JP4018063B2 - Imaging system and method - Google Patents

Description

[0001]
(Field of Invention)
The present invention relates generally to an imaging system for analyzing and detecting moving objects or particles, and more particularly to an imaging system and method for determining and analyzing the morphology of moving objects such as cells. The present invention relates to an imaging system and method for detecting the presence and composition of a fluorescence in-situ hybridization (FISH) probe in a cell.
[0002]
(Background of the Invention)
Due to the limitations of cell and particle analysis techniques, there are many biological and medical applications that are currently infeasible. Examples of such biological applications are battlefield monitoring of known airborne toxins and monitoring of cultured cells to detect the presence of both known and unknown toxins. Medical applications include non-invasive prenatal genetic testing and routine cancer screening by detection and analysis of rare cells (ie cells with a low incidence) in peripheral blood. All these applications require an analytical system with the following key characteristics:
1. Measurement speed is fast.
2. Very large samples or continuous samples can be processed.
3. High spectral resolution and wide bandwidth.
4). Good spatial resolution.
5). High sensitivity.
6). Measurement fluctuation is small.
[0003]
Target cells in prenatal testing are fetal cells that cross the placental barrier and enter the mother's bloodstream. In cancer screening, target cells rot in the bloodstream from nascent cancer tumors. In any of these applications of this technology, target cells are present in the blood at a concentration of 1-5 cells per billion. This concentration results in about 20-100 target cells in a typical 20 ml blood sample. If the target cells are extremely rare, any detection and analysis system used for these applications will process approximately 100 million cells of the enriched sample within a few hours, corresponding to a minimum throughput of 10,000 cells per second. It must be possible. Cell processing includes accurate measurement of cell morphology parameters such as overall size, nucleus size, nucleus shape, and optical density, detection and characterization of numerous fluorescent markers and FISH probes, Quantification of the total amount of DNA and detection of other cellular components such as fetal hemoglobin is included. In order to accomplish these processing tasks, the system must be able to collect cell images with a spatial resolution of about 1 micron. Similarly, to discriminate between four or more fluorescent colors, the system must have high spectral resolution and wide bandwidth. Some probes label important cellular features with only a few thousand fluorescent molecules, so the system is sensitive and good to discriminate very weak signals Must have consistent measurement.
[0004]
The dominant laboratory protocol for non-invasive prenatal diagnosis includes gradient centrifugation to remove non-nucleated cells, high-speed cell separation to enrich fetal cells, and fetal cell identification and genetic analysis A series of complex processing steps are used, including fluorescence microscopy to do this. Because fetal cells are lost little by little at each step of the protocol, these protocols often result in very few or no fetal cells being provided for analysis. Nevertheless, the protocol is not simplified due to limitations of existing analytical techniques. Ideally, fetal cell identification and analysis is performed within a few hours by a high-speed cell separator with the required speed and sample throughput. Traditional cell separators have the necessary imaging power, sensitivity, and reproducibility to reliably identify fetal cells and enumerate the number and color of FISH probes used for diagnosis. Because of the lack, this ideal is not possible with conventional systems. Thus, in current protocols, cells must be isolated and examined on slides using fluorescence microscopy to establish fetal origin and perform genetic diagnosis. The combination of fewer fetal cells and long processing times has hindered clinical applications for non-invasive fetal testing with existing technologies.
[0005]
Prior to the present invention, there was no technology that incorporated all six major characteristics for the effective fetal cell analysis system or cancer analysis system described above. Although advanced conventional techniques can be applied to these applications, there are still significant limitations.
[0006]
In an article published by Ong et al. [Anal. Quant. Cytol. Histol., 9 (5): 375-82], a Time Delay and Integration (TDI) detector for an imaging flow cytometer is described. The use is described. A TDI detector is any pixelated device that can move a signal generated in response to radiation directed at the device in a controlled manner. Typically, the TDI detector pixels are arranged in rows and columns, and the signals move from row to row in synchronization with the moving image projected onto the device, allowing long integration times without blurring. . The approach disclosed by Ong et al. Advances the technology by addressing the need for spatial resolution and sensitivity to flowing cells, but this approach does not address the remaining key characteristics. The author of this document cites an operating speed of 10 cells per second that is at least an order of magnitude slower than that required for non-invasive fetal testing and a theoretical speed limit of 500 cells per second. Also, the system has no spectral resolution and laser scattered light and fluorescent light are collected indiscriminately by the imaging system.
[0007]
A very recently developed US Pat. No. 5,644,388 discloses an alternative to an imaging flow cytometer. This patent discloses the use of a frame-based image acquisition technique in which cells in which a video camera flows are examined in a frozen frame manner. This approach requires that the image acquisition system be synchronized with the cells present in the image area, unlike the TDI detector where the detector readout speed and cell speed are synchronized. When cells are imaged in a frame-based manner, the integration period must be very short to avoid blurring. The integration time is shortened by using a continuous light source combined with a strobe light source or a shutter detector, but in any case, the signal to noise ratio is reduced by reducing the integration time, and thus the above The sensitivity of the method is reduced. Further, in the case of a frame-based camera, it takes time to transfer data from the camera, and during that time, an image cannot be obtained, and detection of the cell may be missed. Finally, like the Ong et al. Patent, this patent does not provide for data acquisition over a wide spectral bandwidth, but has sufficient spectrum to resolve multiple fluorescent probes and FISH spots colored in different colors simultaneously. Has resolution.
[0008]
Spectral discrimination is addressed in US Pat. No. 5,422,712, where the spectrum of particles suspended in the fluid is collected as the spectrum of particles flowing through the detection region. However, the spatial representation of the object in the system is not disclosed in this patent because the object is out of focus at the detector portion. In this system, light from an object is collected and an image is generated at an intermediate opening. The light is continuous through the aperture to the spectral dispersion element. The spectral dispersion element disperses the spectrum of light along the flow axis. The dispersed light is applied to an optical amplifier and amplified. The optical signal output of the optical amplifier is finally guided to a frame-based detector. The image at the intermediate aperture represents the spatial distribution of light in the object space prior to spectral dispersion. The spatial distribution is blurred as light passes through the image plane and propagates through the spectral dispersion element to the optical amplifier. Since there is no provision for reimaging the intermediate aperture in the optical amplifier part, the resulting signal distribution in the optical amplifier part only represents the spectral distribution of the light, and the spatial distribution of the light from the object Is not maintained. The loss of spatial information limits the utility of the invention for applications such as fetal cell analysis. If there are multiple identical FISH spots in the cell, their spectrum can be confirmed using this technique, but the number of spots cannot be measured accurately. This technique also disperses the wavelength spectrum parallel to the flow axis. When two particles are illuminated along the flow axis, the spectra of the two particles overlap on the detector. To circumvent this problem, the above invention discloses the use of irradiation from a very low height along the flow axis. Irradiation from a low height shortens the integration time and necessitates the use of an optical amplifier. Also, when irradiating from a low height, throughput is limited because simultaneous imaging of multiple cells along the flow axis must be avoided.
[0009]
Thus, it will be apparent that an improved technique is desirable that overcomes the limitations of the proposed conventional approach. New approaches that have been developed to address these problems of traditional technology also include applications that analyze other types of moving objects other than cells, and have specific requirements for disparate technical applications There is an expectation that it can be implemented in different configurations to meet the requirements.
[0010]
(Summary of Invention)
The present invention is directed to an imaging system adapted to determine one or more characteristics of an object from an image of the object. There is relative motion between the object and the imaging system, and either (or both) are intended to move, but the object is moving and the imaging system is fixed Is preferred. Also, although the following summary and most of the corresponding claims are described with reference to “a single object”, the present invention is preferably intended for use with multiple objects, and in particular, It should also be understood that it is clearly intended to be useful for imaging a flow.
[0011]
The present invention provides a method and apparatus for determining at least one property of an object moving relative to a detector through a field of view utilizing a plurality of detectors. Although the detector is preferably stationary, it is a critical aspect that there is relative motion between the object and the detector, and therefore the present invention provides that the object is stationary and the detector is It should be understood that the embodiment for moving is foreseen. The data provided by the present invention includes simultaneous spatial and spectral images covering a wide bandwidth with high resolution, and the present invention provides a spatial origin of spectral information collected from the object. Note that it is provided. In particular, the use of multiple detectors ensures that image distortion due to emission bandwidth, i.e., convolution, is avoided, thus eliminating the need for deconvolution to correct the image. Sufficient detectors are used to provide an independent detector for each of the spectrally resolved images.
[0012]
Several different embodiments of a multi-detector imaging system are provided. The first series of embodiments is directed to systems with imaging lenses combined with individual detectors, and the second series of embodiments is directed to systems with single imaging lenses. Yes.
[0013]
In general, an imaging system includes a condensing lens arranged so that light traveling from an object becomes parallel by passing through the condensing lens and travels along a converging optical path. Yes. At least one lens for receiving the light passing through the condenser lens and forming an image is disposed. The arrangement of one or more such imaging lenses is whether there is one imaging lens used in the imaging system or whether individual lenses are coupled to individual detectors as mentioned above. It depends on. The relative arrangement will be described in more detail below. A plurality of light reflecting elements receive light that has passed through the condenser lens, reflect light having a predetermined characteristic, and pass light having no predetermined characteristic. The light passing through the condenser lens is preferably in a plane that is substantially perpendicular to the direction of relative movement between the object and the imaging system. As described above, the object and / or imaging system can be moved relative to each other. Hereinafter, for the sake of brevity, this relative movement is simply referred to as “movement”. Each of the light reflecting elements includes a detector arranged to reflect light in different directions and receive the reflected light. Each of the detectors can generate a signal representative of at least one characteristic of the object. Each of the detectors is preferably a TDI detector arranged to receive an image imaged by at least one imaging lens. When movement occurs, the image of the object imaged by the imaging lens moves from row to row between the ends of the TDI detector. Each TDI detector generates an output signal representative of at least one characteristic of the object by integrating light from at least a portion of the object over time.
[0014]
In this embodiment, all the light emitted from the first point of the object travels in parallel rays due to the collimation of the light by the condenser lens. Further, the light emitted from the second point of the object also travels in parallel rays, but the angle with respect to the light from the first point is different. By this method, the spatial information of the object is converted into angle information in the condensed light path by the condenser lens. A plurality of different reflective elements reflect light of different characteristics, so that different spectral components are substantially from a plurality of different reflective elements, preferably substantially relative to the direction of movement between the object and the imaging system. Acts on the collimated light so as to be separated in different directions in a plane perpendicular to the plane. By this method, both spatial information and spectral information of the object are converted into angle information. At least one imaging lens acts on the collimated light, and various light angles are converted to various positions on each detector. Spatial information is stored by the system because light from various locations on the object is projected to various locations on the detector for both axes. Also, light with different spectral components emitted from the object is preferably projected onto different detectors along an axis that is substantially perpendicular to the movement. In this way, spatial information from the object is preserved and spectral information covering a wide bandwidth is simultaneously collected with high resolution. Using a single detector for each of the different spectral components allows each detector to individually focus each color, thereby eliminating the longitudinal color correction constraints required for a single detector system and optical It means that the design is simplified. Yet another advantage is that the quantum efficiency of each of the detectors can be individually optimized for that particular color band, thereby improving the overall sensitivity of the system.
[0015]
Each of the light reflecting elements is preferably a dichroic filter or dichroic mirror adapted to reflect light within a predetermined bandwidth at a predetermined angle. Unlike prisms, where all wavelengths are separated by various angles, all light within a predetermined bandwidth enters the dichroic element at a common angle and leaves a given dichroic element at the same angle Therefore, no convolution occurs between the emission spectrum of the light leaving the object and the image of the object. When using such a reflective element, light of the first spectral bandwidth is reflected at a nominal angle predetermined by the first dichroic element. The light of the second spectral bandwidth passes through the first dichroic element to the next dichroic element where it reflects at another predetermined nominal angle. The light of the third spectral bandwidth passes through the first and second dichroic elements toward the third dichroic element, where it is reflected at a third predetermined nominal angle. The dichroic element is selected to cover the desired light spectrum and has an appropriate spectral passband. Each angle of the dichroic element is set so that light within the spectral bandwidth corresponding to the spectral bandwidth of the dichroic element reflected by the dichroic element is focused onto a different detector. Yes.
[0016]
In at least one embodiment, a single image lens is disposed in the collection optical path. The position of the detector is manipulated so that the distance from each of the detectors to the single image lens is substantially equal. Each of the reflective elements is disposed between the image lens and its corresponding detector.
[0017]
In another embodiment, each detector is provided with one image lens disposed between the reflective element and its corresponding detector. The image lens is arranged so that the light reflected by the reflecting element passes through the image lens before reaching the detector.
[0018]
In still other embodiments, each of the dichroic reflective elements is a cube substrate, and in other embodiments, each of the dichroic reflective elements is a pellical. In another embodiment, each of the dichroic reflective elements is a plate substrate. Note that in most embodiments, light from the object passes only once through each of the light reflecting elements.
[0019]
Particularly in embodiments using a single lens, the light distortion increases after the light passes through each of the dichroic reflective elements. In one embodiment, the use of a cube substrate reduces such distortion, the numerical aperture associated with each of the cube substrates is sufficiently small, and coma and astigmatism are substantially eliminated. In another embodiment, a correction plate is disposed between each successive light reflecting element. Each of the correction plates is oriented so that any astigmatism imparted by the immediately preceding light reflecting element is substantially removed relative to the immediately preceding light reflecting element. The orientation of the correction plate is preferably substantially perpendicular to the axis on which the immediately preceding light reflecting element rotates to direct the reflected light toward one of the detectors.
[0020]
In order to properly focus the light of a particular color directed to each of the TDI detectors by corresponding light reflecting elements to the individual TDI detectors, it is used to reflect or pass the light Preferably, the predetermined characteristic of the light reflecting element is color, and each TDI detector preferably focuses a specific color of light individually. Most preferably, each individual TDI detector is individually optimized for a particular color directed towards that TDI detector.
[0021]
It should be understood that additional optical elements can be incorporated into the present invention. One embodiment includes an aperture stop disposed adjacent to and immediately in front of at least one image lens. The aperture stop allows control of the numerical aperture associated with the at least one image lens. Other embodiments include an objective lens and an imaging slit disposed along a light collection optical path between the object and the collection lens. A light source is provided for providing incident light that illuminates the object.
[0022]
Note that the use of the TDI detector in the present invention expands the imaging area along the axis of movement, which in turn increases the integration time. Multiple light sources can be simultaneously projected onto the imaging area to increase the amount of light incident on the object in the imaging area. In addition, the extended imaging region and the orthogonal orientation of the spectral dispersion axis with respect to the movement axis combine to enable simultaneous imaging of a plurality of objects. The long integration time and parallel image acquisition according to this embodiment is highly sensitive and allows a consistent combination of imaging performance and high throughput.
[0023]
There are several alternative ways to provide light from the object, and in one embodiment the light from the object consists of non-stimulated emission of the object. That is, the object emits light without requiring a light source for inducing the emission. In another embodiment, a light source is provided for providing incident light that illuminates the object. In this embodiment, the object scatters the incident light so that at least part of the light scattered by the object passes through the condenser lens, or the incident light that irradiates the object guides the object. The light passing through the condenser lens is emitted. Further, at least a part of the incident light is absorbed by the object, and therefore the light passing through the condenser lens does not include a part of the light absorbed by the object. Finally, incident light from the light source is reflected from the object toward the condenser lens. The one or more light sources used preferably comprise at least one of a coherent light source, a non-coherent light source, a pulsed light source, and a continuous light source.
[0024]
The object can flow into the fluid stream that moves the object through the condenser lens, or alternatively, the object can be carried on the support. Alternatively, it can be simply moved without the use of a support or fluid medium. Further, the present invention is not limited to microscopic imaging, that is, microscopic object imaging.
[0025]
The TDI detector preferably reacts to the image of the object by generating a signal that propagates across the TDI detector. Although the pixels of a typical TDI detector are arranged in rows and columns and the signal is propagating from row to row, the present invention uses a TDI detector (eg, a microchannel plate-based TDI) that uses a linear pixel array. It is not limited to a detector. The signal propagation speed between the ends of the TDI detector can be synchronized to the movement of the image of the object on the TDI detector resulting from the movement, or can be asynchronous with the movement.
[0026]
Another aspect of the invention is directed to a method for imaging an object. These methods perform steps generally consistent with the imaging system discussed above.
[0027]
The foregoing aspects and many of the attendant advantages of this invention will be more readily appreciated and better understood by reference to the following detailed description, taken in conjunction with the accompanying drawings, in which:
[0028]
(Description of Preferred Embodiment)
The present invention provides significant advantages over prior art systems used for cell and particle analysis. These advantages are due to the use of the present invention in combination with a light dispersion system and a TDI detector that produces an output signal in response to images of cells and other objects directed to the TDI detector. It is. Multiple objects can be imaged simultaneously on the TDI detector. Also, for analysis, the image of each object can be spectrally resolved and the features of the object can be distinguished by absorption, scattering, reflection or probe emission using a common TDI detector.
[0029]
Using the present invention to measure optical signals including light scattering, reflection, absorption, fluorescence, phosphorescence, luminescence, etc., morphological, photometric and spectral properties of cells and other objects Can be determined. Morphological parameters include nuclear area, perimeter, composition or spatial frequency component, centroid position, shape (ie, circle, ellipse, barbell shape, etc.), volume, and any ratio of these parameters. The present invention can also be used to determine similar parameters for the cell trait of a cell. Photometric measurements according to the present invention determine nuclear optical density, cytoplasmic optical density, background optical density, and any ratio of these values. An object that is imaged using the present invention is either an object that emits light by inducing fluorescence or phosphorescence, or an object that generates light without requiring induction. May be. In any case, a cell or other object in which light from the object is imaged on a TDI detector according to the present invention and the presence and magnitude of the emitted light, one or more optical signals are generated The number of discrete locations within, the relative placement of the signal sources, and the color (wavelength or wavelength band) of light emitted from each location in the object are determined.
[0030]
The first application of an imaging system with the present invention is the use of cells entering a fluid flowing through the imaging system as a cell analyzer to determine one or more parameters as described above. While contemplated, it should be understood that the present invention can be used to image other moving objects.
[0031]
<First Preferred Embodiment>
1-3 schematically illustrate a first preferred embodiment of an imaging system 20 according to the present invention, along with the imaging of moving objects such as cells carried by a fluid stream 22 flowing through the imaging system. Is. In FIG. 1, an object 24 (such as a cell, but alternatively a microparticle) is flowed into a fluid stream 22 to carry the object through an imaging system. The direction of the fluid flow in FIG. 1 is the direction toward (or comes out of) the drawing paper. In FIGS. 2 and 3, the direction of the flow is from top to bottom as indicated by the arrows on the left side of the drawing. It is the direction toward. The light 30 from the object 24 passes through condensing lenses 32a and 32b that collect the light and generate condensed light 34 that is substantially focused at infinity. That is, the light rays of the condensed light from the condensing lens 32b are substantially parallel. The condensed light 34 enters a prism 36 that disperses the light and generates a dispersed light 38. The dispersed light then enters an imaging lens 40a and 40b that focuses the light 42 onto the TDI detector 44.
[0032]
As can be seen from FIG. 2, assuming that the imaging of the object 24 is always shown, as the object moves with the fluid stream 22, the object will be shown at both position 26 and position 28. As a result, an image of the object 24 is generated at two discrete spatial positions 26 'and 28' on the detector, as shown on the right side of FIG. Alternatively, if FIG. 2 shows a single moment, positions 26 and 28 represent the positions of two individual objects imaged simultaneously at positions 26 'and 28' on the detector.
[0033]
With respect to imaging system 20 and all other imaging systems shown herein, it should be understood that the lenses and other optical elements shown in FIG. 2 are shown only in a relatively simple form. Therefore, the condensing lens is shown as a compound lens comprising only condensing lenses 32a and 32b. As will be appreciated by those skilled in the art, imaging systems can be constructed using lens elements of different designs, whether simpler or more complex, to provide the desired optical performance. . The actual lens or optical element used in the imaging system will vary depending on the particular type of imaging application in which the imaging system is used.
[0034]
It should be understood that for each of the embodiments of the present invention, there is a relative movement between the object to be imaged and the imaging system. In most embodiments, it is more convenient to move the object than to move the imaging system, but in some embodiments, the object is kept stationary and the imaging system is moved relative to the object. Some embodiments are intended to be moved. As another alternative, both the imaging system and the object can be moved in different directions or at different speeds.
[0035]
The TDI detector used in various embodiments of the present invention preferably comprises a rectangular charge coupled device (CCD) using a dedicated pixel readout algorithm, as described below. Non-TDI CCD arrays are widely used for two-dimensional imaging of cameras. In a standard CCD array, photons incident on the pixel generate charge that is trapped in the pixel. Photon charges from each pixel are read from the detector array by shifting the charge from pixel to pixel. The charge read from the detector array is then provided to the output capacitor, producing a voltage proportional to the charge. The capacitor is discharged between pixel reads and the process is repeated for all pixels on the chip. During readout, the array must be shielded from any exposure to prevent charge generation in pixels that have not yet been read out.
[0036]
In one type of TDI detector 44 with a CCD array, the CCD array remains exposed while the pixels are being read. Reading is performed from top to bottom of the array, one row at a time. When the first row is read, the remaining rows are shifted by one pixel in the direction of the row that has just been read. As the object to be imaged on the array moves in synchronism with the movement of the pixels, the light from the object is integrated over the duration of the total readout period of the TDI detector without blurring the image. The intensity of the signal generated by the TDI detector increases linearly with an integration period proportional to the number of TDI rows, but noise only increases with the square root of the integration period, so the signal is only the square root of the number of rows. The overall noise to noise ratio increases. One TDI detector suitable for use in the present invention is the Dalsa Type IL-E2 image sensor, although other equivalent or better image sensors could alternatively be used. The Dalsa image sensor has 96 stages, or 96 rows, each with 512 pixels. The present invention may use other types of image sensors having different row and column configurations, or other types of image sensors in which pixels are arranged non-linearly. The sensitivity and signal-to-noise ratio of the Dalsa sensor is about 96 and 10 times that of a standard CCD array, respectively. Also, since the integration time associated with TDI detection is long, temporal and spatial illuminance fluctuations are averaged and the consistency of measurement is improved.
[0037]
In an imaging system 20 that uses fluid flow to carry objects through the imaging system, and in other embodiments of the invention, a flow-through cuvette or jet (not shown) analyzes cells or other The object is included. Fluid velocity and cell concentration are controlled using a syringe pump, gas pressure, or other pumping scheme (not shown) that drives the sample solution through the system to match the pixel readout rate of the TDI detector. However, it should be understood that the TDI detector readout rate can be selectively controlled as needed to match the movement of the sample solution.
[0038]
Various optical magnifications can be used to achieve the desired resolving power of the object imaged on the light sensitive area (pixel) of the TDI detector. In most embodiments, the optical magnification range is between 1: 1 and 50: 1, and for the number of photosensitive areas on the TDI detector on which the object image is formed, and of course Although intended to provide a substantial range depending on the actual size of the object being imaged and the distance of the object from the imaging system. The present invention is intended to have applications ranging from the analysis of cells and other microscopic objects to the imaging of objects such as stars.
[0039]
It is emphasized that the present invention is not limited to CCD type TDI detectors. Other types of TDI detectors such as complementary metal oxide semiconductor (CMOS) and multi-channel plate imaging devices can also be used in the TDI detector of the present invention. Any pixelated device (ie, a device having multiple photosensitive areas) that can move a signal generated in response to radiation directed at the device in a controlled manner is the TDI detection of the present invention. It is important to understand that it is suitable for use as a vessel. Normally, the signal moves in synchronization with the moving image projected on the device, thereby increasing the time to integrate the image without causing blurring, but it is necessary to achieve the desired effect Accordingly, the signal movement can be selectively desynchronized with respect to the radiation image movement.
[0040]
<Second Preferred Embodiment>
FIG. 4 shows an imaging system 45, which is a second preferred embodiment of the present invention, which is similar in many respects to the imaging system 20, but the imaging system 45 is an external light TDI. It is a confocal embodiment provided with a slit 52 that substantially prevents reaching the detector 44. In the imaging system 45, the light 46 from the object 24 is focused on the slit 52 by the objective lens 48. As shown in FIG. 4, the slit 52 is sufficiently narrow to prevent passage of the slit by light that is not focused on the slit by the objective lens 48. The light 30 ′ that has passed through the slit is collected by a condenser lens 32 as discussed above in connection with the imaging system 20. The condensed light 34 is spectrally dispersed by the prism 36 and imaged on the TDI detector 44 by the imaging lens 40 as described above. By eliminating the arrival of light other than the light from the object 24 to the TDI detector 44, the TDI detector 44 is affected by the already excluded external light corresponding only to the actual image of the object. Output signal can be generated. If not eliminated by this method, ambient light reaching the TDI detector 44 will generate “noise” in the output signal from the TDI detector.
[0041]
Note that the light source is not shown in each of the imaging systems 20 and 45. The first two embodiments make it clear that if the object is a light emitter, i.e. if the object generates light, a separate light source is not required to generate an image of the object. These are shown in the most general form of these two embodiments. However, many of the applications of the present invention require the use of one or more light sources that provide light incident on the object to be imaged. The position of the light source substantially affects the interaction between the incident light and the object, and in turn affects the information obtained from the image on the TDI detector.
[0042]
FIG. 5 shows a plurality of various light source positions that can be used to provide light incident on the object 24, but many other positions than those shown in FIG. It should be understood that they can be arranged in The position of each of the one or more light sources used will vary depending on the object being imaged and the object data derived from the signal generated by the TDI detector. For example, as shown in FIG. 5, the use of a light source 60a or 60b provides light 58 incident on the object 24 and scattered from the object along the optical axis of the condenser lens 32. The optical axis of the condenser lens 32 forms an angle of about 90 ° with respect to the direction of light incident on the object 24 from either the light source 60a or 60b.
[0043]
On the other hand, the light source 62 is disposed so that the light 58 emitted from the light source 62 travels in a direction substantially aligned with the optical axis of the condenser lens 32 toward the object. Therefore, the image formed on the TDI detector 44 does not include light absorbed by the object 24. Therefore, the light absorption characteristics of the object can be determined by irradiating the object using the light source 62.
[0044]
The light source 64 is arranged to irradiate the object 24 with light guided to the object along an optical path deviated by about 30 to 45 ° from the optical axis of the condenser lens 32. When this light 58 enters the object 24, it is reflected (scattered) by the object 24, and the reflected or scattered light forms an image on the TDI detector 44. More direct reflected light is provided by the external light source 66. The external light source 66 is arranged so that the light 58 of the external light source 66 is guided toward the partially reflecting surface 68. The partially reflecting surface 68 is arranged so that a part of the light is reflected on the object 24 through the condenser lens 32. The light that reaches the object is reflected by the object and returns along the optical axis of the condenser lens 32, at least part of which passes through the partially reflecting surface 68, thereby causing an image of the object on the TDI detector 44. Is formed. Another method is to use a dichroic mirror instead of the partially reflective surface 68 to direct the light from the external light source 66 at the location of the partially reflective surface 68 to cause fluorescence or other stimulated emission from the object 24. It can also be excited. In this case, at least a part of the emission from the object 24 is collected by the condenser lens 32, passes through the dichroic mirror and the spectrum is dispersed and detected by the TDI detector.
[0045]
In addition to imaging the object using light incident on the object, a light source can also be used to induce the emission of light by the object. For example, a FISH probe inserted into a cell fluoresces when excited with light and generates a corresponding characteristic emission spectrum that is imaged on the TDI detector 44 from any excited FISH probe. In FIG. 5, the light source 60a, 60b, 64 or 66 is alternatively used to excite the FISH probe of the object 24 so that the TDI detector 44 causes the FISH spot generated by the FISH probe to be reflected by the prism 36. As a result of the spectral dispersion of the light from the provided object, it can be imaged at different locations on the TDI detector. The placement of these FISH spots on the TDI detector surface depends on the emission spectrum of the FISH spot and the position of the FISH spot in the object. The use of a FSF probe in connection with image generation of FISH spots on a TDI detector according to the present invention will be described in further detail below.
[0046]
Each of the light sources shown in FIG. 5 generates either coherent, non-coherent, broadband or narrowband light 58, depending on the desired imaging system application. Thus, tungsten filament light sources can be used for applications that do not require narrow band light sources. In the case of an application that induces fluorescence emission from the FISH probe, it is possible to generate a spectrally resolved undistorted image of the object from the light scattered by the object. . This scattered light image can be resolved individually from the FISH spots generated on the TDI detector 44 as long as the emission spectra of all FISH spots are at different wavelengths other than the wavelength of the laser light. The light source may be either a continuous wave (CW) type or a pulse type. When using a pulse type illumination source, the integration period associated with TDI detection is long, so that signals from multiple pulses can be integrated. Also, there is no need to pulse the light in synchronization with the TDI detector.
[0047]
In the present invention, a pulsed laser provides several advantages over a CW laser as a light source, including small size, high efficiency, high reliability, and the ability to deliver multiple wavelengths simultaneously. Another advantage of the pulsed laser is its ability to achieve a saturation level of fluorescence excitation of the fluorescent probe used in the cell. Fluorescence saturation occurs when the number of photons that encounter a fluorescent molecule exceeds its absorption capacity. Saturated excitation provided by a pulsed laser is inherently less noisy than non-saturated CW laser excitation because changes in excitation intensity between pulses have less effect on fluorescence emission intensity.
[0048]
Any of the prisms 36 of the imaging system described above can be replaced with a diffraction grating because they can spectrally disperse the optical signal from the cells onto the pixels of the TDI detector. Using spectral dispersion not only provides useful data from cells or other objects, but also reduces measurement noise. If the wavelength of the light source and the emission spectrum of the fluorescent probe are different, the light from the light source scattered in the collection system is spatially isolated from the fluorescent signal. If the wavelength of the light source and the emission spectrum of the fluorescent probe overlap, the pixel of the TDI detector that hits the light of the wavelength of the light source is isolated from the pixel that hits the remaining fluorescent signal. Also, by dispersing the fluorescent signal over multiple pixels, the overall dynamic range of the imaging system is expanded.
[0049]
<Third Preferred Embodiment>
The third preferred embodiment is a three-dimensional structure 70 of the first preferred embodiment, as shown in FIG. With this structure, an object can be imaged from two different directions, and features that would overlap when viewed from a single direction can be distinguished. The third preferred embodiment can be used for objects on a moving substrate such as a microscope slide, but is particularly useful for analyzing multi-component objects in solution, such as FISH probes containing cells. Such probes appear as point light sources throughout the three-dimensional nucleus of the cell. In some cases, multiple FISH probes may appear in an overlapping fashion along the optical axis of the imaging system. In such a case, it is difficult to accurately measure the number of probes present in the cell because one of the FISH probes interferes with the other FISH probe. Accurate measurement of the number of probes is an important factor in determining genetic abnormalities such as trisomy 21 known as Down syndrome. Single perspective systems address this problem by “panning through” the object along the optical axis, resulting in multiple image planes in the object. Although this method is effective, it requires a considerable amount of time to collect a plurality of images and cannot be easily applied to flowing cells. The stereoscopic imaging system 70 shown in FIG. 6 includes two TDI detectors 44a and 44b and associated optical elements as described above in connection with the imaging system 20.
[0050]
The optical axes of the condenser lenses 32 of the two TDI detectors were imaged on at least one TDI detector 44a or 44b from a plurality of FISH probes, for example by placing them at 90 ° intervals. FISH spots can be resolved individually. If multiple FISH probes overlap with the image generated on one detector, the FISH probes are individually separated in the spectral dispersion image generated on the other TDI detector. It will be resolved. In addition, by using two TDI detectors in an imaging system 70 that can be referred to as a “stereo or three-dimensional configuration”, parameters such as relative TDI readout speed, axial orientation, tilt, focal plane position and magnification can be set. Each leg of the including system can be configured flexibly. Multiple cells or other objects can be simultaneously imaged vertically on each detector. Since an object moves in synchronization with a signal on TDI, a gate, that is, a shutter for preventing blurring of an image is unnecessary. As already mentioned, the present invention can use a pulsed light source or a CW light source without the need for a timed trigger mechanism to match the pulse to the arrival of the particle in the field of view. When using a pulsed light source, the field of view along the movement axis coupled to the TDI detector is widened, so that a moving cell, ie, an object, can be irradiated with a plurality of pulses during its longitudinal section. In contrast to a frame-based imaging device, a TDI system can generate a single, unblurred image of an object that integrates signals from multiple pulses. When using a CW light source, the signal generated by the object is collected over the entire period of time that the object traverses through the field of view, as opposed to the time of the few minute segments in which the shutter is open. Can do. Thus, in the present invention, the amount of signal collected and imaged on the detector is substantially greater than prior art frame-based imaging systems. Thus, the present invention can operate at an extremely high throughput rate with an excellent signal-to-noise ratio.
[0051]
Also shown in FIG. 6 are a plurality of exemplary light source positions that are useful for various purposes associated with the imaging system shown. With respect to the TDI detector 44a, the light source 62 provides illumination of the object 24 from a direction in which the absorption characteristics of the object can be determined from the image generated on the TDI detector. At the same time, light scattered by the object 24 provided by the light source 62 can be used to generate a scattered image and a spectrally dispersed image on the TDI detector 44b. The light source 74 can be used to generate spectrally dispersed and scattered images for both TDI detectors 44a and 44b. If the light sources 62 and 72 have different wavelengths and are equipped with appropriate filters to block the wavelengths from the light sources aligned with the optical axis of each condenser lens 32, then the two light sources are used, Light scattered by the object can be generated. For example, assume that the light source 72 generates light of wavelength A that is scattered by the object 24 and directed toward the TDI detector 44a. By providing a filter (not shown) that blocks the wavelength B generated by the light source 62, the light of the wavelength B does not directly affect the image generated on the TDI detector 44a. Similarly, a suitable filter (not shown) may be used from the light source 72 to avoid interfering with the imaging of light scattered by the light source 62 from the object 24 onto the TDI detector 44b. Can block light.
[0052]
Also, the external light source 66 shown in FIG. 6 can be used to generate an image on the TDI detector 44a along with the partial reflector 68. The light source 64 can be used to generate reflected light that produces an image on the TDI detector 44a. Scattered light from the external light source 66 is guided toward the TDI detector 44. For the location of these light sources and other possible positions suitable for providing incident light on the object, necessary to achieve imaging in response to information about the specific application and object desired, see It will be obvious to engineers.
[0053]
<Imaging slide or object transferred by slide>
Turning now to FIG. 7, an imaging system 80 is shown. Imaging system 80 is similar to imaging system 20 except that it is used to image object 24 on slide 82. The object 24 is supported by a slide 82 that moves relative to the imaging system, as shown in FIG. Alternatively, the slide 82 may be an object to be imaged. Since the object is imaged using the reflected incident light, the object may be a semiconductor wafer, paper, or other object of interest.
[0054]
A light source located at one of a plurality of different locations is used to provide light incident on either the slide 82 or the object 24 supported by the slide 82. The exemplary light sources 62, 64, and 66 illustrate some locations where light sources useful in the present invention are located. The light 58 emitted from any one of the light sources may be coherent light, non-coherent light, pulse, or CW. The light 58 is directed from the light source 62 through the slide 82 (if the slide is transparent) or is reflected by the object or slide if the light source 64 or 66 is used. As already mentioned, the external light source 66 illuminates the object together with the partially reflective surface 68.
[0055]
<Fourth Preferred Embodiment>
FIGS. 8A and 8B show two different views of an imaging system 90 that generates a scatter pattern image of the object 24 on a TDI detector 44, which is a fourth preferred embodiment. As in the embodiment described above, the light 30 from the object 24 passes through the condenser lenses 32 a and 32 b, and the condensed light 34 is guided onto the cylindrical lens 92. The cylindrical lens 92 focuses light 94 onto the TDI detector 44 along a line that is generally aligned with the central axis 96 of the cylindrical lens 92. Although a central axis 96 is shown in FIG. 8B, it will be apparent that the object 24 is perpendicular to the direction in which the object 24 moves through the imaging system. When the object 24 moves downward relative to the arrangement shown in FIG. 8A, the focal point of the cylindrical lens 92 on the TDI detector 44 moves upward. Accordingly, the cylindrical lens 92 distributes the image of the object along the photosensitive area of the TDI detector 44, ie, one or more rows of pixels.
[0056]
<Fifth Preferred Embodiment>
Referring now to FIG. 9, there is shown a fifth preferred embodiment, an imaging system 100 that forms both a scatter pattern image and a spectral dispersion image of the object 24 on a TDI detector 44. In the imaging system 100, light 30 from the object 24 passes through condenser lenses 32 a and 32 b that generate infinity focused light 34 that is directed toward the dichroic filter 102. The dichroic filter 102 reflects light having a specific wavelength incident on the object 24, for example, light having a wavelength of a light source (not shown). All other wavelengths of light are transmitted through the dichroic filter 102 toward the diffraction grating 112. The diffraction grating 112 spectrally disperses the light that is transmitted through the dichroic filter 102, typically generated by the fluorescence of the FISH probe of the object 24, thereby providing a plurality of FISH spots corresponding to the number of different FISH probes. And an object to be imaged is formed on the TDI detector 44.
[0057]
The light 104 reflected by the dichroic filter 102 passes through the cylindrical lens 106 and is focused along the line into a region 110 on the TDI detector as a scattered pattern image. A FISH spot or other aspect of the spectral dispersion image of the object 24 having a wavelength different from that reflected by the dichroic filter 102 is reflected on the region 118 of the TDI detector as light 116 by the imaging lenses 114a and 114b. Is imaged. Thus, both signals corresponding to the scatter pattern image and the spectral dispersion image are generated by the TDI detector 44.
[0058]
<Sixth Preferred Embodiment>
As shown in FIG. 10, the sixth preferred embodiment uses a dichroic filter 102 ′ that is angled in a different direction toward the second TDI detector 44b, so The imaging system 120 is slightly different from the embodiment. In this embodiment, the dispersion pattern image indicated by the light 108 'is generated by the cylindrical lens 106'. Just as in the imaging system 100, the light transmitted through the dichroic filter 102 'is focused on the TDI detector 44a. The operation of the imaging system 120 is substantially the same as that of the imaging system 100 except that two separate TDI detectors located on different sides of the imaging system are used, but the third Since two separate TDI detectors are used just as in the preferred embodiment, each leg of the system can be flexibly configured including parameters such as relative TDI readout speed, axial orientation, tilt, focal plane position and magnification. can do. It should also be noted that the imaging system 100 can be configured with two individual TDI detectors instead of a single TDI detector, if desired.
[0059]
<Processing of spectral dispersion image on TDI detector>
When used for cell analysis, the present invention provides a substantial utility for resolving FISH spots on TDI detectors, even when the FISH probes are placed in close proximity in the cell. Is done. When performing spectral imaging using the present invention, the spatial distribution of light in the object is convolved with the spectral distribution of the light and an image of the object is generated on the TDI detector. This convolution causes blurring along the dispersion axis depending on the spectral bandwidth of the light. When the spectral bandwidth is narrow, the blur due to the spectral resolution of the system is small or not at all. Although the present invention contemplates that the spatial resolution of the object space is about 1 micron and a spectral resolution of about 3 nm per pixel, the spatial resolution and spectral resolution are adjusted to meet the needs of a particular application. be able to.
[0060]
FIG. 11 shows the present invention with a spectral resolution of about 10 nm per pixel and a spatial resolution of about 0.5 microns. FIG. 11 further illustrates the use of the present invention to image a cell 140 having a nucleus 142 in which two FISH probes 144a and 144b having the same emission spectrum are disposed. In FIG. 11, the emission spectra 146 of FISH probes 144a and 144b have a width of about 10 nm and are generated with “quantum dots” or narrow band fluorescent dyes. Narrow bandwidth spectral light convolution minimizes blurring of FISH spots 148a and 148b and allows them to be easily resolved on TDI detector 44.
[0061]
In FIG. 12, a cell 150 having a nucleus 152 with FISH probes 154 and 156 having different emission spectra is shown. FISH probes are designed so that different emission spectra correspond to different DNA sequences. Each of the emission spectra of FISH probes 154 and 156 is relatively narrow, as shown by wavelength bands 158 and 160, and therefore, as in FIG. 11, blurring of FISH spots 162 and 164 is minimized. Also, due to the spectral dispersion according to the present invention that maps the wavelength to a lateral position on the TDI detector 44, the relative proximity of the FISH spots 162 and 164 despite the close proximity of the intracellular FISH probes 154 and 156. A wide physical displacement is provided. Considered together, FIGS. 11 and 12 illustrate a method for discriminating FISH probes of the same or different colors according to the present invention so that multiple genetic features can be listed simultaneously. Those skilled in the art will appreciate that the present invention is well suited to the requirements of fetal cell analysis, where more than 10 different colored probes are present in the cell at a time. It will also be appreciated by those skilled in the art that the present invention is not limited to fetal cell analysis using FISH probes.
[0062]
FIGS. 13 and 14 also show that the present invention can be used with light having a wide spectral bandwidth. In this case, an additional signal processing step is executed to correct the lateral blur due to the wide emission spectrum. In FIG. 13, a cell 140 having a nucleus 142 is shown. In the nucleus, FISH probes 170a and 170b having a common emission spectrum are arranged. FISH probes 170 a and 170 b have been characterized by generating a relatively broad emission spectrum 172. When optically convolved with the spectral dispersion provided by the present invention, FISH spots 174a and 174b are generated on the TDI detector 44, but the emission spectrum of the FISH spot is relatively wide, so The image is blurred in the horizontal direction between both ends. In order to more clearly resolve the separation of FISH spots 174a and 174b, a known FISH emission spectrum is used to perform a deconvolution on the signal generated by TDI detector 44, thereby providing an accurate FISH spot. Representations 178a and 178b are generated on display 176. The deconvolution step enhances the ability to enumerate the number of FISH spots.
[0063]
FIG. 14 is a diagram showing the correspondence between the FISH probes 180 and 182 arranged in the nucleus 152 of the cell 150. FISH probes 180 and 182 have been characterized by generating relatively broad emission spectra 184 and 186, respectively, as shown. FISH spots 188 and 190 are generated on the TDI detector 44 by the optical convolution of the fluorescence emitted by the spectrally dispersed FISH probe. Again, the corresponding images of FISH spots 192 and 194 shown on display 176 have been recovered by deconvolution over the known emission spectrum using the signal generated by TDI detector 44. Again, due to the spectral dispersion according to the present invention that maps the wavelength to a lateral position on the TDI detector 44, the FISH spots 192 and 194 are in close proximity even though the intracellular FISH probes 180 and 182 are in close proximity. A relatively wide physical displacement is provided. By this method, these FISH spot images generated by FISH probes that are relatively wide and have different emission spectra can be resolved.
[0064]
FIG. 15 illustrates a system 230 for analyzing the signal generated by the TDI detector 44 and performing the deconvolution step described above. In FIG. 15, the TDI detector 44 signal is sent to an amplifier 232 that buffers and amplifies the signal from the TDI detector 44 to achieve the level required by the analog-to-digital (AD) converter 234. Applied. This A-D converter converts the analog signal from the amplifier 232 into a digital signal and inputs it to the TDI line buffer 236. The TDI line buffer 236 temporarily stores the digital signal until the digital signal is processed by the CPU 238. In order to perform the deconvolution described above, the signal stored in the TDI line buffer 236 can be used to perform a deconvolution on the emission spectrum of the FISH probe being used. A spectrum is loaded into the spectrum buffer 240 for each FISH probe. The CPU 238 is a high speed processor programmed to perform deconvolution and other analysis procedures, allowing identification of desired properties, i.e. parameters, of the imaged object. The output of the CPU 238 is temporarily stored in an image line buffer 242 that can display an image or record it for later analysis.
[0065]
FIG. 16 is a diagram illustrating a practical application for identifying male cells 200 and female cells 208 and generating corresponding scattered images 212 and 220 according to the present invention. The male cell 200 includes a nucleus 202 colored with a yellow fluorescent dye. FISH probe 204 also provides orange fluorescence emission indicating the presence of the X chromosome in the nucleus, while FISH probe 206 provides fluorescence emission indicating the presence of the Y chromosome. By spectrally resolving fluorescence emission from male cells 200 irradiated with green laser light, a series of images separated as a function of the wavelength of the light to be imaged is provided on the TDI detector 44. Since the wavelength band of the laser light incident on the cell is extremely narrow, the convolution by the spectral decomposition processing on the image 212 of the male cell 200 generated by laser scattering is very small. A green laser scattering image 212 of the cell 200 and its nucleus 202 appears on the left side of the TDI detector, and the fluorescent spot 214 corresponding to the yellow fluorescence emitted by the nucleus 202 is in the next several rows on the TDI detector. Has appeared. In addition, FISH spots 216 and 218 as functions of different wavelengths of fluorescence emitted by the FISH probes 204 and 206 appear at spaced positions on the detector, depending on the width of their respective emission spectra. , Slightly blurred over several rows of TDI detectors 44. By analyzing the signal generated by the TDI detector, the FISH probe in response to the X and Y chromosomes is detected, so that the user contains both X and Y chromosomes because the cell contains them. It can be determined that cell 200 is a male cell. Similarly, the spectrally resolved female cell 208 also contains the characteristic yellow fluorescence of the nucleus 210, but unlike the male cell, it indicates the presence of two X chromosomes corresponding to the FISH probe 204. Two FISH spots 216 are included. Since the TDI detector 44 also distinguishes between the spatial positions of the male cell 200 and the female cell 208, when both cells pass through the imaging system in the direction indicated by the left arrow in FIG. Can be easily resolved individually. Again, it should be noted that by applying deconvolution to the signal generated by the TDI detector 44, the corresponding FISH spot shown in the figure is better resolved.
[0066]
<Strain-free spectral dispersion system>
The present invention can include a spectrally dispersive filter assembly that does not convolve the image with the emission spectrum of the light that forms the image to eliminate the need for deconvolution of the light-emitting spectrum from the image. FIG. 17 illustrates a seventh preferred embodiment of the present invention and corresponds to such a distortion-free spectral dispersion system 250 using a five-color stack wedge spectral dispersion filter assembly 252. This seventh embodiment is substantially similar to the embodiment shown in FIGS. 1-3 except that the spectral dispersion prism element 36 (FIGS. 1-3) is replaced with a spectral dispersion filter assembly 252. FIG. Yes. The spectral dispersion filter assembly divides light into a plurality of light beams having different bandwidths. Thus, each generated light beam is directed at a different nominal angle and illuminates a different area of the TDI detector 44, respectively. The nominal angular separation between each bandwidth produced by the spectral dispersion filter assembly 252 exceeds the viewing angle of the imaging system in the object space, thereby allowing different bandwidth field images on the detector. Overlap is prevented.
[0067]
The spectral dispersion filter assembly 252 includes a plurality of dichroic stack wedges including a red dichroic filter R, an orange dichroic filter O, a yellow dichroic filter Y, a green dichroic filter G, and a blue dichroic filter B. It consists of a filter. The red dichroic filter R is disposed in the optical path of the condensed light 34 and is oriented at an angle of about 44.0 ° with respect to the optical axis 253 of the condenser lenses 32a and 32b. Light above the red wavelength, ie> 640 nm, is reflected at the surface of the red dichroic filter R at a nominal angle of 1 ° measured counterclockwise from the vertical optical axis 257. FIG. 18 plots a typical spectral reflectance characteristic R ′ of a red dichroic filter R along with typical spectral reflectance characteristics corresponding to the other dichroic filters used in the spectral dispersion filter assembly 252. FIG. In FIG. 18, O ′ represents the spectral reflectance characteristic of the orange dichroic filter O. Y ′ represents the spectral reflectance characteristic of the yellow dichroic filter Y, and so on. The light reflected by the red dichroic filter R leaves the spectral dispersion filter assembly 252 and passes through imaging lenses 40a and 40b that image the light in the region of the TDI detector 44 that receives the red light. The region for receiving red light is arranged toward the right end of the TDI detector, as shown in FIG.
[0068]
The orange dichroic filter O is disposed behind the red dichroic filter R at a slight distance and is oriented at an angle of 44.5 ° with respect to the optical axis 253. Light above the orange wavelength, ie> 610 nm, is reflected by the orange dichroic filter O at a nominal angle of 0.5 ° with respect to the vertical optical axis 257. Since part of the condensed light 34 having a wavelength longer than 640 nm is already reflected by the red dichroic filter R, the light reflected by the surface of the orange dichroic filter O is orange between 610 nm and 640 nm. The band is effectively passed through the colored area. This light travels at a nominal angle of 0.5 ° from the vertical optical axis 257, and also receives red light from the central region of the TDI detector toward the right side of the TDI detector 44, as shown in FIG. An image is formed by imaging lenses 40a and 40b arranged to irradiate a region that receives orange light and is disposed between the regions to be illuminated.
[0069]
The yellow dichroic filter Y is disposed behind the orange dichroic filter O at a slight distance and is oriented at an angle of 45 ° with respect to the optical axis 253. Light having a yellow wavelength, that is, a wavelength of 560 nm or more is reflected by the yellow dichroic filter Y at a nominal angle of 0.0 ° with respect to the vertical optical axis 257. The wavelength of the light reflected by the yellow dichroic filter Y effectively passes through the yellow region between 560 nm and 610 nm and irradiates the region that receives yellow light toward the center of the TDI detector 44. An image is formed by the imaging lenses 40a and 40b in the vicinity of the vertical optical axis 257.
[0070]
The dichroic filters G and B are regions in which the green light wavelength band and the blue light wavelength band receive the green light and the blue light of the TDI detector 44, respectively, in the same manner as the dichroic filters R, O, and Y. It is configured to be imaged above and is oriented. The region for receiving green light and blue light is arranged toward the left side of the TDI detector. By stacking dichroic filters at different pre-determined angles, the spectral dispersion filter assembly 252 collectively functions so that light within a defined wavelength band of the optical spectrum can be pre-determined in a predetermined region of the TDI detector 44. Focus on top. Those skilled in the art will appreciate that the filters used in the spectral dispersion filter assembly 252 can have spectral characteristics different from those described above in connection with FIG. The spectral characteristics for achieving the desired dispersion characteristics are arbitrary, and filters other than dichroism can be used.
[0071]
Due to the wedge shape of the dichroic filter described above, the spectrally dispersive filter assembly 252 can be formed by placing the filters in close contact or in contact, and possibly joining them together. it can. The angle of the wedge shape produced in the substrate of the dichroic filter allows the spectral dispersion filter assembly 252 to be easily assembled, forming a monolithic structure with a wedge-shaped substrate sandwiched between adjacent dichroic filters. can do. When the filters are brought into contact with each other or joined together, the composition of the material determining the spectral performance of the filter can be different from the composition of the non-contacting material. Those skilled in the art will appreciate that the spectrally dispersive filter assembly 252 can be manufactured using a flat, non-wedge shaped substrate. In that case, other means such as mechanical filter attachment can be used to maintain the angular relationship between the filters.
[0072]
In addition to the configuration described above, the undistorted spectral dispersion system 250 optionally includes a detector filter assembly 254 for further attenuating unwanted signals in each light beam depending on the amount of rejection required for out-of-band signals. be able to. FIG. 19 is a diagram showing the structure of an exemplary detector filter 254 corresponding to the above-described five color bands. As shown in FIG. 19, the blue spectral region 256, the green spectral region 258, which are all arranged next to each other, A yellow spectral region 260, an orange spectral region 262 and a red spectral region 264 are provided. 20A to 20E show spectral characteristics corresponding to the spectral regions, that is, wavelength bands of blue, green, yellow, orange, and red, respectively. The detection filter assembly shown in FIG. 19 is constructed by joining individual filters in a side-by-side arrangement on a common substrate, or by other means well known to those skilled in the art. Is. It will also be appreciated by those skilled in the art that instead of placing the filter in front of the TDI detector 44, it can alternatively be placed in the intermediate image plane.
[0073]
In the embodiment shown in FIG. 17, the light has passed through each of the dichroic filters in the spectral dispersion filter assembly 252 twice before exciting the spectral dispersion filter assembly 252. This condition further attenuates out-of-band signals, but also attenuates in-band signals. FIG. 21 is a view showing an eighth embodiment of the present invention, and the light after reflection does not pass through another dichroic filter. In this embodiment, multiple cube dichroic filters comprising a red cube filter 266, a yellow cube filter 268, a green cube filter 270, and a blue cube filter 272 ensure that light does not pass through any cube filter more than once. In order to do so, they are sufficiently spaced apart. As in the embodiment shown in FIG. 17, the cube dichroic filter is oriented at an appropriate angle to image light within the defined bandwidth onto a discrete area on the TDI detector 274. As light reflects off each of the cube dichroic filters 266, 268, 270 and 272, it is directed towards the imaging lenses 40a and 40b and different bandwidth portions of the light are formed on the light receiving surface of the TDI detector 274. Are respectively focused into segments or regions that receive the corresponding red, yellow, green and blue light. If desired, an optional detector filter assembly 276 similar in structure to the detector filter assembly 254 (except for the orange spectral region) can be used to enhance out-of-band signal rejection. Those skilled in the art will appreciate that spaced apart discrete plates or peripheral elements can be used for this application instead of cube filters. In the eighth embodiment shown in FIG. 21, the imaging lenses 40a and 40b must be located sufficiently away from the cube filters to minimize the requirement for a clear aperture for the lenses 40a and 40b. I must. Those skilled in the art will appreciate that the clear aperture in a plane perpendicular to the page increases with increasing distance between the lens and the cube filters. Therefore, the placement of lenses 40a and 40b must be selected to properly accommodate the clear aperture in both planes.
[0074]
The foregoing description of the seventh and eighth preferred embodiments has shown the use of four and five color systems. In order to build a system that covers a wider spectral region, or a narrower spectral region, or different passbands within a given spectral region, spectral components with more or fewer filters can be configured Those skilled in the art will understand what can be used. Similarly, those skilled in the art have also noticed that by appropriately selecting the number and spectral characteristics of the dichroic and / or bandpass filters used, the spectral resolution according to the present invention is improved or reduced. Will be understood. It will also be appreciated by those skilled in the art that the angle or orientation of the filter can be adjusted to direct light of a given bandwidth to any desired point on the TDI detector. Furthermore, it is not necessary to focus the light in the order of increasing wavelength or decreasing wavelength. For example, in fluorescence imaging applications, filters that support excitation and emission wavelengths are more spatially separated between these wavelengths by changing the angle at which they are oriented with respect to the optical axis of the system. Can be generated on a TDI detector. Finally, it will be apparent to those skilled in the art that the collected light can be dispersed based on non-spectral characteristics including angle, position, polarization, phase or other optical characteristics.
[0075]
As with the embodiments already described above, many applications of the seventh and eighth preferred embodiments require one or more light sources that are used to provide light incident on the object to be imaged. It is. Accordingly, the various light sources shown in FIGS. 5-7 and located at the various positions described above can be used to improve the quality of the image produced by each of these embodiments. For simplicity and clarity of explanation of these embodiments, the light source is omitted in FIGS. 17 and 21, but based on the description of the use of the light source in connection with the above-described embodiment, these light sources are omitted. One skilled in the art will recognize the use of such light sources in embodiments.
[0076]
FIG. 22 is a diagram showing an image distribution on the TDI detector 44 corresponding to an image of a plurality of cells 200 when the undistorted spectral dispersion system 250 is used. As can be seen by comparing FIG. 22 and FIG. 16, the resulting image on the TDI detector is similar in many respects, but can be seen from FIG. 22 when using a distortion-free spectral dispersion system. In addition, there is no image spread caused by convolution of the emission spectrum and the object. Instead of convolution, all wavelengths within the predetermined bandwidth of each dichroic filter are reflected at the same nominal angle by the filter, so that the image components in that passband cause positional distortion on the detector. It does not occur. FIG. 22 shows a viewing angle perpendicular to the flow of the object space. In this particular configuration, the viewing angle in the object space is less than +/− 0.25 °. Those skilled in the art will appreciate that the viewing angle can be made larger or smaller. For example, the field of view at the detector in proportion to the number of colors used, by increasing the viewing angle to image cells over a larger area on the slide, or cells in a wide flat stream. The angle increases. Those skilled in the art will appreciate that a commercially available flow cell can be used to easily generate a wide, flat flow, as shown in FIG. 25 with the incorporated flow cell 306 incorporated. The flow cell 306 has an elongated cross section along an axis that is perpendicular to both the flow and optical axes. Wide flat flow generation is discussed in many references, including US Pat. No. 5,422,712. By using the flow cell 306, a wide flat flow is obtained. In an embodiment incorporating a flow cell 306 or other means for providing a wide flat flow, any object flowing into such a wide flat flow is imaged and the image is captured by a detector. The viewing angle is preferably large enough to be able to do. Therefore, if the width of the flow rate becomes wider, when the object passes through the field of view, in order to reliably image all the objects in the flow rate, the field angle is increased in proportion to the increase in the width. There must be.
[0077]
FIG. 22 shows an image projected on the detector when three cells 280, 282 and 284 pass through the field of view. Light scatter images of cells 280, 282 and 284 appear on the left side of the detector, shown as the blue region. An image of the cell nucleus 202 colored with the green fluorescent dye appears in the green region of the detector. In addition, genetic probes 204, 205 and 206 colored in three different colors are used to analyze intracellular sex chromosomes. The probe 204 colors the X chromosome with an orange fluorescent dye, and the probe 205 colors the Y chromosome with a yellow fluorescent dye. The probe 206 colors the inactive X chromosome in the female cell with a red fluorescent dye. Cells 282 are imaged on a detector as shown in FIG. An image 286 of the probe 204 from the cell 282 appears in the orange region of the detector. Similarly, the image 288 of the probe 205 appears in the yellow area of the detector. The signal on the detector is processed to determine the presence and location of these images on the detector to determine that cell 282 is a male cell. Similarly, cells 280 and 284, which contain probes 204 and 206 that produce images 290 and 292 in the orange region of the detector and images 294 and 296 in the red region of the detector, respectively, It shows that it is a female.
[0078]
<Multiple TDI detector embodiment of a distortion-free spectral dispersion system>
FIGS. 25, 26 and 28 illustrate alternative embodiments of the present invention utilizing multiple detectors for spectral dispersion and imaging. Spectral decomposition is generally performed using the dichroic filter described above, but independent imaging lenses and detectors are used for each of the spectral regions, as shown in FIG. Dichroic filters 301 to 305 are disposed in an infinite space with respect to the object, and the light from the object is spectrally resolved in order to minimize optical aberration. Individual imaging lenses 311 to 315 are used after each dichroic filter to form an image of the object on the corresponding detectors 321 to 325. With this configuration, the number of pixels in each detector is less than in the embodiment described above, so that this embodiment can be operated at high pixel line speeds. The image projected onto each of the detectors appears as shown in one of the detector zones shown in FIG. The image on the detector that is adapted to receive light in the red part of the spectrum appears in the same way as the image that appears in the rightmost zone of FIG.
[0079]
Since light from the object passes through each of the dichroic filters only once, the embodiments shown in FIGS. 25, 26 and 28 have advantages over other embodiments in terms of light efficiency. Yes. Another advantage of the multi-detector embodiment is that the individual detectors focus individual colors individually, thus eliminating longitudinal color correction constraints and simplifying optical design. Yet another advantage is that the quantum efficiency of each detector can be individually optimized for that particular color band. Those skilled in the art will readily recognize that such optimization is achieved by doping the semiconductor material utilized in the detector. As shown in FIG. 25, when using a plurality of imaging lenses, the focal length of one or more lenses (as exemplified by lens 311) is different from the other lenses, Can simultaneously collect images using differential magnification. In this case, the detector 321 clock rate increases in proportion to the focal length to maintain synchronization, so one of the channels is used at a higher magnification to collect bright field images, thereby It is expected to be useful when analyzing details more accurately. Also, with the configuration shown in FIG. 25, the numerical aperture can be controlled independently for each channel, as shown by the arrangement of the optical aperture stop 330. Note that due to the characteristics of flow cell 306, object plane 348a shown in FIG. 25 is wider than object plane 348 shown in the other figures. As described above, the flow cell 306 creates a wide, flat flow in which multiple objects passing through the object plane 348a are imaged simultaneously as long as each of the images covers a sufficiently large viewing angle. Obtainable. When the imaging system according to the present invention is used with such a wide flat flow, the viewing angle is set to that of the object plane (such as the object plane 348a) so that the generated image surrounds substantially all the object plane. Must be matched to size. Note that the object plane is formed by the circumference of the fluid passage used.
[0080]
FIG. 26 is a diagram illustrating another embodiment of a multiple detector technique. Although similar to the embodiment shown in FIG. 25, the embodiment shown in FIG. 26 has the advantage of reducing the number of imaging lenses required to project an image onto the detector. In the embodiment shown in FIG. 26, an image lens 340 is disposed in front of the dichroic filters 345 to 347. Those skilled in the art will appreciate that the functions of the condenser lens 32 and the image lens 340 can be performed in a single element. The detectors 341 to 344 are arranged at appropriate positions along the optical path, and form an object plane 348 on the surface of each detector. The detectors 341 to 343 are arranged along the optical path of light from the object reflected by the dichroic filters 345, 346 and 347, and the detector 344 receives light from the object that passes through the dichroic filter 347. Are arranged along the optical path. The dichroic filter is placed in the focusing space for the image of the object, so each filter is astigmatism, coma, spherical aberration and chromatic aberration in each downstream detector image, depending on its design. Is granted. Each subsequent filter progressively adds many of each of these aberrations. In an exemplary embodiment of the invention, the numerical aperture in the filter space (ie the product of the refractive index and the sine of the illumination half cone angle) is about 0.03. Therefore, when the cube substrate is used for a dichroic filter, coma and astigmatism can be ignored, and spherical aberration is substantially eliminated and is less than 0.15 wave height. Longitudinal chromatic aberration is effectively offset by moving the detector to the best focal plane for each of those color bands. Also, a pellicle having excellent theoretical optical performance can be used instead of a cube for the substrate of the dichroic filter.
[0081]
When a plate substrate is used for the dichroic filters 345 to 347, the dominant aberration is astigmatism. Astigmatism is imparted to the transmitted wavefront through the dichroic plate filter. As shown in FIG. 27, the astigmatism is effectively obtained by inserting a transparent correction plate 360 having substantially the same thickness, incident angle and glass type. Offset. However, the correction plate 360 must be rotated 90 ° around the axis Z with respect to the dichroic filter 361. The correction plate 360 and the dichroic filter 361 impart an equal and opposite amount of astigmatism to the transmitted wavefront, thereby canceling each other out of astigmatism. Therefore, there is no astigmatism in the light that irradiates the detector 342. This configuration leaves some residual coma, but the optical performance is very close to the diffraction limit. Those skilled in the art will appreciate that the corrector plate can be placed in many alternative positions by adjusting its thickness, material and / or angle for light propagation. Additional objective lens 48 and slit 52 can be used to construct any undistorted spectral dispersion embodiment to form a confocal stop structure as shown in FIG. FIG. 28 shows an embodiment similar to FIG. 25 using a plurality of imaging lenses, but most of the detectors are arranged along the transmitted light path of the dichroic filter. Both multiple detector embodiments detect light transmitted through a dichroic filter, light reflected by a dichroic filter, or a combination of transmission and reflection, as shown in both FIGS. Is constructed to receive light.
[0082]
<Wide-field decomposition image>
By using a segmented TDI detector 300 as shown in FIG. 24, the present invention can be used to image a wide field of view and improve throughput. This method allows cells or other objects found in a wide flat stream or on microscope slides and microplates to be oriented side by side. With this configuration, more objects can be imaged simultaneously than possible when the objects are aligned in a single row orientation.
[0083]
FIG. 23 is a diagram showing an embodiment of the present invention that can easily image a wide field of view. In FIG. 23, the substrate 73 has moved in a direction generally parallel or aligned with the axis of spectral resolution provided by the dichroic element 252. An optional external illuminator 60a with a laser or other type of illumination source can be used to illuminate the object being transferred on the substrate 73. There is also a relative movement between the substrate and the imaging system in the direction indicated by the double arrows. Optionally, another illuminator 60b can be provided, and the light reflected by the reflective surface 77 can be used to provide bright field illumination of objects on the substrate. The light from the object on the substrate 73 that has passed through the lens 71 is reflected by the reflecting surface 69, passes through the dichroic (or partial reflection) mirror 67, and is focused on the slit 55 by the lens 57. The condenser lens 32 collimates the light from the slit and guides it onto the dichroic element 252. The dichroic element 252 disperses the light passing through the lens 40 individually on various regions of the detector 44.
[0084]
The segment detector 300 (FIG. 24) is used in the detector 44 shown in FIG. 23, and the spectral dispersion filter assembly 252 decomposes the light along an axis parallel to the direction in which the image travels between the ends of the detector 44. Oriented to do so. As described above, the visual field of the substrate 73 shown in FIG. 23 is irradiated in the bright field using the bright field illuminator 60b or using external illumination by the illuminator 60a. In either case, the illuminated field is the same size as one of the segments of detector 300 when imaged by the optical system.
[0085]
As already described, when using the spectral dispersion filter assembly 252, the light is split into multiple light beams with different bandwidths. Thus, each of the generated light beams is directed at a different nominal angle and illuminates a different segment of detector 300, respectively. The nominal angular separation between each bandwidth produced by the spectral dispersion filter assembly 252 exceeds the viewing angle of the imaging system in the object space, thereby allowing different bandwidth field images on the detector. Overlap is prevented. Thus, each detector segment sees the same field of view, but sees light with a different spectral bandwidth. The slit 55 is provided to remove any stray light from outside the intended field of view and prevent them from passing through the system and illuminating inappropriate zones of the detector 300.
[0086]
In the illustrated embodiment, the segmented detector 300 consists of four segments or zones 302a-302d, each receiving light of different characteristics. The detector is segmented into these zones so that the charge corresponding to the incident image flows between the ends of the segment, matching the image moving between the ends of the segment. This charge is read out of the segment and adjacent light of different characteristics is not allowed to enter the segment or zone being imaged. Optionally, the charge corresponding to the image received by each zone is integrated over the length of the zone and read from the taps provided in the zone. Also, optionally, the rate at which charges are read from each zone can be individually controlled. In summary, according to this last embodiment of the present invention, a wide field of view is imaged and analyzed on the detector so that multiple characteristics of light can be collected and analyzed simultaneously.
[0087]
The present invention has been described above with reference to preferred embodiments for practicing the present invention. However, many modifications can be made within the technical scope of each claim recited in the claims. It will be understood by engineers. Therefore, the scope of the present invention is not limited to the above description, and can be determined only by referring to the claims.
[Brief description of the drawings]
FIG. 1 is a plan view of a first embodiment of the present invention in which particles are carried by a fluid flow depicted as flowing into a drawing sheet.
FIG. 2 is a side view of the first embodiment shown in FIG.
FIG. 3 is an isometric view of the first embodiment of FIG.
FIG. 4 is an isometric view of a confocal embodiment with a slit used for spatial filtering of external light.
FIG. 5 is an isometric view showing various positions of the light source together with the first embodiment.
FIG. 6 is a first embodiment alternative and light source alternative location with a second set of imaging elements and a TDI detector for monitoring light from particles to avoid interference between FISH probes. FIG.
FIG. 7 is an isometric view of an embodiment in which an object is supported by a slide that moves through a condenser lens or includes a slide that moves through a condenser lens; FIG.
8A is an alternative plan view of the embodiment of FIG. 7 used to generate a scattering pattern on a TDI detector. FIG.
FIG. 8B is an alternative side view of the embodiment of FIG. 7 used to generate a scattering pattern on a TDI detector.
FIG. 9 is a plan view of yet another embodiment in which the light forming the scatter pattern image and the spectrally dispersed light from the object are imaged on separate portions of the TDI detector.
FIG. 10 is a plan view of yet another embodiment in which light forming a scatter pattern image and spectrally dispersed light from an object are imaged by two different TDI detectors.
FIG. 11 is a schematic diagram showing the optical convolution of a narrow FISH emission spectrum according to the present invention for resolving two FISH probes in a cell.
FIG. 12 is a schematic diagram illustrating the optical convolution of two different colors of narrow FISH emission spectra to resolve an image of a FISH probe on a TDI detector.
FIG. 13 is a schematic diagram showing how the present invention provides deconvolution for resolving images of two single color FISH probes when the FISH emission spectrum is wider.
FIG. 14 is a schematic diagram showing the deconvolution of a relatively wide two-color FISH spectrum for resolving an image of a FISH probe.
FIG. 15 is a block diagram of an imaging system used to process signals generated by a TDI detector according to the present invention.
FIG. 16 is a schematic diagram illustrating a method of using the present invention to determine whether a cell is from a male or female.
FIG. 17 is a plan view of an alternative embodiment using a spectral dispersive element with multiple dichroic stack filters used to spectrally separate light.
FIG. 18 is a graph XY plotting some typical passbands of the dichroic filter used in the embodiment shown in FIG.
FIG. 19 is a schematic diagram showing a detection filter assembly optionally placed in front of the TDI detector of the embodiment of FIG. 17 to further suppress out-of-band light.
20A is a graph XY plotting transmittance versus wavelength for a corresponding passband of a filter segment of a detection filter assembly optionally placed in front of a TDI detector. FIG.
FIG. 20B shows another graph XY plotting transmittance versus wavelength for the corresponding passband of the filter segment of the detection filter assembly optionally placed in front of the TDI detector.
FIG. 20C is a graph XY plotting transmittance versus wavelength for a corresponding passband of a filter segment of a detection filter assembly optionally placed in front of a TDI detector.
20D is another graph XY plotting transmittance versus wavelength for a corresponding passband of a filter segment of a detection filter assembly optionally placed in front of a TDI detector. FIG.
FIG. 20E shows another graph XY plotting transmittance versus wavelength for the corresponding passband of the filter segment of the detection filter assembly optionally placed in front of the TDI detector.
FIG. 21 is a plan view of another embodiment of the configuration of FIG. 17 where the spectral dispersion filter system comprises a plurality of dichroic cube filters oriented at various angles to produce a spectral dispersion effect. .
FIG. 22 shows an exemplary set of images projected on a TDI detector when using the spectral dispersion filter system of FIG.
FIG. 23 is a schematic isometric view of yet another embodiment in which spectral decomposition occurs along an axis generally parallel to the direction of movement of the substrate carrying the object.
FIG. 24 is a schematic plan view of a spectral segment detector used for the detection and imaging of light of a plurality of different spectral configurations.
FIG. 25 is an isometric view of an alternative embodiment using a separate TDI detector and a separate imaging lens for each spectrally resolved image.
FIG. 26 is an isometric view of an alternative embodiment using a separate TDI detector and a common imaging lens placed in front of the spectral decomposition element.
FIG. 27 is an isometric view showing a correction plate added to correct astigmatism induced by a plate beam splitter disposed in a convergence space.
FIG. 28 is an isometric view of an alternative embodiment using an individual TDI detector that receives both light transmitted through and reflected from the spectral decomposition element.

Claims (65)

An imaging system adapted to determine one or more characteristics of an object from an image of the object,
(A) a condensing lens arranged to pass light traveling from the object and travel along a condensing optical path;
(B) It is disposed along the condensing optical path, reflects light having a predetermined characteristic, passes light having no predetermined characteristic, and transmits the light having the predetermined characteristic to another light reflecting element. Is reflected at a different angle with respect to the converging light path, and the light reflected by all the light reflecting elements except the first light reflecting element is then at least one. A plurality of light reflecting elements positioned adjacent to the preceding light reflecting element so as to pass through the two preceding light reflecting elements;
(C) is arranged to receive the light reflected by the light reflecting element, generates an output signal representing at least one characteristic of the object, and sets the output signal to a time according to the formed image of the object. An imaging system comprising: a detector that integrates with respect to. The wedge-shaped substrate forms an angular difference between each of the light-reflective elements, and the light-reflective surface of the light-reflective element is sandwiched between the wedge-shaped substrates, thereby forming a monolithic structure. The imaging system of claim 1 , characterized in that: The imaging system according to claim 1 , wherein the light reflecting element reflects light based on a spectral component of light. The imaging system of claim 1 , wherein the light reflecting element reflects light as a function of light polarization characteristics. It said plurality of arranged to receive light reflected by the light reflecting element, the imaging system according to claim 1, characterized in that it comprises an imaging lens for forming the light onto the detector. Imaging system according to claim 1 from each of said plurality of light reflecting elements, the light traveling in different directions, characterized in that incident on different regions of the detector. The imaging system of claim 1 , wherein the detector comprises a time delay integration detector. A filter disposed between the plurality of light reflecting elements and the detector, for attenuating light having no predetermined characteristic of light reflected by at least one of the plurality of light reflecting elements; The imaging system of claim 1 , characterized in that: The filter reflects the light reflected by each of the plurality of light reflecting elements combined with each of the predetermined characteristics, and enters the corresponding spectral region of the filter. A plurality of spectral regions having different ones of the predetermined characteristics, the spectral regions passing light having the predetermined characteristics, and the predetermined characteristics of the spectral regions 9. The imaging system of claim 8 , wherein light that is not present is blocked. The imaging system of claim 1 , wherein the detector comprises a pixelated detector. The imaging system of claim 1 , wherein there is a relative movement between the object. The imaging system according to claim 1 , wherein the plurality of light reflecting elements form a dispersive element. The plurality of light reflecting elements includes a plurality of beam splitters adapted to reflect light having a predetermined spectral characteristic at different predetermined angles, and all light from each of the beam splitters corresponds to an individual light beam. and, imaging system of claim 1, each of the light beam, characterized in that the leaving the spectral dispersion element at different nominal angles. The detector includes a plurality of taps, and a signal representing a different one of a plurality of images received by the detector is output from a corresponding different one of the plurality of taps. The imaging system of claim 1 . The detector has a certain length and width, the width is divided into a plurality of regions, each of the divided regions has a corresponding one of the plurality of taps, and each region 15. The imaging system of claim 14 , wherein when receiving one of the plurality of images, a charge is generated in each region and integrated over the length of the detector. The object to be imaged is flowed into a wide flat flow and the condenser lens is sufficient to collect light traveling from the object in the wide flat flow in the object space. The imaging system of claim 1 , comprising a magnitude viewing angle. A method for determining one or more characteristics of an object based on light from the object, comprising:
(A) focusing the light from the object along a collected light path;
(B) a step of reflecting light having a predetermined characteristic at each of a plurality of consecutive points arranged along the light collection optical path, and a step of allowing light having no predetermined characteristic to pass through And so that different ones of the plurality of different predetermined characteristics are reflected at each successive point in a direction different from the direction at the other points, and at the successive points. The different predetermined characteristics are combined with each of the plurality of points such that light reflected at all points except the first point then passes through at least one preceding point of the successive points. Step and
(C) receiving reflected light with a detector, wherein the detector integrates an output signal with respect to time according to the formed image of the object;
(D) analyzing the output signal of the detector to determine at least one characteristic of the object. Each of the plurality of light reflecting elements coupled to a particular different one of the different characteristics is disposed at each successive point, integrally coupling the plurality of light reflecting elements and the intermediate wedge-shaped substrate; 18. The method of claim 17 , comprising forming a monolithic structure that disperses light in different directions as a function of the different predetermined characteristics coupled with a plurality of light reflecting elements. The method of claim 17 , wherein the step of reflecting light in a different direction comprises reflecting light in a particular direction based on a spectral component of the reflected light. The light traveling along the converging optical path has a plurality of different polarization characteristics, and the step of reflecting the light includes the step of reflecting the light in a specific direction based on the polarization characteristics of the reflected light The method according to claim 17 , wherein: The method according to claim 17 , further comprising the step of imaging light traveling in different directions using a lens. Corresponding to the intensity of light traveling through one of the different directions, and claims, characterized in that it comprises the step of generating a plurality of signals having one of said plurality of different predetermined characteristic 17 The method described in 1. The method of claim 17 , wherein the detecting step comprises providing a time delay integrating detector for detecting reflected light. The step of detecting comprises the step of detecting the intensity of light traveling in one of different directions on each of a plurality of regions spaced apart on the detector. Item 18. The method according to Item 17 . 18. The method of claim 17 , further comprising the step of attenuating light reflected at each of the successive points that do not have the predetermined property coupled to successive points along the light reflected path. Method. The step of attenuating the light comprises the step of filtering so that only light having the predetermined characteristic combined with the successive points reflected by the light is passed toward the detector. 26. The method of claim 25 . The detector includes a plurality of taps and has a certain length and width, and the width is divided into a plurality of regions, and each of the divided regions corresponds to a corresponding one of the plurality of taps. Each region receives light from one of the points, a charge is generated in each region, integrated over the length of the detector, thereby producing an output signal, Analyzing the output signal of the detector comprises analyzing the output signal from at least one of the plurality of taps of the detector to determine at least one characteristic of the object; The method according to claim 17 , wherein: An imaging system adapted to determine, from an image of the object, one or more characteristics of the object flowing into a fluid flow when there is relative movement with respect to the object; There,
(A) a condensing lens arranged to pass light traveling from the object and travel along a condensing optical path;
(B) In order to receive light that has passed through the condenser lens, the light is arranged along the condenser optical path, the light is dispersed into a plurality of individual light beams, and each of the dispersed light beams has a different predetermined direction. A dispersion element guided by
(C) is arranged to receive the light beam from the dispersive element, generates a plurality of images corresponding to each of the light beams, and each of the generated images is projected toward a different predetermined position. An imaging lens
(D) at least one feature of the object arranged to receive the plurality of images generated by the imaging lens and during the relative movement between the object and the imaging system; The output signal is expressed by integrating the light from at least a part of the object with respect to time, and the output signal is generated. An imaging system comprising: a time delay integration detector as formed above. The dispersive element comprises a spectral dispersive element having a plurality of beam splitters adapted to reflect light having a predetermined spectral characteristic at different predetermined angles, and all the light from each of the beam splitters is an individual light. 29. The imaging system of claim 28 , corresponding to a beam, each of the light beams leaving the spectral dispersion element at a different nominal angle. The beam splitter is disposed along a condensing light path adjacent to each other to receive light that has passed through the condensing lens, and is used in all beam splitters except the first beam splitter in the spectral dispersion element. 30. The imaging system of claim 29 , wherein the reflected light is then passed through at least one previous beam splitter. Wedge substrate, defines the angular difference between each of the beam splitter and the beam splitter is interposed between the wedge substrate, according to claim 30, thereby characterized in that the monolithic structure is formed The imaging system described in 1. The beam splitter is disposed along the condensing optical path so as to receive the light that has passed through the condensing lens, and the light reflected by any of the beam splitters then passes through the spectral dispersion element. 30. The imaging system of claim 29 , wherein the imaging systems are separated from each other by a sufficient distance so that they do not pass through any other beam splitter. A band-pass filter assembly disposed between the time-delay integral detector and the imaging lens, the band-pass filter assembly comprising a plurality of adjacent filter segments, each filter segment having the spectral dispersion; Light that is positioned to receive a different light beam from an associated beam splitter in the element and that has a wavelength within a predetermined wavelength band passes through the filter segment, and light having a wavelength other than the wavelength band. 30. The imaging system of claim 29 , having spectral transmission characteristics that are attenuated by the filter segment. 29. The imaging system according to claim 28 , wherein light that has passed through the condenser lens is dispersed in a plane perpendicular to the direction of relative movement between the object and the imaging system. . An image of the object generated by the imaging lens moves between both ends of the time delay integration detector in response to the relative movement between the object and the imaging system; 30. The imaging system of claim 28 . 30. The imaging system of claim 28 , comprising a light source arranged to provide incident light that illuminates the object. The imaging system according to claim 36 , wherein the incident light is scattered by the object to generate scattered light, and at least a part of the scattered light passes through the condenser lens. 37. The imaging system according to claim 36 , wherein the object is guided by the incident light that irradiates the object, and at least a part of the light emitted by the guidance passes through the condenser lens. 37. The light according to claim 36 , wherein at least a part of the incident light is absorbed by the object, so that light passing through the condenser lens does not include a light portion absorbed by the object. Imaging system. 37. The imaging system according to claim 36 , wherein the incident light is reflected by the object toward the condenser lens. The light source is
37. The imaging system of claim 36 , comprising at least one of (a) a coherent light source (b) a non-coherent light source (c) a pulsed light source (d) a continuous light source. 42. The imaging system of claim 41 , wherein the object is flowed into a fluid stream that moves the object past the condenser lens. 42. The imaging system of claim 41 , wherein the object is transported past the condenser lens on a support. 30. The imaging system of claim 28 , wherein the time delay integration detector is responsive to the image of the object by generating a signal that propagates through the time delay integration detector. The propagation speed of the signal through the time-delay integral detector is an image of the object on the time-delay integral detector obtained as a result of the relative movement between the object and the imaging system. 45. The imaging system of claim 44 , wherein the imaging system is synchronized with movement. The propagation speed of the signal through the time-delay integral detector is an image of the object on the time-delay integral detector obtained as a result of the relative movement between the object and the imaging system. 45. The imaging system of claim 44 , wherein the imaging system is not synchronized with movement. An objective lens disposed between the object and the condenser lens and having a focal point on which the object forms an image, and aligned in the direction of relative movement between the object and the imaging system; and A light slit disposed at a focal point of the objective lens between the objective lens and the condenser lens, and the slit condenses light from the object focused on the slit by the objective lens. 29. The imaging system of claim 28 , wherein transmitting the lens substantially prevents external light from reaching the condenser lens.   A light dispersing element having a plurality of light reflecting elements positioned along the optical axis, each of the light reflecting elements reflecting light having a predetermined characteristic and not having the characteristic Each of the light reflecting elements is positioned at a different angle with respect to the optical axis to direct light of the predetermined characteristic in a direction different from the direction in which the other light reflecting elements guide, The range of different angles is from about 44 degrees to about 46 degrees with respect to the optical axis, and each of the light reflecting elements is all light reflecting elements except the first light reflecting element in the light dispersing element. A light dispersion element, wherein the light dispersion element is positioned adjacent to the preceding light reflecting element so that the reflected light then passes through at least one preceding light reflecting element. The wedge-shaped substrate forms an angular difference between each of the light-reflective elements, and the light-reflecting surface of the light-reflective element is sandwiched between the wedge-shaped substrates, thereby forming a monolithic structure. The light dispersing element according to claim 48 , characterized in that: A method for determining one or more characteristics of a moving object from an image of the object when there is a relative movement between the object and an imaging system comprising:
(A) focusing the light from the object along a collected light path in a direction different from the direction of the relative movement between the object and the imaging system;
(B) dispersing the light traveling along the condensed light path into a plurality of light beams;
(C) focusing each of the light beams to produce individual images corresponding to the individual light beams;
(D) providing a time delay integration detector arranged to receive individual images and generating an output signal in response to the formed image of the object;
(E) analyzing the output signal of the time delay integration detector to determine at least one characteristic of the object. The light is spectrally dispersed by a plurality of beam splitters adapted to reflect light of a predetermined spectral characteristic at different defined angles, and all light from each of the beam splitters corresponds to a separate light beam, each of the light beams 51. The method of claim 50 , wherein leaves the spectrally dispersive element at a nominal angle. Multiple beam splitters adapted to reflect light of different polarization characteristics at different predetermined angles distribute the light as a function of the polarization characteristics of the light, and all light of each polarization characteristic is at the nominal angle 51. The method of claim 50 , corresponding to an individual light beam leaving the dispersive element. The image of the object generated by the focusing step moves between both ends of the time delay integration detector while the relative movement occurs between the object and the imaging system. 51. The method of claim 50 . Providing a filter comprising a plurality of different spectral bandpass regions, each of the spectral bandpass regions passing light of a wavelength within a predetermined passband and outside the predetermined bandpass region. 51. The method of claim 50 , wherein the wavelength of light is attenuated. further,
(A) providing a light source;
51. The method of claim 50 , comprising: (b) irradiating the object with incident light from the light source while the object is moving. 56. The method of claim 55 , wherein the incident light is scattered by the object and at least a portion of the light scattered by the object passes through the condenser lens. 56. The method of claim 55 , wherein the object is guided by the incident light that illuminates the object, and the light emitted by the guidance is focused along the collected light path. 56. The light absorbed by the object is not included in the light that is absorbed by the object so that at least a portion of the incident light is thereby converged along the condensed light path. The method described. 56. The method of claim 55 , wherein the light focused along the collected light path is incident light generated by the light source and reflected from the object. 51. The method of claim 50 , comprising the step of flowing the object into a fluid stream that moves the object. 51. The method of claim 50 , comprising transferring an object on a substrate during the step of focusing light from the object along the collected light path. 51. The method of claim 50 , wherein the time delay integration detector responds to the image of the object by generating a signal that propagates through the time delay integration detector. While the image of the object is moving on the time delay integration detector, the movement of the object image on the time delay integration detector is changed to a signal propagation speed via the time delay integration detector. 64. The method of claim 62 , comprising the step of synchronizing. While the image of the object is moving on the time delay integration detector, the movement of the object image on the time delay integration detector is changed to a signal propagation speed via the time delay integration detector. 64. The method of claim 62 , comprising the step of desynchronizing. 51. The method according to claim 50 , further comprising the step of avoiding the arrival of external light to the time delay integration detector by transmitting substantially only light from the object along the condensed light path. The method described in 1.

Images