Image analysis – Image enhancement or restoration – Intensity – brightness – contrast – or shading correction
Reexamination Certificate
2001-04-17
2004-10-12
Patel, Kanji (Department: 2625)
Image analysis
Image enhancement or restoration
Intensity, brightness, contrast, or shading correction
C250S287000, C436S173000
Reexamination Certificate
active
06804410
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Field of the Invention
The invention relates to a method and apparatus for improving mass spectrometry analysis of samples. Specifically, the invention relates to a method and apparatus for mass spectrometry analysis which allows for more precise alignment of a laser with samples, as well as for selection of impingement point(s) on each sample depending on the crystalline structure and other characteristics of the sample, to improve the quality of the data collected by a mass spectrometer.
B. Background Information
Mass spectrometry devices measure the molecular mass of a molecule by measuring the molecule's flight path through a set of magnetic and electric fields. Such devices are well known and are widely used in the field of bio-molecular research. In proteomics research, for example, mass spectrometry is used to identify proteins.
Proteins are typically separated from one another by electrophoresis, such as the techniques described and claimed U.S. Pat. No. 5,993,627 to Anderson et. al. (hereinafter referred to as the Anderson et. al. patent), which is incorporated herein by reference in its entirety. For instance, as set forth in the Anderson patent, a tissue sample is first subjected to a first dimension electrophoresis process where groups of proteins are separated linearly within a tubular gel filled column. The first dimension separation of proteins is then inserted along an edge of a flat planar gel slab and subjected to a second dimension of electrophoresis, thereby generating a two dimensional pattern of spots formed by clusters of proteins that have moved to respective iso-electric focusing points. Thereafter, selected proteins are excised from the second dimension gel slab for further study. The selected excised spots are next prepared for analysis using, for instance, mass spectrometry.
An increasingly popular technique for studying biological molecules is the use of a matrix-assisted laser desorption ionization (MALDI) mass spectrometry apparatus wherein a biological sample such as an above-referenced excised spot is embedded in a volatile matrix which is subsequently vaporized by an intense laser emission. One such MALDI mass spectrometry apparatus is a MALDI-TOF apparatus (TOF is time-of-flight spectrometry). In the field of proteomics, mass spectrometry, and in particular, MALDI-TOF techniques are used to determine the molecular weight of peptides produced by digestion of isolated proteins. One such MALDI-TOF apparatus is VOYAGER DE STR Biospectrometry Workstation manufactured and sold by APPLIED BIOSYSTEMS.
The drawbacks of conventional methods for analyzing samples using mass spectrometry such as in proteomics research will become apparent from the following description of a conventional MALDI-TOF apparatus.
FIG. 1
depicts a generic MALDI-TOF apparatus that includes a frame
1
that supports the electronic and computer equipment necessary to control a laser
5
. The laser
5
is aimed at a fixed location in a positioning mechanism
10
. The positioning mechanism
10
includes means (not shown) for positioning a sample in the line of fire of the laser
5
. Typically, in a MALDI-TOF apparatus, the laser is fixed in place and the sample is moved into position for analysis.
The MALDI-TOF apparatus comprises a small removable sample plate
15
, shown in
FIGS. 2 and 3
, that fits into the positioning mechanism
10
. Typically, the sample plate
15
is insertable into a slot
20
in the positioning mechanism
10
of the MALDI-TOF apparatus and is thereafter held in a specific orientation within the positioning mechanism
10
for sample analysis. The sample plate
15
typically holds a plurality of discrete samples
16
on one surface thereof, with the samples
16
being spaced apart from one another, as shown in FIG.
3
. The sample plate
15
includes guide members
15
a
, guide holes
15
b
and alignment pin
15
d
that are used by corresponding members (not shown) within the positioning mechanism
10
.
The MALDI-TOF apparatus generally comprises a camera (not shown) in the positioning mechanism
10
, which includes the sample plate
15
in its field of view, as well as the video monitor
25
depicted in FIG.
1
. Thus, the MALDI-TOF apparatus can generate an analog output corresponding to the field of view to generate a display of the sample plate
15
. Using the display, an array of Cartesian coordinates (X,Y) can be generated which corresponds to respective target sample areas on the sample plate
15
. The sample plate
15
can then be moved automatically with respect to the line of fire of the laser
5
using these coordinates.
The samples
16
are loaded onto the sample plate
15
by a separate device or robotic apparatus that is typically manufactured and sold with each specific mass spectrometry apparatus. The robotic apparatus includes a recess that retains the sample plate
15
in position for sample loading, a first arm that moves back and forth along an X axis, and a second arm that moves along a Y axis defined along the length of the first arm. The second arm supports a pipette tip that is used to spot samples on the sample plate
15
as it is moved by the first and second arms.
Typically, an array of samples
16
are spotted on the sample plate
15
at predetermined locations, as depicted in FIG.
3
. After the array of samples
16
are loaded onto the sample plate
15
, the sample plate
15
is inserted into the slot
20
of the MALDI apparatus. Using the imaging system provided by the computer as indicated at
25
, which is focused on the sample plate
15
within the MALDI apparatus, in combination with the positioning mechanism
10
, the laser beam from the laser
5
can be aimed, one by one, at the sample(s) on the sample plate
15
in accordance with the array of coordinates.
In accordance with conventional methods for mass spectrometry analysis, the locations of the samples
16
are pre-programmed into the computer that controls the MALDI-TOF apparatus so that during the analysis of the samples, the positioning mechanism
10
automatically repositions the sample plate
15
into the line of fire of the laser
5
. For example, a user enters via a mouse, keyboard or other input device an array of X-Y coordinates corresponding to sample positions on a sample plate. Thus, if any of the samples
16
on the sample plate
15
were not properly deposited in the target positions by the robotic apparatus, the laser
5
is not likely to hit those samples. More specifically, on the sample plate
15
depicted in
FIG. 3
, a 10×10 array of samples is positioned on the upper surface at spaced apart intervals. The positioning mechanism
10
moves into a target position with respect to centers of the desired or target location of each sample or spot. The desired location of each spot assumes that center of each of the spots in the 10×10 array is constant and therefore coincides with the centers
20
of the target areas
18
, as depicted in FIG.
4
.
Unfortunately, there are several shortcomings associated with the above-described robotic apparatus. Although the positioning mechanism
10
within the MALDI apparatus has positional accuracy with respect to movement of the sample plate
15
, the robotic apparatus typically sold with a MALDI apparatus is not as precise with respect to accurate spotting or depositing of samples on the sample plate
15
. Specifically, the spots
16
in a 10×10 array of samples are not centered on the desired center
20
targeted by the positioning mechanism
10
. The array of 10×10 samples may have some samples (e.g., the sample
16
a
in
FIG. 4
) that are substantially accurately centered, and other samples (e.g., samples
16
b
and
16
c
) that are off center by as much as half the width of the sample. In addition, the crystalline structures of the samples can affect the manner in which they are deposited on the sample plate and therefore cause a certain degree of offset from the actual area of deposit for a sample and the target area for the sample plate.
During mas
Lennon John J.
Makusky Anthony James
Michael Samuel G.
Large Scale Proteomics Corporation
Patel Kanji
Robbins John C.
Tarcza John E.
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