Image analysis – Applications – Biomedical applications
Reexamination Certificate
1999-04-19
2001-07-17
Stucker, Jeffrey (Department: 1648)
Image analysis
Applications
Biomedical applications
C382S129000, C382S172000, C382S195000, C382S203000, C435S005000, C435S006120, C436S516000, C436S518000, C436S172000, 36
Reexamination Certificate
active
06263095
ABSTRACT:
FIELD OF INVENTION
This invention concerns methods and apparatus for detecting the presence and position of materials labelled with a substance which emits radiation in the visible range of the electromagnetic spectrum on a sample.
The invention is of application in the field of diagnostics and analysis and for performing measurements and analysis on labelled immunoassays, tissue sections, microbiological specimens, cellular specimens, cellular monolayers, reporter genes, DNA and/or protein gels and blots.
U.S. Pat. No. 4,979,221 Perryman disclosed a photon event detection system in which a regular pattern or array of pixels is used as the window within which photon events on a CCD array are to be investigated. The system is suited to the application for which it was designed where in general it could be known in advance that a 3×3 array of pixels would always in at least one position, encompass all the pixels attributable to an event of interest. That is not the case with chemiluminescence arrays, labelled immonuassays and other low light level emitting specimens such as listed above particularly where the affected pixels can be spaced over irregularly shaped areas encompassing far more than a 3×3 matrix of pixels. It is an object of the invention to provide a pixel investigation technique which can accommodate irregularly shaped regions of affected pixels in a CCD array.
The invention is also of particular application to diagnostic analysis of samples in which the concentration of light emitting material and therefore the quantity of light from different regions of the sample can vary considerably from one region to another in the filed of view.
It is an object of the present invention to provide an improved method and apparatus by which light from a plurality of different points in a sample can be detected and mapped in two dimensions and quantified.
It is a further object to provide a method and apparatus as aforesaid which is nevertheless capable of detecting and mapping points from which light is being emitted at low levels and from such points when present in the same sample as points from which considerably higher levels of light are being emitted, due to events which may have nothing to do with the sample, such as ion events in an image intensified CCD camera. In this connection, the system described in U.S. Pat. No. 4,979,221 Perryman does not allow such events to be distinguished.
DESCRIPTION OF THE INVENTION
According to one aspect of the present inventor a method of detecting the presence and position of labelled material in a sample in which the labelled material either gives off light or can be stimulated to do so comprises the steps of repetitively imaging the sample onto a CCD camera, scanning the camera CCD array and interrogating the charge pattern thereon following each exposure, performing measurements on the data signals obtained to identify clusters of data values from adjacent regions of the array caused by light emitted from the sample onto those regions and comparing the measurements with at least one threshold so as to distinguish clusters resulting from light emitting regions of labelled material from the remainder of the sample, and computing the centroid of each light produced cluster of data values so identified with reference to the camera array, and storing the centroid coordinates in a memory together with the centroid coordinates of any other light produced clusters identified during the same interrogation.
The method may include the further step of storing the coordinates from each of a succession of interrogations of the same sample in an accumulation store, which can be read out to give a list of the recorded light emissions and/or used to produce a display of the events which may also be superimposed on an outline of the sample.
According to another aspect of the present invention a method of detecting the presence and position of light emitting labelled material in an area of sample comprises the steps of:
1. imaging the sample onto a CCD camera,
2. repetitively scanning the CCD array of the camera, each scan corresponding to an interrogation of the charge pattern on the array and being preceded by an exposure period and followed by a resetting step, which initiates the beginning of the next following exposure period,
3. generating during each interrogation data signals whose values described the charge pattern on the array,
4. performing measurements on the data signals to identify clusters of data values from adjacent regions of the array indicative that photon emission from the sample has impinged thereon, and
5. identifying a center position of each identified cluster of such data values and storing the center coordinates in a memory together with the centre coordinates of any other identified clusters occurring during the same interrogation.
Preferably the data signal values generated for each interrogation are stored and subsequently read out by computing and data processing means for performing the said measurements thereon.
Preferably coordinates from each of a succession of interrogations of the same sample are stored in an accumulation store.
The invention also lies in the further step of reading out the stored coordinates and producing a list thereof.
The method also lies in the further step of operating a visual display system so as to reproduce therein the scanned area of the CCD array and modulating the light producing element of the display system so as to generate visually distinguishable features in the display at positions defined by the stored coordinates.
Either a cooled CCD camera or an image intensified CCD camera may be employed.
Where an image intensified CCD camera is employed, a so-called first generation image intensifier is preferred.
Noise reduction can be enhanced if a low noise material is selected for the photocathode of the image intensifier.
Preferably a first generation image intensifier is selected having a low noise bi-alkali material photocathode.
CCD Camera operation
The CCD camera may be operated in so-called “inverted mode” so that contributions to background from fluctuations in thermal noise in tis CCD array (so called “shot noise”), at room temperatures, can be reduced, but in any case is cooled, typically cryogenically.
To reduce dead time, the duration of each interrogation of the charge pattern of the CCD array and the associated resetting of the CCD array, is preferably as short as possible and will normally be much shorter than the duration of the preceding exposure period.
According to a preferred feature of the method, the addressing of the CCD array is organized so as to define a plurality of sub-regions which together make up the optically sensitive region of the camera array. The quantity of photon energy incident on each sub-region during an exposure period determines the charge to be found on that sub-region during the following interrogation, so that an electrical signal indicative of the photon energy incident on each sub-region can by obtained and stored as an electrical information signal for each sub-region.
Display of CCD camera signal
For display purposes a light producing element of a scanning display system may be modulated by the said electrical information signal obtainable for the interrogation of the CCD array, so as to generate a visual difference between one part of the display and another depending on the level of photon activity on the corresponding sub-regions of the CCD array during the preceding exposure period.
If instead the display system is modulated by signals obtained by repeatedly reading out the coordinate accumulation store previously referred to, the display will present a continually updated picture of the positions from which light has been detected in the sample.
A threshold may be applied to the information signals so that in the simplest case the modulation is two-state and sub-regions which have received photon energy greater than a value for example K, are displayed in one colour and all other sub-regions are displayed in a contrasti
Ansorge Richard Eric
Hooper Claire Elizabeth
Neale William Wray
Rushbrooke John Gordon
Cambridge, Imaging Ltd.
Lee, Mann, Smith, McWilliams, Sweeney and Ohlson.
Stucker Jeffrey
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