Direct detection of x-rays for protein crystallography employing a thick, large area CCD

An apparatus and method for directly determining the crystalline structure of a protein crystal. The crystal is irradiated by a finely collimated x-ray beam. The interaction of the x-ray beam with the crystal produces scattered x-rays. These scattered x-rays are detected by means of a large area, thick CCD which is capable of measuring a significant number of scattered x-rays which impact its surface. The CCD is capable of detecting the position of impact of the scattered x-ray on the surface of the CCD and the quantity of scattered x-rays which impact the same cell or pixel. This data is then processed in real-time and the processed data is outputted to produce a image of the structure of the crystal. If this crystal is a protein the molecular structure of the protein can be determined from the data received.

BACKGROUND OF THE INVENTION 
This invention relates to the use of a charged coupled device (CCD) for the 
direct detection of scattered x-rays used in the determination of the 
structure of a protein crystal. 
Currently, the scattered x-rays used to determine the structure of a 
crystalline material are detected using film or in the alternative a 
scintillating or phosphorescent material which when illuminated by a 
scattered x-ray gives off a visible light. In the latter case the phosphor 
is coupled with a device which is capable of detecting the light given off 
by the phosphor and converting it into electronic data which is used to 
determine the position and intensity of the scattered x-rays. Often a CCD 
is used in conjunction with the phosphor to determine the position and 
frequency of the x-rays which are scattered off of a crystallized version 
of a molecule. With a CCD a single photon can generate a measurable 
electrical charge. The CCD employs a two dimensional array of cells or 
pixels, each of which acts as an independent x-ray detector, thus, 
allowing many scattered x-rays to be stored in the cells of the CCD and 
then read and recorded. 
Generally, CCD-based imaging devices compromise sensitivity and resolution 
due to smearing and spatial distortion of the image caused by the presence 
of the scintillating or phosphorescent material between the crystal and 
the CCD. This is due to the alteration of the path of the x-rays upon 
interacting with the scintillating or phosphor material. Since, after the 
x-ray interacts with the light emitting material, the photons emitted do 
not necessarily follow the exact path of the initial x-ray. This results 
in a decrease in the resolution or a smearing of the measured point of 
impact on the CCD of two successive x-rays traveling along the same or 
close to the same path. 
As related above, the prior art CCD-based imagery employs an intermediate 
light generating transducer, such as a phosphor, to transform the incident 
x-rays to a light source. The photons from the light source are converted 
by the CCD to electrical signals. The conversion step of using the 
phosphor was instituted with the x-rays since the majority of x-rays 
striking a typical CCD would pass through the CCD without detection. This 
results from the CCD being too thin. 
An alternate method of detection was proposed by Antonuk et al. as 
described in U.S. Pat. No. 5,262,649, in which, the use of a CCD is 
avoided in lieu of a thin film amorphous silicon device. Antonuk's patent 
teaches a thin-film, flat panel, pixelated detector array which serves as 
a real-time digital image and dosimeter for x-rays or gamma rays. The 
detector is a plurality of photodiodes made of hydrogenated amorphous 
silicon arrayed in columns and rows on a glass substrate. Each photodiode 
is connected to a thin film field effect transistor also located on the 
glass substrate; the combination of which forms one pixel. For megavoltage 
beams, a photon to electron conversion layer is located directly above and 
in contact with a phosphor or scintillating layer. Since each sensor is 
adjacent to and connected to its corresponding field effect transistor, 
the area available for detection in reference to the total area of the 
detector is drastically reduced. 
Applicants in their invention provide for the direct detection of the 
scattered x-rays without an intermediate phosphor or scintillation layer. 
By using a large area, thick CCD device, applicants can detect the 
scattered x-rays directly and on a real-time basis. Use of a thick CCD as 
a direct detection device for use in detecting breast cancer is described 
in U.S. patent application entitled "High Resolution Mammography", Ser. 
No. 08/697,536, filing date Aug. 26, 1996, now U.S. Pat. No. 5,742,659, 
which is incorporated herein in its entirety by reference. 
One object of this invention is provide a device which is capable of 
directly discerning, in real-time, the frequency and position of x-rays 
scattered by a protein crystal initially subjected to an incident x-ray 
beam. 
Another object of this invention is the employment of a large area, thick 
CCD to provide a high resolution image of the pattern generated by the 
scattered x-rays resulting from the interaction of the protein crystal and 
the incident x-ray beam. 
Additional advantages, objects and novel features of the invention will 
become apparent to those skilled in the art upon examination of the 
following and by practice of the invention. 
SUMMARY OF THE INVENTION 
To achieve the foregoing and other advantages, this invention comprises an 
apparatus for real-time, direct detection of x-rays impacting on the 
surface of a large area, thick charge coupled device (CCD). The CCD is 
positioned to detect the scattered x-rays resulting from the interaction 
of an initial incident x-ray beam and a protein crystal. By employing CCD 
technology, the frequency and position of the scattered x-rays can be 
detected on a real-time basis and with high resolution. 
The CCD is functionally divided into storage cells or pixels, each of which 
stores an electrical count which indicative of the number of x-rays 
detected by the cell. A pattern generator receives the electrical signal 
from the CCD in the form of a stored count. The display device then 
displays the image representation of the count data which has been 
processed by the image processing system.

DETAILED DESCRIPTION OF THE INVENTION 
FIG. 1 depicts the x-ray crystallography apparatus, 10, for the 
determination of the structure of protein crystals. A x-ray source, 11, is 
used to generate a finely collimated beam of x-rays, 12, which irradiates 
a protein crystal, 14. By measuring the position and intensity of the 
scattered x-rays, 16, resulting from the interaction of the protein 
crystal and the collimated incident x-ray beam, 12, the structure of the 
protein crystal, 14, is determined. The scattered x-rays, 16, are detected 
by a thick, large area CCD, 30, located at a known position relative to 
the position and orientation of the protein crystal, 14, and the incident 
x-ray beam, 12. The pattern generator, 18, is electrically coupled to the 
CCD, 30. The pattern generator, 18, controls the output of the cells 
making up the CCD. The stored count of each cell of the CCD can be 
outputted individually or, in the alternative, the counts from multiple 
cells can be combined prior to output. This practice is referred to as 
"binning". By employing varying degrees of binning of the cells, the 
resolution of the output can be controlled. The front end electronics, 20, 
control the operation of the apparatus through the microprocessor, 22. The 
microprocessor, 22, is coupled to programmed computer, 24, and display, 
26, which provides a visual display of the data detected by the CCD, 30 
and with a resulting hard copy. The computer, 24, is capable of directing 
the storage of the data on disk or tape. 
FIG. 2 depicts a top plan view of the charge coupled device (CCD), 30. The 
CCD, 30, is divided into a matrix of individual cells, 32, arranged in a 
series of rows, 34, and columns, 36. Each cell, 32, temporarily stores an 
electrical signal or count which is representative of the number of 
scattered x-rays incident on and detected by the CCD cell. Upon completion 
of the irradiation process, the data is downloaded and used to produce an 
image of the structure of the irradiated protein crystal. 
An alternate configuration can be established by partitioning the CCD. For 
example, the CCD can be partitioned into quadrants, 37-40. The output 
registers can be arranged in such a manner that each group of cells 
comprising the quadrant can output through a specific register, 40-43, so 
that, for example, the data from quadrant 37 outputs through output 
register 41. 
In FIG. 3, for the subject embodiment, a buried channel charge coupled 
device is employed, 31. In the present case, a p-type substrate, 44, is 
utilized with an n-type buried channel, 45. A silicon oxide layer, 46, 
resides on top of the p-type substrate. Output electrodes, 47 and 48, are 
located in the gaps in the silicon oxide layer, 46. Transfer electrodes, 
49, rest on the silicon oxide layer, 46. Selective electrical potentials 
are applied to electrodes, 49, to define the cells, 32. Applying a bias to 
transfer electrodes, 49, causes stored electrical counts to shift between 
cells to the output electrodes 47 and 48. The transfer electrodes, 49, 
employed to form the cells or pixels of the CCD allow the passage of 
x-rays, thus, making the entire upper surface of the CCD receptive to 
x-rays. As x-rays interact with each cell, 32, of the CCD, the energy of 
the x-ray is absorbed to provide an electron hole pair within the cell. 
Thus, the number of electron hole pairs within a cell is proportional to 
the number of incident, detected x-rays. When this information is 
transferred to the imaging system, the light intensity of the image, of 
the imaging system, corresponds to the count in a corresponding cell. 
However, not all of the x-rays incident on the surface of the CCD are 
detected and converted into electrons. The conversion efficiency of the 
CCD is proportional to its thickness, 50, FIG. 3, of the CCD, 31. The 
conversion efficiency increases as the CCD thickness increases. Preferably 
the thickness of the CCD varies between 30 and 500 microns. An optimum 
range would be between 60 and 300 microns. The conversion efficiency is 
65% for a CCD having a thickness of 60 microns. A typical CCD may include 
a matrix of cells, 32, 2,048 by 2,048 where each cell has surface 
measurements of 24.times.24 microns. 
The CCD employed with the subject invention has a large dynamic range 
allowing it to detect and count a large number of incident x-rays. This 
results from the cells being capable of storing a large number of electron 
holes without becoming saturated. The large dynamic range enhances the 
contrast experienced between cells during the detection process. 
The CCD processor 60, FIG. 4, comprising items 18, 20, and 22 of FIG. 1 is 
shown in more detail in FIG. 4. FIG. 4 includes a CCD controller, 62, a 
dewar module 64, a camera interface, 66, and a multi-channel signal 
processing board, 68. The CCD controller, 62, sets the CCD timing patterns 
and the voltage levels. It, also, controls the CCD operating temperature 
and monitors the voltage levels of the camera electronics. To control 
thermal electron noise, the CCD operates at a temperature of approximately 
-110 degree C. Although, this temperature is allowed to vary somewhat in 
response to the optimal thermal electron current. The subject temperature 
control apparatus found in the CCD control, 62, controls the temperature 
to within one degree C. The camera interface, 66, includes processor, 70, 
which remotely controls the CCD controller, 62. The camera interface, 66, 
also, includes RAM, 72, which provides memory for the system, and PROM, 
74, stores the processor program, the reset routine and the camera default 
program. The EEPROM, 76, contains the camera programs. 
The CCD controller, 62, includes a line shifter, a pattern generator, 63, 
and a level setting digital to analog converter. The pattern generator, 
63, is in communication with the DEWAR module, 64. The DEWAR module, 62, 
includes the CCD, 30, a CCD driver and a pair of two channel amplifiers. 
The CCD driver is electrically connected to the transfer electrodes, 49, 
of the CCD and functions to create cells within the CCD and to shift 
counts within the CCD in response to a signal received from the pattern 
generator, 63. 
On the application of power, the processor, 70, loads a set of operating 
parameters from the PROM into the RAM. The pattern generator, 63, 
generates a pattern of signals for the CCD driver to control the CCD. The 
pattern generator is set to a subsection mode when only a subsection of 
the pixels or cells of the CCD are to be read out. 
In the alternative, materials other than silicon can be used to form the 
CCD. These materials include Gallium Arsenide (GaAs), Gallium Nitride 
(GaN) and the like. Since these materials are denser than silicon, the 
conversion efficiency of the CCD with respect to x-rays is increased. 
The subject invention provides a high resolution, real-time readout of the 
position and intensity of the scattered x-rays produced when a crystal is 
irradiated by an incident beam. By changing the position of the CCD or by 
providing encircling three dimensional coverage, the position and 
intensity of scattered x-ray can be determined. This information allows 
one to quickly determine the structure of the crystal or in the case of a 
protein crystal, the structure of the protein. The data can be viewed 
directly on a monitor or stored for further compilation. The thick CCD is 
sized so that the efficiency of detection is between 65%-70%. 
This invention is not limited to the determination of the structure of a 
protein crystal. The structure associated with the interaction of an 
organic molecule with the protein can be determined from its crystallized 
structure by implementation of this apparatus. Also, the structure of 
other biological molecules with or without the interaction of an organic 
molecule can be determined. 
The foregoing description of a preferred embodiment of the invention has 
been presented for purposes of illustration and description. It is not 
intended to be exhaustive or to limit the invention to the precise form 
disclosed, and obviously many modifications and variations are possible in 
light of the above teaching. The embodiments described explain the 
principles of the invention and practical applications and should enable 
others skilled in the art to utilize the invention in various embodiments 
and with various modifications as are suited to the particular use 
contemplated. It is intended that the scope of the invention be defined by 
the claims appended hereto.