WO2010091311A2 - A nanowire afm probe for imaging soft materials - Google Patents

Abstract

The invention provides for a device comprising a support and a nanowire attached to the support designed for use with an atomic force microscope (AFM).

Description

A nanowire AFM probe for imaging soft materials

RELATED PATENT APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 61/150,307, filed February 5, 2009, which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

[0001] The invention described and claimed herein was made utilizing funds supplied by the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The government has certain rights in this invention.

FIELD OF THE INVENTION

[0002] The present invention is in the field of atomic force microscopy.

BACKGROUND OF THE INVENTION

[0003] All cellular functions are accomplished by proteins and protein complexes which are 10 to 100 nm in size. Our understanding of cellular and molecular biology would be greatly enhanced if we had an imaging technology with high enough resolution to watch individual proteins as they worked. While Atomic Force Microscopy (AFM) readily images most surfaces with subnanometer resolution, its ability to image cells has been limited because the minimum force required for detection causes deformation. As a result, most researchers using AFM to investigate biological systems use strong cells such as plants or bacteria or they destroy the cell by removing a membrane fragment and supporting it with a firm surface to enable imaging. Examples of this work include the imaging the structure of cellulose fibrils in the plant cell wall to enable the creation of degradation mechanisms facilitating the production of bio fuels, or the imaging of protein crystals in the coats of bacterial spores and their change in morphology upon rehydration and germination. AFM images of supported membranes from destroyed cells reveal the structure and organization of proteins in fine detail. Individual domains of each light harvesting complex in a chloroplast are resolvable along with other examples like ATPases in the mitochondria and rhodopsins in the retina. [0004] The AFM is able to measure material properties of a surface (topography, magnetism, specific chemical interactions, etc.) with high spatial resolution by sensing the force the surface applies to the instrument. However, using this instrument to measure the surface of soft materials such as living cells has been very elusive because the minimum force required by the instrument is enough to deform the cell, reducing the resolution and possibly causing damage. Researchers have been attempting to reduce the minimum force required by the instrument. The main means of doing this is by reducing the size of the spring used to measure the force. The current state of the art uses springs which are a few microns in width and length and the size is limited by the detection mechanism which requires reflection of a laser beam so the spring must be optically flat and larger than the diffraction limit of light. No detection mechanism exists to measure smaller cantilevers with high enough precision.

SUMMARY OF THE INVENTION

[0005] The present invention provides for a device comprising a support and a nanowire attached to the support designed for use with an atomic force microscope (AFM). The nanowire is a cantilever of the AFM.

[0006] The present invention also provides for an AFM comprising the device of the present invention. When in uses, the nanowire is presented perpendicular to the sample surface (shear-mode). When in use, the nanowire is at times in contact with target of interest, wherein the target is soft, such as a living or viable cell or a gel. When in use, the nanowire is at times in the beamwaist of a focused laser beam. When in use, at times, the resulting scatter pattern of the focused laser beam on the nanowire is incident upon a split-photodiode. The split-photodiode detects change in oscillation frequency/amplitude of the nanowire, which corresponds to a signal on the split-photodiode. In some embodiments, when the target is a living or viable cell, the target is in a liquid, such as a solution.

[0007] The present invention also provides for a method of using an AFM comprising a device of the present invention, comprising observing a target of interest that is soft, such as a living or viable cell or a gel.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

[0009] Figure 1 shows an embodiment of the invention.

[0010] Figure 2 shows an embodiment of the invention.

[0011] Figure 3 shows an embodiment of the invention fabricated from VLS silicon nanowire growth.

[0012] Figure 4 shows the calculated thermal force noise for a device of the invention. The device of the invention has significantly less force noise which will enable imaging of soft materials.

[0013] Figure 5, Panel A shows the theoretical difference signal from a split- photodiode when a conductive, 152 nm radius nanowire passes through a 532 nm wavelength Gaussian laser with a 1 μm spotsize, in air. Both the polarization and the photodiode-split are oriented along the length of the nanowire. Figure 5, Panel B shows the images of the near- field absolute electric field, with the beam propagating from left to right.

[0014] Figure 6 shows the theoretical sensitivity plot showing its dependence on the nanowire's radius and laser spot size. This analysis is for a 633 nm laser, with the polarization along the axis of the nanowire, in air.

[0015] Figure 7 shows the split-photodiode signal measured from the setup described in Example 2.

[0016] Figure 8 shows the measurement of the spectrum of the nanowire's thermal fluctuation from the setup described in Example 2. Based on the dimensions and material properties of the cantilever in air, one expects a resonant frequency of 37.5 kHz and a Q of 4.42. ONe measures a frequency of 36.6 kHz and a Q of 2.4. The discrepancy in Q is being investigated and in particular whether it is due to internal losses in the nanowire that are not modeled. The fit DC thermal noise is 0.1 A/VHZ.

[0017] Figure 9 shows the Mie scatter analysis.

[0018] Figure 10 shows a process for producing Ag₂Ga nanowires. DETAILED DESCRIPTION OF THE INVENTION

[0019] Before the invention is described in detail, it is to be understood that, unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes, host microorganisms, or processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

[0020] As used in the specification and the appended claims, the singular forms "a,"

"an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "target of interest" includes a single target of interest as well as a plurality of targets of interest.

[0021] The principal constraint to AFM imaging of soft materials in fluids is that the noise associated with cantilever-fluid interactions requires a stronger probe-sample signal than soft materials provide. Much of the noise originates from viscous damping and as such is strongly minimized by reducing the size of the cantilever. However the current deflection detection approach imposes limits on the cantilever's minimum size. In the present invention, an optical scattering-interference detection scheme (commonly used in optical tweezers) is adapted to detect the motion of cantilevers that are smaller than optical wavelengths. What is developed is a rigorous model of Mie scattering from a Gaussian beam off of a cylinder that indicates this detection approach is appropriate and has sufficient sensitivity to measure thermal vibrations of highly damped nano wires.

[0022] The present invention makes it possible to measure the position of a nanowire with high precision. The nanowire can be used as an AFM spring which would reduce the force between the surface and spring. The measurement of position is performed by putting the nanowire in the beamwaist of a focused laser beam. The nanowire will scatter the laser light differently depending upon the position of the nanowire within the beamwaist. The differences in the scattered light can be measured as intensity changes on a position sensitive photodiode (quadrant photodiode). This detection mechanism is similar to that used for measuring the position of beads in laser tweezers but applied to cantilevers used in AFM. In some embodiments, the photodiode is configured such that it gathers the forward scattered light along with the non-scattered light. In some embodiments, the scattered light is perpendicular to the beam axis so that it does not have the background signal of the non- scattered light. In some embodiments, the nanowire can further comprise a luminescent element, such as a quantum dot, for example at the end of the nanowire, so that a shift the color of the detected light would indicate detection of the quantum dot. In some embodiments, the nanowire can further comprise a molecule, such as an antibody, capable of specifically binding a target molecule at high affinity.

[0023] New cantilever fabrication techniques and deflection detection mechanisms are required to reduce the forces associated with AFM imaging. Thermal excitation forces define the fundamental lower limit for AFM. These forces derive from the interaction of the cantilever with its hydrodynamic environment. Smaller cantilevers have lower damping and better noise characteristics. However, current research has reached a limit in reducing the cantilever dimensions because the detection mechanism requires specular reflection of the laser beam. The smallest cantilevers are on the order of the size of the focused laser beam waist.

[0024] The device of the present invention can be constructed from epitaxially grown silicon nanowires and uses interference of forward scattered light as the detection mechanism. VLS silicon nanowire growth provides a convenient, high yielding, and reproducible method for fabricating nanoscale cantilevers. The width and length of silicon nanowires are tuned easily by controlling the size of the initial catalyst particle and growth conditions. Also, growth is epitaxial with predictable orientations. Nanowires of diameter larger than 20nm grow in the <111> direction. Conveniently, the growth direction of nanowires with diameters less than 20nm switches to <110> which is perpendicular to the <111> direction. By performing a second growth step with a smaller catalyst particle deposited at the end of the originally grown nanowire, a sharp tip can be fabricated which only requires a single cut by an FIB. An embodiment of a device of the present invention is shown in Figure 3. The detection mechanism for optical tweezers similarly uses interference between forward scattered light and the non-scattered beam to achieve 1 pm/rtHz baseline noise. The high index of refraction and large scattering cross section for the gold catalyst particle at the end of the wire will increase the sensitivity 100 times over typical laser tweezers measurements allowing the use of much smaller diameters. The significant reduction in the dimensions of the cantilever leads to an enormous decrease in the force noise associated with thermal excitation (Figure 4). Thus, the device of the present invention provides the opportunity to probe soft materials significantly more gently than the smallest AFM probes currently being utilized.

[0025] The nanowire size can be of any suitable size. Such a size can be at least 5 or

10 nm in diameter and/or at least 100, 200, or 300 nm in length. Such a size can be up to 55, 100, 200, 300, 400, or 500 nm in diameter and/or up to 1.5, 10, 50, or 100 μm in length. In some embodiments, the size can be from 10 nm in diameter to 500 nm in diameter and/or from 300 nm in length to 100 μm in length. In some embodiments, the size can be from 10 nm in diameter to 55 nm in diameter and/or from 300 nm in length to 1.5 μm in length. The cross-sectional symmetry can be circular or any of the faceted growth patterns such as trigonal, square, rectangular, hexagonal, octagonal, or the like.

[0026] The nanowire can comprise of any suitable material, such as a metal, semiconductors, insulator, or the like. Suitable metals include gold, silver, silver gallium alloy, and iron. Suitable semiconductors include silicon, germanium, gallium arsenide, gallium phosphide, Indium Arsenide, Indium Phosphide, Zinc Sulfide, Zinc Selenide, Zinc Telluride, Cadmium Sulfide, and Cadmium Selenide. Suitable insulators include glass- siliconoxide, Quartz, and aluminumoxide.

[0027] The nanowire cantilever must be supported on a support, such as a relatively stiff base. The method of attaching the nanowire to the support can vary. In many instances, the nanowire can be grown from the support using nanowire growth mechanisms such as laser ablation, VLS (vapor-liquid-solid), chemical vapor deposition. Those same methods along with SLS (solution-liquid-solid) and assembly from colloidal nanocrystals can be used to make independent nanowires that are then mounted to a support.

[0028] Methods for making a nanowire are well known in the art. Yazdanpanah, M., et. al., JAP, 98, 073510 (2005) teaches the making of a silver gallium alloy whisker. Wagner, R. S., et. al., APL, 4, 89 (1964), Wu, Y., et. al. Nano Letters v4 p433 (2004), and Wang, D., et. al. Nano Letters v4 p871 (2004) teach the VLS growth of silicon nanowire.

[0029] It is expected that holding the nanowire perpendicular to the surface and measuring the changes in shear force associated with proximity to the surface will be the best means of imaging very gently. Conventional AFM operation holds the cantilever a few degrees from parallel to the surface such that cantilever oscillations are normal to surface. This configuration may work well for the device of the present invention also. Any angle between parallel and perpendicular can be made to work. [0030] The detection mechanism for measuring the force applied to the nanowire is a crucial element of this design. Present AFM methods reflect the laser from the cantilever. A reflection is the same as 180° scattering. The intensity of the scattering drops off precipitously as the nanowire diameter becomes less than the diffraction limit of light. For small diameters, most of the light falls within small scattering angles (less than 20°) with some of it within wide angles. There are many configurations which can be used to measure the position of the cantilever within the beamwaist.

[0031] In some embodiments, the method comprises collecting the forward scattered light with the non-scattered light and measuring the spatial shift in intensity of the scattered light as the cantilever is deflected using a position sensitive photodiode (quadrant detector).

[0032] In some embodiments, the method comprises providing the collection optics perpendicular to the axis of the acceptation optics and collecting the wide angle scattered light and none of the non-scattered light.

[0033] In some embodiments, the method comprises collecting the total light intensity of the forward scattered and non-scattered light. This is a method of measuring the wide angle scattered light as a reduction in the total light intensity in the forward direction.

[0034] In each of the above embodiments described, one can use a nanowire that has a special scattering center. Metal scatters better than dielectrics. Some example of this type of wire may be a silicon semiconducting nanowire which has a gold particle at the end. The special scattering center may be a luminescent material which emits photons in a different color than the excitation beam. Such that filters can be used to remove the non-scattered light. Since the direct bandgap semiconductors are highly luminescent, the whole wire can be used as a different color scattering center.

[0035] Interference and intensity are intimately related. Both methods measure intensity but interference influences the intensity pattern on the detector which can be harnessed for higher sensitivity. When in use, there are two possible detection mechanisms: scattering intensity and scattering interference. In scattering intensity, scattered light perpendicular to the beam propagation is gathered so that there is backgroundless detection. In scattering interference, interference between the scattered and unscattered light is measured, and it utilizes stronger forward scattering intensity. Both methods require a full Mie scattering analysis. Mie scattering is a theoretical model we use to help us guide parameter selection for nanowire fabrication. It is not part of the invention. Mie scattering analysis is described in Example 1.

[0036] The invention can be applied in the investigation of protein function, with the ultimate goal of manipulating the cellular machinery, by gently imaging the cell with scanned probe microscopy. Indeed, engineered protein structures offer enormous potential as a new material because of their great versatility. Such proteins of interest include proteins involved in cell adhesion, motility, and proliferation, such as large membrane spanning proteins, for example integrins. Because of their crucial role in cell adhesion and signaling, understanding these basic functions would potentially provide the key to the future use of protein machinery in the selective assembly of nanostructures. Such proteins of interest include proteins found on the surface of cell membranes. The AFM of the present invention is the only imaging technology which can image intact cell surfaces with nanometer scale resolution. High resolution images provided by AFM of the present invention can uniquely address a number of outstanding and pressing questions related to protein function and signaling.

EXAMPLE 1

[0037] Numerical analysis indicates that laser light focused on a nanowire produces a pattern that can be used to infer the nanowire's displacement with a split-photodiode. The most sensitive region of the diode's difference signal occurs when the nanowire is at the focal spot, where the response is roughly linear for several hundred nm. This makes this approach suitable as a detection scheme for nanowire deflection. Additionally, smaller focal spots (high N.A.) significantly improve the deflection sensitivity.

[0038] The relevant length scales of the system do not lend themselves readily to a large or small/Rayleigh size scattering approximations. Thus a rigorous Mie scattering model of a cylinder in a Guassian beam is adapted from literature to determine feasibility of the technique and identify key engineering parameters. Adaptation is necessary as traditional Mie scattering is determined by solving the wave equation with spherical boundary conditions in a uniform electric field, whereas here the field is not uniform, but corresponds to a focused, Gaussian beam. The analysis is further complicated in that the focal spot is offset from the cylinder's axis. An approximate solution to the governing equations is determined with a Matlab model that calculates the far field scattered intentisty of a single geometry, and determines the signal it would make on a split-photodiode (see Figure 6). Nanowire deflection is simulated by calculating a set of geometries that correspond to a nanowire that is translated laterally with respect to the focal spot simulates. Because the code is parameterized, standard non-linear least-square fiting routines can be used to solve for uncertain experimental parameters (such as axial mis-focus). See Figure 5.

[0039] The Mie scattering analysis is as follows:

[0040] Key parameters:

ni : Medium ref. index a: Nanowire radius n₂: Nanowire ref. index λ: Laser wavelength β: Focussed spot- size y₀: Lateral displacement z₀: Axial displacement n: Number of terms

OO

E(ρ,θ) = Σ fⁿ J^nθ J_n(kρ) A_n

[0041] rør-oo ^)

CO

Scattered (ββ)

tⁿ J^nθ

B_n

[0043] ^^⁰⁰ (III)

[0045] The analysis is expedited by using the inverse Fourier integral of the electric field (around α), and working in cylindrical coordinates.

EXAMPLE 2

[0046] The transmission-detection scheme is validated by translating the nanowire through the laser focus with a nanopositioniong flexure stage. The resulting split-photodiode signal reveals a functionally odd profile characteristic of the numerical simulations (see Figure 7). By positioning the nanowire in the most sensitive part of the profile (at the center of the laser focus) the spectrum of the nanowire 's thermal fluctuations can be measured (see Figure 8).

[0047] In the experimental setup, the laser polarization is set by rotating tight loops of the single-mode fiber. It is subsequently focused onto the nanowire, which is attached to a nano-positioning flexure stage. As the nanowire is translated through the beam the transmitted light is measured by a split-photodiode directly beneath the base-plate.

[0048] A variety of methods are available to create nano wires, including focussed ion beam etching, vapor-liquid-solid (VLS) growth and Ag₂Ga alloy pulling. The Ag₂Ga nanowires are suitable for use in the present invention due to their growth eometry and their commercial availability (from NaugaNeedles LLC, Louisville, KY).

[0049] NaugaNeedle Ag₂Ga nanowires are made by pulling silver-coated AFM tips from gallium droplets using a method adapted from M.M. Yazdanpanah et al, "Selective self-assembly at room temperature of individual freestanding Ag₂Ga alloy nanoneedles," J. Appl. Phys. 98, 2005, hereby incorporated by reference. See Figue 10.

[0050] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.

[0051] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

What is claimed is:

1. A device comprising a support and a nanowire attached to the support designed for use with an atomic force microscope (AFM).

2. The device of claim 1 , wherein the nanowire is a cantilever of the AFM.

3. The device of claim 1, wherein the nanowire comprises a special scattering center comprising a metal, semiconductor, or insulator.

4. The device of claim 3, wherein the metal is gold, silver, silver gallium alloy, or iron.

5. The device of claim 3, wherein the semiconductor is silicon, germanium, gallium arsenide, gallium phosphide, Indium Arsenide, Indium Phosphide, Zinc Sulfide, Zinc Selenide, Zinc Telluride, Cadmium Sulfide, or Cadmium Selenide.

6. The device of claim 3, wherein the insulator is glass-siliconoxide, Quartz, or aluminumoxide .

7. The device of claim 1 , wherein the nanowire has a size from 5 nm to 500 nm in diameter and has a length from 100 nm to 100 μm.

8. The device of claim 7, wherein the nanowire has a size from 10 nm to 500 nm in diameter and has a length from 300 nm to 100 μm.

9. The device of claim 8, wherein the nanowire has a size from 10 nm to 55 nm in diameter and has a length from 300 nm to 1.5 μm.

10. An AFM system comprising the device of claim 1, wherien during use of the AFM the nanowire is in contact to a target of interest.

11. The AFM of claim 10, wherein the nanowire is presented about perpendicular to the target of interest.

12. The AFM of claim 10, wherein the target of interest is living or viable cell, gel, or the target of interest is in a liquid.

13. The AFM of claim 10, wherein during use of the AFM the nanowire is at times in the beamwaist of a focused laser beam.

14. The AFM of claim 10, wherein during use of the AFM the resulting scatter pattern of the focused laser beam on the nanowire is incident upon a split-photodiode, wherein the split-photodiode is capable of detecting a change in oscillation, frequency, or amplitude of the nanowire.

15. A method of using an AFM to observe a target of interest, comprising:

(a) providing the AFM comprising a laser source, a nanowire on a support, split- photodiode, and a target of interest,

(b) contacting the nanowire to the target of interest,

(c) directing a laser provided by the laser source to the support whereby at least a portion of the light of the laser is scattered, thereby producing scattered light and non-scattered light, and

(d) measuring the scattered light and/or non-scattered light.

16. The method of claim 15, wherein the target of interest is living or viable cell, gel, or the target of interest is in a liquid.

17. The method of claim 15, wherein the measuring step comprises: (i) collecting forward scattered light with non-scattered light, and (ii) measuring the spatial shift in intensity of the scattered light as the cantilever is deflected using a position sensitive photodiode

18. The method of claim 15, wherein the measuring step comprises: (i) providing a collection optics perpendicular to the axis of the acceptation optics, and (ii) collecting the wide angle scattered light and none of the non-scattered light

19. The method of claim 15, wherein the measuring step comprises: (i) collecting total light intensity of forward scattered light and non-scattered light, wherein the wide angle scattered light is measured as a reduction in the total light intensity in the forward direction.