Measuring and testing – Surface and cutting edge testing – Roughness
Reexamination Certificate
1998-11-09
2002-03-19
Raevis, Robert (Department: 2856)
Measuring and testing
Surface and cutting edge testing
Roughness
C073S149000
Reexamination Certificate
active
06357285
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
The present invention relates generally to methods and apparatus for acquiring and quantifying data, and more particularly to a method and apparatus for acquiring and quantifying high-resolution, three-dimensional (3D) data. This invention is particularly well-suited for classification or typing based on quantitative data acquired from biological cells and macromolecules using atomic force microscopy.
2. Discussion of the Related Art
Determining the morphology of biological cells and macromolecules (hereinafter biological structures) is important for a number of tasks, including (1) cell typing (e.g., typing blood cell into red blood cells, lymphocytes, platelets, etc.) and (2) classifying cells and other biological structures into normal and abnormal cells (e.g., classifying benign, premalignant, and malignant cells). Morphological determination of biological structures has traditionally been accomplished using microscopy, first with light microscopy and later with electron microscopy. More recently, local sensitive force detectors, such as atomic force microscopes (AFMs), have been used for obtaining data measurements.
Confocal microscopes have the highest resolution of all light microscopes, but have a lateral (X-Y) resolution of only about 200 nm and a vertical (Z) resolution of only about 650 nm. In contrast, electron microscopes (EMs) and AFMs have a much higher resolution. Specifically, AFMs have a lateral (X-Y) resolution of 1 nm. Theoretically, AFMs have a vertical (Z) resolution of 0.01 nm (i.e., 0.1 angstrom (Å)). Because of environmental noise, however, in practice AFMs have a Z-axis resolution of around 1.0 Å. In comparison, AFMs provide two orders of magnitude better resolution than light microscopes and comparable resolution to EMs.
While the lateral resolution of EMs is sufficient to discriminate subtle surface features, EMs have many practical limitations for use as an analytical tool for biological material. The main problem is that the typical biological material has low electron density and conductivity. Therefore, to permit good visualization and to prevent sample damage from electronic radiation, EM samples are typically dried and metal-coated. Additionally, EM samples often require sectioning and chemical fixation. Consequently, sample preparation for EM is time-consuming. Furthermore, due to sample preparation requirements, EMs cannot be used to study biologically active samples.
In contrast, imaging with an AFM is relatively fast because the sample does not require drying, sectioning, metal coating or chemical fixing. Thus, AFMs may be used with samples that require very little sample preparation, including (1) samples that are biologically active and (2) samples in both ambient air (including dried samples) and liquid.
The typical AFM has a probe comprising (1) a flexible cantilever and (2) a tip disposed on the free end of the cantilever. Interactions between the tip and the sample influence the motion of the cantilever, and one or more parameters of this influence are measured to generate data representative of one or more properties of the sample. AFMs can be operated in different modes including contact mode, TappingMode, light TappingMode, (Tapping and TappingMode are trademarks of Digital Instruments, a Division of Veeco Instruments Inc.), and non-contact mode. In contact mode, the cantilever is not oscillated, and cantilever deflection is monitored as the tip is dragged over the sample surface. In TappingMode, the cantilever is oscillated mechanically at or near its resonant frequency so that the probe tip repeatedly taps the sample surface, thus reducing the tip's oscillation amplitude. The change in oscillation amplitude indicates proximity to the sample surface and may be used as a signal for feedback. Changes in other oscillation parameters, such as phase, may also be monitored. U.S. patents relating to Tapping and TappingMode include numbers 5,266,801; 5,412,980; and 5,519,212, by Elings et al., all of which are hereby incorporated by reference. In the non-contact mode, attractive interactions between the tip and the sample (commonly thought to be due to Van der Waals' attractive forces) shift the cantilever resonance frequency when the tip is brought within a few nanometers of the sample surface. These shifts can be detected as changes in cantilever oscillation resonant frequency, phase, or amplitude, and can be used as a feedback signal for AFM control.
Whether operating in contact mode, TappingMode, or non-contact mode, feedback is typically used during AFM scanning to adjust the vertical position of the probe relative to the sample so as to keep a probe operational parameter, such as the tip-sample interaction, constant. A measurement of surface topography or another sample characteristic may then be obtained by monitoring a signal such as the voltage used to control the vertical position of the scanner. Alternatively, independent sensors may monitor the position of the tip during scanning to obtain a map of surface topography or another measured sample characteristic. Measurements can also be made without feedback by monitoring variations in the cantilever deflection as the probe moves over the surface. In this case, recording the cantilever motion while scanning results in an image of the surface topography in which the height data is quantitative. Additionally, the positioning of the AFM probe can be enhanced by compensating for drift. U.S. patents relating to drift compensation include 5,081,390 and 5,077,473 by Elings et al., both of which are hereby incorporated by reference.
As described above, the AFM typically provides up to 1 Å resolution for the Z-axis and 1 nanometer resolution for the X- and Y-axes for samples in air or liquid. Traditionally, AFM data recorded from all three axes of a biological structure are displayed as computer generated, space filling images with height-encoded scaling. These images are analyzed directly from the computer-rendered two-dimensional (2D) or three-dimensional (3D) images. Additionally, the most widely used practice for further analysis of AFM results is simply a subjective description of the AFM image, such as height, depth, width, distance between features, Fourier averaging, volume, surface area roughness, force versus distance curves, and 2D section profiles.
While many measurements exist for AFM imaging, AFMs are currently not used as a clinical diagnostic tool for several reasons. One reason is that samples cannot be marked for differentiation by AFMs. For instance, AFMs cannot differentiate a dyed sample from an undyed sample, whereas confocal microscopes can. One example of dyeing samples for differentiation purposes is employing dye exclusion to count viable cells. Viable cells are impermeable to naphthalene black, trypan blue, and a number of other dyes. After these dyes are added to cells, the cells can be examined by light microscopes to determine the proportion of viable cells to non-viable cells. In contrast, AFMs are not typically used to detect such dyes.
Another reason that atomic force microscopy is currently not used as a clinical diagnostic tool is that there is a lack of objective methods to analyze the AFM data beyond displaying a space filling image of the biological structure based on two or three axes of the biological structure. In rare instances, proposals have been made to plot the AFM data as a function of another experimental variable, resulting in more unique graphical forms. For instance, Radmacher et al., “Direct Observation of Enzyme Activity with the Atomic Force Microscope,” Science, Vol. 265, Sep. 9, 1994 (Radmacher), proposes placing an AFM tip in stationary mode on an enzyme, and plotting the height fluctuations that the enzyme undergoes during an enzymatic reaction versus time in seconds. Another proposed use is disclosed in Allen et al., “Extent of Sperm Chromatin Hydration Determined by Atomic Force Microscopy,” Molecular Reproduction & Development, Vol. 45, 1996 (Allen).
Nilles & Nilles S.C.
Raevis Robert
Veeco Instruments Inc.
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