cochlear hair cells or
smooth muscle cells of the jejunum. When combined with patch
clamping, the mechano-transduction of signals and sensitivity
of mechano-sensitive ion channels to membrane strain and tension
can be studied.
Figure 7: Patch Clamping Process Illustration
Optical Manipulation
Optical tweezers use the energy of light to trap and manipulate
microscopic particles without mechanical contact. The ability
to remotely trap, manipulate and track particles has wide-ranging
applications in different disciplines of science. A few vigorously
pursued biological applications in recent years include studies
of nanoscale mechanics of biological motors, inter-particle
interactions, and protein folding and unfolding.
Typical optical manipulation setups consist of a laser beam
focused through a high numerical aperture microscope objective
onto the specimen of interest. The “trapped” particle
can now be easily manipulated and its mechanical properties
studied. Incorporating a laser through an objective, however,
has several disadvantages: drastic modifications of the original
setup are necessitated; we can typically use only a single
beam through the laser and, hence, obtain a single trap; reconfiguration
or recalibration of the system is typically cumbersome. Holographic
and beam-splitting methods produce multiple traps, but add
complexity to the optical setup.
To overcome some of these limitations, and to enable non-contact
manipulation of sub-micron sized particles, we are developing
optical trapping functionality as an add-on option for the
Zyvex L200. The basic idea is to direct a fiber optic laser
and focusing optics through the L200’s nanopositioner
head to trap the particle of interest in the specimen plane
[8].
Shining lasers from above the sample means that no modification
of benchtop optics is necessary to add this feature; the optical
trapping system essentially functions as a “plug-and-play”
module. Other inherent advantages include the ability to run
multiple lasers through a single optical fiber to enable multiple
beam optical manipulation and trapping. Also, the trapped
particles will now move with the optical fiber (not by moving
the specimen or the objective). This decoupling of the optical
trapping module from the microscope objective will allow for
better viewing of the sample.
Some advantages resulting from combining fiber optical trapping
with the Zyvex L200 Nanomanipulator system are the obtainable
precise positioning capability (down to 5 nm) and the possibility
of combining optical trapping with the other modalities enabled
by the L200. For example, one could now simultaneously trap
a subcellular organelle with the fiber optic through one positioner,
monitor its electrochemical properties with an electrode attached
to the end of a second positioner, and simultaneously monitor
its mechanical properties using a probe attached to a third
positioner. Other applications include stretching DNA
molecules, localized photochemistry, studying the kinetics
of cell motility, understanding molecular motors, intracellular
surgery, and dissection of sub-micron intracellular structures,
etc.
Figure 8: Illustration of optical tweezers
with one L200 positioner. This illustration also highlights
the multifunctional nature of the L200 where each of the positioners
is used for a different application.
Cellular Nanosurgery
On the micro scale, sharp probes are routinely used to excise
cells from a tissue or cell group growing on a soft membrane
this is accomplished by cutting a pattern around the cells,
releasing them for harvesting. The probe could also be a “cookie
cutter” to cut out pre-defined patterns.
On the nanoscale, a probe could be used as a “nano-scalpel”
to cut subcellular organelles within living cells. Nuclei
or lysosomes are reasonable test systems because of their
size. For example, it should be readily possible to observe
the consequences of cutting a lysosomal or nuclear membrane
[9]. For instance, the contents of individual lysosomes can
be systematically released and the effects monitored in real
time.
If small enough needles are available, removing subcellular
organelles by applying suction on the needle is possible.
In the reverse, injecting fluid or particles directly into
subcellular organelles is also possible.
Figure 9: Inserting a probe into a sucrosome
of an NRK cell. Scale bar equals
10 mm.
Nanowriting, Nanoetching, and Other Applications
Because of the L200’s acurate positioning capabilities,
nanowriting or dip pen lithography proves a very simple task.
This allows controlled deposition of DNA or proteins at defined
locations on a surface, and printing of very high density
DNA microarrays.
An intuitive spin-off from nanowriting is nanoetching which
is realizable through Zyvex Corporation’s nanoprobes
and nanoeffectors. This opens up a wide range of biomedical
applications ranging from cutting or dissecting biomolecules,
to creating nanochannels in substrates by laying down etchant
solutions.
In combination with other microscopy techniques such as AFM,
SPM, and SICM, the Zyvex L200 makes controlled imaging and
manipulation of single proteins possible allowing the investigation
of several molecular interactions.
back to top
| ©
Copyright 2009, Zyvex Instruments. All Rights Reserved. |
|
