MXPA01001887A - Novel optical architectures for microvolume laser-scanning cytometers - Google Patents

Abstract

Methods and instrumentation for performing charge coupled device (CCD)-based confocal spectroscopy with a laser spot array are provided. The methods and instruments of the invention are useful in any spectroscopic application, including, but not limited to, microscopy and microvolume laser scanning cytrometry (MLSC).

Description

NEW OPTICAL ARCHITECTURES FOR CITO METERS OF MICROVOLUMEN LASER SQUEEGEE.

BACKGROUND OF THE INVENTION Microvolume laser scanning cytometry (MLSC) is a method to analyze the expression of biological markers in a biological fluid. See example of Patents number: 5,547,849 and 5,556,764; Dietz et al., Cytometry 23: 177-186 (1996); US Provisional Application 60 / 144,798, filed July 21, 1999, each of which is incorporated herein by reference. A sample, such as blood, is incubated in a capillary with one or more probes with phosphorescent labels that specifically bind to biological markers, such as the membrane proteins deployed on the surface of the bead. The sample is then analyzed with an MLSC instrument that scans the excitation light of a laser on the sample along a capillary axis, while it moves on an orthogonal axis in an automated stage. The phosphorescent probes in the sample emit alternating pulses of light in response to the excitation light and this light is collected by the cytometer and used to form an image of the sample. In these images, the cells or particles that are attached to the phosphorescent probes can be identified and quantified by the algorithms of image analysis. The information resulting in the expression of biological markers in the sample can be used for medical diagnostic and prognostic purposes.

The current laser scanning cytometers are based on the confocal flying point laser scanner. Said systems scan a point of laser excitation light in one dimension through the sample using a reciprocal or rotating mirror, such as a mirror mounted on a galvanometer. The sample is moved orthogonal to the scanning direction.

The collimated excitation laser light follows an epi-illumination path through the microscope objective and focuses on the specimen and the scanning center of the mirror is reflected as an image to the entrance pupil of the microscope objective. _ ^ - ^ fl ^ The light emitted from the sample is then gathered by the microscope objective and retracts the path of the excitation light back to the scanning mirror in which it is scanned again. The emitted light passes through a dichroic filter and a long-pass filter to separate the reflected excitation light, and is then focused on an optical detector by means of an aperture, which serves as a spatial filter and reduces the amount of light. Out of focus light that is introduced to the detector. The wider the aperture, the deeper the focus of the system will be. The detector generates a signal that is proportional to the intensity of the incident light. In this way, at the moment in which the laser scans the sample, the image is assembled pixel by pixel. This optical architecture is usually referred to as confocal fluorescence detection.

To detect multiple fluorescence probes, the scanning cytometry system may also include dichroic filters that separate the light emitted at their component wavelengths. Each distinct wavelength is reflected as an image in a separate detector by means of a separate aperture. In this way, an image of the sample is assembled pixel by pixel for each wavelength emitted. Individual images are channels with terms and the final multicolored image is obtained by mixing individual channels.

The use of the 4-channel confocal fluorescence detection for MLSC is illustrated in FIGURE 1 and is described in U.S. Provisional Patent Application 60/144798 entitled "Improved System for Microvolume Laser Scanning Cytometry", presented on July 21, 1999, here incorporated completely for your reference. In this, the light of a laser is scanned on a capillary arrangement 8 in which each contains a sample with species with phosphorescent labels. Specifically, the collimated excitation light is provided by a He-Ne 10 laser. The collimated laser light is reflected by means of a dichroic excitation filter 12. At the moment of reflection, the light is incident on a scanning mirror 14 driven by a galvanometer. The scanning mirror can be swung rapidly over a range of angles fixed by the galvanometer. For example, +/- 2.5 degrees. The scanning mirror reflects the incident light to two repeating lenses (repeater lens 1 _ (16) and repeating lens 2 (18)) that reflect the image of the scanning mirror to the entrance pupil of the objective 20 of the microscope. This optical configuration converts a specific scanned angle in the mirror to a specific field position at the focus of the microscope objective. The angular rubbing is typically selected to result in a scan width of 1 mm at the focus of the lens. The relationship between the scanning angle and the position of the field is essentially linear in this configuration and along this range of angles. In addition, objective 20 of the microscope focuses the incoming collimated beam at a point in the focus plane of the objective. The diameter of the point that establishes the optical resolution is determined by the diameter of the collimated beam and the length of the target. The fluorescence samples located in a capillary array 8 in the path of the excited excitation ray emit lights transferred to the pulsations. This light is gathered by objective 20 and the colima. This collimated light emerges from two repeating lenses (16 and 18) still collimated and collides with the scanning mirror that reflects and re-scans it. The light transferred to the pulses then passes through a dichroic excitation filter 12 (the greater excitation light reflected inside the optic at this point is reflected by this dichroic filter) and then through the long pass filter 1 (22) which it also serves to filter any reflected excitation light. The dichroic fluorescence filter 1 (24) then divides the two bluer colors of fluorescence from the two reddest. Then, the two bluer colors are focused through the focusing lenses 1 (26) to aperture 1 (28) to significantly reduce any out-of-focus fluorescence signal. After passing through the opening, the fluorescence dichroic 2 (30) further separates the individual blue colors from one another, passing these to two photomultipliers 1 (32) and 2 (34). After being divided from the two bluer colors by the fluorescent dichroic 1 (24), the two reddest colors are passed through the long pass filter 2 (36) and mirrored from the mirror (38) then focusing the lens 2 (40) to the aperture 42. After passing through the aperture 2, the reddest colors are separated from one another by means of the fluorescent dichroic 3. The individual red colors then pass to the photomultipliers 3 (46) and 4 ( 48). In this way, four fluorescence signals separated from the sample held in the capillary can be transmitted simultaneously to photomultiplier light detectors (PMT1-4).

^^ --- ^^ Each photomultiplier generates electronic current in response to the flux of the incoming fluorescence photon. These individual currents are converted into voltages separated by one or more preamplifiers in sensing electronics. The voltages are displayed at regular intervals by means of an analog or digital converter to determine the intensity values of the pixel for the scanned image.

Other ways are known in the art of obtaining multichannel information in the context of microscopy. For example, in the art of fluorophores it is known that light emits emission spectra superimposed, but with different time-of-emission constants. Time-resolved microscopy systems typically use very fast laser pulses and speed detection circuits to solve, in the time domain, fluorophore signals of nanosecond scale time. Alternatively, measurement can be achieved in the frequency domain with amplitude modulated laser sources and detection circuits that measure phase translation and modulation amplitudes. Both of these techniques add significant complexity to the phosphorescent measurement system.

Typical MLSC instruments use photomultiplier quotas (PMTs) as light detectors. PMTs are affordable and have high data read rates that allow the sample to be scanned with speed. However, a major drawback of PMT is its low quantum efficiency.

For example, in the red near the infrared region of the electromagnetic spectrum, the PMT have quantum efficiencies of less than 10%, that is, less than one photon in ten that impacts the PMT are actually detected.

To have high sensitivity at high speed of measurement it is desirable to use high power laser sources. For each fluorophore there is a proportional relationship between the intensity of the excitation illumination and the intensity of the emitted light.

This proportionality is only applicable to the point at which the fluorophore is saturated.

- By this time, the fluorophore state is essential to run out and all fluorophores exist in the excited electronic configuration. The laser power increasing beyond the saturation point does not increase the intensity of the light emitted. This effect is especially pronounced with fluorophores having a long fluorescent half-life, such as inorganic fluorophores and for quantum dot nanocrystals. These molecules saturate at densities of low relative power given their long-term fluorescence emission constants. Other unwanted processes may occur at higher laser power, including photo-destruction and cross-systems. In many applications it is desirable to operate at power densities below saturation.

In an attempt to increase the speed with which confocal images can be acquired, microscope systems have been developed so that a continuous laser excitation light is scanned through the sample, rather than at a single point. The line of emitted light produced by the sample in response to the excitation is reflected as an image to a slot-shaped opening. Since light is distributed over a line of pixel speed limitations due to fluorophore, photophysics is avoided. However, the depth of the field, or the change in the lateral resolution with depth of focus of the line scanner is inversely proportional to the numerical aperture of the lens. MLSC applications require a large depth of field to accurately reflect the image of the cells in a deep blood suspension. High sensitivity and speed require a numerical aperture lens, but would result in a prohibitively small depth of field. This exchange ultimately results in limited speed and sensitivity.

Given the limitations with the use of a PMT as a light detector, much research is currently being directed towards the development of high efficiency detectors that will allow the rapid acquisition of images at power densities below saturation. One of said light detectors is the coupled charge device (CCD). See the example, G. Holst, CCD Arrangements, Camera and Deployments, 2nd Edition, JCD Publishing and SPIE Optical Engineering Press 1998.

CCD consists of an interlinked array of sensitive photodetectors, each of which can have a quantum efficiency greater than 80%. Despite their high efficiency, CCDs are not ideal for use in the MLSC context. One reason is that the CCD is usually used as an imaging device in which the entire field of view is excited and the CCD captures all the light emitted in the field of view. Used in this way, the depth and sensitivity of the field are joined in pairs like a line scanner. Moreover, the illumination and collection of the full field means a substantial amount of out-of-focus light excited and received by the CCD detector. Even when a CCD is used in an off-image mode in combination with a scanned laser point and a tiny aperture-which is used in a manner very similar to that of a PMT-additional problems are encountered. First, when PMTs are replaced with CCD for simultaneous multichannel image acquisition, a separate CCD is required for each channel. The cost to provide a high efficiency CCD separately for each channel adds a large amount to the cost of the instrument. Moreover, CCDs take much longer to read than PMTs, so by placing a significant limitation on the speed with which information can be acquired.

The present invention is directed towards optical architectures for spectroscopy that can acquire information with more speed than previous art systems. The instruments of the invention can also be used for measures solved by time or emission of fluorophore. The methods and instruments of the invention are useful for any application in which the spectroscopic information of a sample is required. In preferred embodiments, the methods and instruments of the invention are used for the MLSC.

The invention uses CCDs in which containers are used to subdivide a CCD into groups of pixels that simultaneously gather information from a number of different regions of a sample. Preferred embodiments of an invention use multiple laser excitation points combined with CCD light detectors. In some embodiments, the individual vessels are subdivided further to provide spectral information for each region of the sample. The Pixel intensity values for each container are assembled by computer to give seamless images of the sample on each channel.

Summary of the invention.

Two issues typically limit the performance of the speed and sensitivity of prior art systems. First, PMT detectors, which are commonly used, have low quantum efficiencies, especially in the red to near infrared region of the optical spectrum. Second, these systems typically scan a focused laser spot to excite the phosphorescent emission in the sample. For high sensitivity and speed of measurement, it is desirable to use an excitation source with high power density. However, beyond certain densities of saturating energy (energy per unit area of the laser point), the source of excitation saturates the phosphorescent labels preventing improvements in the speed of measurement and sensitivity.

The preferred embodiment of the present invention uses CCD detectors (instead of PMT) as non-image light detecting devices in a confocal scanning architecture, in which an array of laser spots is scanned through the sample, rather than a dot specific. Two features of this invention solve the limitations described above. First, the high-power laser excitation is divided into multiple points, thus reducing the energy density at each point and minimizing the limitations of laser energy and sensitivity due to fluorophore saturation. Secondly, the CCD is used in a non-image modality, by defining multiple efficient confocal openings as "container" regions of pixels on a two-dimensional surface of the device. Each container region is related to an excitation laser point which is focused on a sample. This architecture retains the controllable depth of field of a confocal point scanner based on PMT, but also takes advantage of the very high quantum efficiencies available with CCD detectors.

L '- * - Brief description of the images.

Note: In all the images, the optical pathways are illustrated schematically. The angles and dimensions are not to scale.

FIGURE 1: Illustrates a single-point multi-channel MLSC system using mechanical confocal openings and photomultipliers. The main beam is traced from each set of rays.

FIGURE 2: Illustrates a two-channel spectroscopy system in which a sample is scanned with three laser excitation points and the resulting emission light is reflected in the CCD light detectors that have been separated into three confocal vessels. The main beam is traced from each set of rays.

FIGURE 3: Illustrates a 512 x 64 pixel CCD detector that separates into three rectangular confocal containers.

FIGURE 4: Illustrates the relative movement of three laser excitation points when scanned on a moving sample.

FIGURE 5: Illustrates an embodiment for performing multi-channel confocal spectroscopy at multiple points using a single CCD and a confocal slot opening. The main beam is traced from each set of rays.

FIGURE 6: Schematically illustrates a 625 x 488 pixel CCD light detector that has been configured for a six-point system with four spectral channels.

FIGURE 7: Illustrates the confocal spectroscopy system resolved by time in which the container CCD gathers the emission light at predetermined times following the excitation of the laser point.

FIGURE 8: Illustrates the time-resolved confocal spectroscopy system in which multiple optical fibers act as confocal openings that are reflected on the sample at predetermined times after excitation of the laser spot.

FIGURE 9: Schematically illustrates a scanning system for non-epi-illuminated points.

Detailed description of the invention.

The present invention provides methods of spectroscopy and instrumentation with a number of new optical configurations that allow multi-channel images to be acquired quickly, and / or with time resolution of individual fluorophore emissions. In the preferred embodiments, the invention uses CCDs as light detectors, in which groups of pixels in the CCD form vessels reflected to a sample when scanning optics. In the preferred embodiments, each container functions as a confocal opening. The size of the container determines the width of the cone of emitted light detected, and therefore, the larger the container the depth of the field will be greater. The light that falls on the CCD outside the container is not detected. In this way, each container functions in the same way as a mechanical confocal opening.

In preferred embodiments, the invention uses a laser dot array to scan the sample simultaneously in multiple locations. The light emitted from each point is reflected in a confocal CCD container and the image is constructed by merging the separate images of each point. In other embodiments a series of containers is reflected to the sample at different times following the excitation so that each container in the CCD represents a different emission time, therefore, allowing time resolved fluorescence measurements to be made.

The advantage of using CCD containers as confocal openings is that the reconfiguration of the instrument is done simply by changing the size and position of the containers to the CCD, rather than by any mechanical operation. The advantage of using multiple points is that lower laser energy is present at each point compared to a single point system. The energy density of the low laser minimizes the saturation of fluorophore, however, the total performance of the system is maintained by simultaneously detecting the emission from multiple excitation points.

Note that in all the additions that follow, the methods and instrumentation of the instant invention are described in the MLSC context. However, those skilled in the art will understand that the methods and instruments of the invention are useful in any spectroscopy or cytometry application, including applications in microscopy. In addition, although a fluorescence reflection application is described, this scanning system can be applied to many modes of light scattering detection. Examples of such possible modalities include: luminescence, phosphorescence, Raman scattering, Ralyeigh scattering and Mié scattering.

Multiple laser point excitation system with container CCD light detection. In an embodiment of the invention, a laser excitation beam is converted into a ray array by a device, such as a Damann network. Alternatively, other point generation devices can be used, such as a microlens array or a fiber group. The system is not limited to the use of a linear array of points and can work with a two-dimensional array of points, which can allow a larger area of the CCD detector to be used and thus increase the reading performance. Other embodiments of the device may not use a single excitation source of the laser beam, but a laser diode array, therefore, omitting the requirement of a lightning generating device. The lightning source is reflected with repeating lenses to the center of the device scanning in a mirror, such as the galvanometer. Other embodiments of the present device include the use of other beam refraction methods.

Such other methods include, but are not limited to, a piezoelectric scanner, polygonal mirrors, acousto-optic reflectors, and holograms. In the present embodiment, the scanning center of the galvanometer is in turn reflected by a second set of repeating lenses to the entrance pupil of a microscope objective. The arrangement of the laser beam is thus focused by means of the objective to the arrangement of points in the focal plane of the objective. At each point in the array, a separate region of a sample is scanned and excited, which moves at a constant speed along the axis orthogonal to the scan axis of the laser dot. The phosphorescent light emitted by the sample is gathered by the objective lens, passes through the dichroic filter and is reflected to the surface of a CCD detector that has been electronically divided into a container of pixels of the desired size. The emitted light that breaches the CCD outside the container is not detected; in this way, the container functions as a confocal opening in which the depth of confocal focus can be controlled only by changing the dimensions of the container. The larger the area of the container, the greater the depth of focus.

The multipoint system can be used for multichannel images by dividing the fluorescent emission into individual wavelengths - using, for example, dichroic filters - and then directing each component wavelength in a separate CCD detector. In this embodiment, a different CCD is used for each channel, however, the container configuration for each CCD is identical. CCDs have a periodic reading in sync to maintain the proper record of individual color images. In addition, this incorporation provides a multi-channel MLSC instrument in which the confocal focus depth is controlled by computer (by changing the dimensions of the container), rather than mechanically controlled (by changing the size of the confocal aperture).

FIGURE 2: Illustrates an embodiment of the invention in which three laser excitation points are generated, then reflect the excitation dichroic to a mirror of the galvanometer. (In this figure three points are selected to illustrate the invention for convenience, as described below, the present invention includes any system with more than one point). Specifically, excitation light 11 provided by a laser 50 is passed through a laser line filter 52 and an optical multi-ray generating device 54 generating three laser excitation points. The three laser excitation points are then passed through a lens 56 and an excitation dichroic 58 and are reflected by a galvanometric scanning mirror (60). The scanning points then pass through the repeating lenses 62 to the entrance pupil of the objective 64 of the microscope and focus on the sample (scanned on the page). The emission light of the sample (three emission rays) is collected by the objective 64 of the microscope, and retraces the path of the excitation black points to the dichroic filter 58. The emission light passes through the excitation dichroic 58, a focusing lens 68 and a long pass filter 70 and separated into two wavelength portions by means of an emission dichroic filter 72. The two portions are then reflected by means of a focusing lens to separate CCDs (CCD1 (74) and CCD2 (76)), while each CCD is separated into three confocal containers 78 and 80. A schematic illustration of a CCD detector of 512 x 64 pixels that is separated into three rectangular confocal containers can be seen in FIGURE 3.

In preferred embodiments, the linear arrangement of the laser points 82 drives the emission of the sample 66. The device of the scanning mirror 60 rotates by scanning the laser points in the direction that is parallel to the line defined by the array of points. The total scan refraction of each point is selected such that each point scans through a sample length of approximately the same length as the distance between the points. The sample is moved along the axis orthogonal to the scan axis 84 of the laser spot. This is illustrated schematically in FIGURE 4A, in which a scan of three points on the vertical axis moves horizontally. In this way, the movement of the relative point tracks a sawed pattern (like the teeth of a saw) 86. When scanning the arrangement of the point through the sample by means of the scanning mirror device, the CCD vessels have a periodic reading . The time of this reading operation . I- determines the pixel size in the scan dimension. Alternatively (FIGURE 4B), the three points 82 are arranged orthogonal to the movement 84 of the sample. The points are scanned (86) through the entire sample 66. When a point reaches the scanning position of the next point, the sample is launched so that the first point begins to scan back into the previous stop position of the last point.

As the scan proceeds, each laser point generates a two-dimensional image that is acquired by means of the corresponding CCD detector vessel. The images of each point, either do not overlap, or overlap only a little. When the scan is complete, the individual images can be linked by means of a computer to provide a seamless image.

Any number of points can be used in the instant invention. The advantage of using multiple points is that the lowest laser density is applied to each point, minimizing fluorophore saturation. For example, each point in a ten point array has l / 10tu of the laser point's power density a single-point system using equal energy sources.

Moreover, each point inhabits ten times more in any region of the sample than the single point system, so that the total flow of the photon per pixel of the image is the same. Since all ten points are excited and detected in parallel, the total scan time for a sample is the same in any case. However, when using CCDs as light detectors, there are significant increases in sensitivity. A CCD can have at least three times more quantum efficiency than a PMT; In addition, with the use of the lower excitation energy density, sensitivity can be significantly increased because it avoids saturation, photodestruction, and other non-linear excitation processes.

Thus, the multipoint system with CCD detection can be between six and ten times more sensitive than a single point PMT system operating at the same scanning speed. This increased sensitivity can be exploited to shorten the time of scanning, so that a multipoint system can produce images comparable to a single point system, but in much less time. Alternatively, the multipoint CCD system can operate at the same scanning scale as the single-point system, but it will be able to detect the emission intensities that a conventional single point PMT system can not detect at that scanning scale. An even greater advantage of using densities at low laser energy is that they allow the use of fluorophores that saturate at comparably low energy densities, such as inorganic fluorophores or quantum dot nanocrystals with constants of long emission times.

The limit on the number of points is determined by means of the required axial response of the system and the amount of interference between the points that can be tolerated. The more points there are in the array, the closer the confocal vessels will be in the CCD. The axial response is proportional to the size of the container, so that it will be reduced effectively. In addition, the closer the containers are, the more the rejected light from one vessel will be vulnerable to the other reducing the sensitivity of the system. The present invention includes any system with more than one point. Preferably there will be between two and a hundred points and optimum would be between five and 400.

The use of CCD vessels as confocal openings is a substantial advance due to the relative simplicity of the change in dimensions of the confocal depth of the field. The mechanical confocal apertures in focused light systems are typically only several J. m in diameter, and thus require precise and sophisticated mechanical systems to control the size of the aperture.

In contrast a CCD container can be changed simply by reconfiguring the CCD array using a computer. The use of a CCD in MLSC is a major improvement over previous MLSC instruments that use PMT for light detection. Biological fluids assayed by MLSC autofluorescence can absorb or disperse excitation light. The use of fluorophores that are excited by red light (such as the 633nm line of a HeNe laser) reduces these problems and allows MLSC assays to be performed on the whole blood. Do not However, PMTs have a quantum efficiency of less than 10% in the red portion of the electromagnetic spectrum, leading to poor sensitivity when these fluorophores are used. In contrast, many types of CCD have quantum efficiencies of more than 80% in the same region of the spectrum.

As mentioned previously, the current CCD detectors have a lower reading capacity than the PMT. If a single point is scanned in a sample and the emitted light reflected to the CCD, the latter would have to read in sequence each of the pixels in the image.

In contrast, the multipoint system provided by the instant invention can simultaneously acquire information for X number of pixels, where X is the number of points. Thus, a ten point system provides information for ten pixels in the image in each reading event, while a single point system provides information for a single pixel in each reading event. In addition, the multipoint system of the instant invention allows for the benefits of increased sensitivity provided by the CCD to be achieved while minimizing the problassociated with the low level of information reading of the CCDs, which with higher levels of reading are feasible to be available in the future.

The use of said improved CCDs will further increase the sensitivity and speed of the instant invention instruments and methods.

Note that all embodiments of the present invention can use conventional PMTs as light detectors, rather than CCDs. In these embodiments, conventional confocal openings require a required rejection of out-of-focus fluorescence. The openings would be arranged in a pattern identical to the laser dot arrangement.

Multiple laser point excitation system with spectral analysis in a single CCD.

In certain embodiments, a single CCD is used to reflect multichannel images of a sample. In these additions, a line of laser points is scanned on the sample and the emitted light is reflected as described in the lines preceding the line of confocal openings. The design of the aperture arrangement equals the array distribution of the laser points, so that the light emitted from each laser point passes through a different aperture. After passing through said opening, each ray of light emitted can pass through scattering optics to distribute the component wavelengths. Dispersing wavelength optics include, but are not limited to, echelle networks, holographic concave networks, transmission networks, or prisms. In addition, the particular wavelengths can be routed mechanically to the detector with the use of a constant deflection dispersion prism, such as a Pellin-Broca prism, or through the use of a resonant filter. Electro-optical methods for selecting wavelengths include the use of an acoustic-optic tunable filter or an electro-optic resonant network filter. Dispersion optics also ...

In a preferred system for this incorporation, the sample is scanned with a laser dotted line and all the light emitted from the sample is reflected in a single confocal aperture of the rectangular "groove" that is parallel to the laser dotted line. The light emitted later passes to scattering optics that spectrally distribute each ray of light emitted to its component wavelengths. The dispersion axis is orthogonal to the long axis of the confocal "slot".

Dispersion optics also reflect the wavelengths of each emission point to a separate rectangular CCD region, while the long axis of each CCD rectangle is parallel to the axis by which the scattering optics distributes the emission point.

Accordingly, the long axis of each CCD rectangle is orthogonal to the long axis of the "slot" opening. Each rectangular region is subdivided by its long axis to the spectral vessels, in such a way that each spectral vessel gathers a wavelength of light resulting from the dispersion of a single emission point. The Ij ^^ rectangular regions of the CCD function as a second "groove" confocal opening oriented in dimension opposite the mechanical "groove" opening. As a result, the final image is confocal in both dimensions. The depth of focus in this system can be controlled by varying in a coordinated manner the width of the opening of the mechanical "groove" and the width of each CCD rectangle.

FIGURE 5: Schematically illustrates an embodiment using a slot opening and a concave network for wavelength dispersion. In this illustration, the laser light generated by the laser 50 is passed through the line filter of the 52 and reflected in the optical device 54 generating points by mirror 1 (88). The three laser points are generated by the optical device generating points, passed through the excitation repeater lenses 1 (90) and 2 (92) to a dichroic filter (94), which reflects the laser points to a galvanometer mirror. scan (60). The points then pass through the repeating galvo lenses 1 (96) and 2 (98) to the mirror 2 (100), so that there is convergence in the entrance pupil of the objective 64 of the microscope. The objective focuses the points on the sample 102 and the light emitted from three points is gathered by the objective 64. The rays of emission light redirect the path of the laser points to the dichroic filter 94. The emission light passes through the dichroic , followed by aperture lenses 104 that focus light at the opening in the slot. The focused light passes through a long pass filter 106 and then through a confocal slot opening 108 which is oriented with the long axis of the slot parallel to the vertical axis. The emission light beams pass through the slot opening and then fall into a concave network 110 which again reflects the dot image and wavelengths to the detector 112. In this illustration, each beam of emission light is scattered in multiple wavelengths along the horizontal axis.

Thus, the horizontal axis of the CCD provides a spectrum 114 of information about each light beam and the vertical axis provides spatial information 116. The direction of the scanning of the points is represented by a double arrow 118 and the direction of the scanning of the sample it is represented by a single arrow 120.

FIGURE 6: Schematically illustrates the 625 x 488 pixel CCD light detector that has been configured for a six-point system with four spectral channels. The CCD is divided into six rectangular regions and each region is subdivided into four spectral vessels. As in FIGURE 5, the horizontal axis of this CCD provides spectral information and the vertical axis provides spatial information when used in combination with a vertically oriented confocal slot opening. The height of each rectangle in the FIGURE 6 can be adjusted to vary the confocality of the image on the horizontal axis. The Pixelvision PlutoTM CCD is a suitable CCD for this application.

As described above, previous attempts to use lighting in MLSC have not produced the desired speed and sensitivity improvement because of the interaction of the numerical aperture collection and the depth of the field system. The instant invention provides for the first time a method to distribute the excitation light in an area larger than a single point while maintaining the depth of field and the axial response of a single confocal flying point laser scanner, which is achieved : a) by scanning the sample with individual points of excitation light instead of a continuous line of light; and b) using confocal openings of the CCD container, with each container corresponding to an excitation point.

This incorporation is also mechanically simpler than the previous incorporation in which the line of the tiny hole openings is used. Changing the size and orientation of a single slot is much simpler than changing a series of smaller tiny hole openings in a comparative manner. In this way, this incorporation retains the real two-dimensional confocality of the architecture of the tiny hole opening, while also possessing the mechanical simplicity of the confocal slot architecture.

An additional advantage of performing spectral analysis on the CCD in the manner stated above is that the CCD vessels in the spectral dimension can be controlled electronically. Each detection color can be optimized for the specific test to be performed. This also eliminates the need in the detection system for many dichroic filters that can vary from unit to unit and with temperature and humidity.

An even greater advantage of the use of the aforementioned CCD systems is that the alignment of the confocal mechanical openings with the CCD can be achieved electronically with ease. Instead of changing the openings physically, the position of the containers in the CCD can be adjusted to ensure that each container is in perfect register with its corresponding opening. This process can be automated using, for example, a test slide containing all the fluorescent labels that will be used in the assay.

In more embodiments of this invention it is possible to use non-epilumination of the sample. In this inlay illustrated in FIGURE 9, the excitation light generated by the laser 50 approaches the sample without passing through the collection target. This method is referred to as off-axis illumination. See example, U.S. Patent Number 5,578,832. As with epi-lighting, a laser exciter is generated. Possible multiple-ray generation optics 122 include, but are not limited to, Damann and other networks, fiber optic groups, micromirror arrays, and acousto-optic and electro-optic devices. The excitation of rays is collected by the first exciter lenses 124 and reconverted in a scanning device, such as the reciprocating mirror 126 with a second exciter lens 128. The rays subsequently pass through a scanning mirror 130 and a final lens. 132 in such a way that each point 82 focuses on the sample 102 (as illustrated in the figure the sample is scanned off the page). The final lens is configured in such a way that the excitation rays are approximately 45 degrees from the sample.

The emitted light is then collected by the collection objective 134 and reflected back to the CCD detector with a tube lens. The unwanted excitation light is removed with a long-pass blocking filter 136. The emitted light is separated into two portions of wavelength by means of a dichromic filter. The two portions are then passed through a lens (tube lens 1 (140) and tube lens 2 (142) to separate array detectors (array detector 1 (144) and array detector 2 (146)). If the points 148 are not de-scanned, then the scanned points will be moved to the detector, however, the correct CCD vessels can be selected so that the reading follows the points.Alternatively, the assembled rays can pass through a second device. reciprocal, such as a mirror in sync with the excitation mirror to scan the dots.A number of different array detectors can be used to gather discrete wavelengths in which the wavelengths are selected by optical filters, by For example, a dichroic filter, just as in epilumination, a dispersive element can be used to spread the wavelengths on the orthogonal of the array detector to the points.

Despite the extra complications involved in scanning points, outside axis has some advantages over epilumination. No dichroicp excitation filter is required and many different excitation wavelengths can be used without having to change the filters.

In addition, extreme excitation, i.e., ultraviolet or infrared wavelengths that are incompatible with the collection of optics can be used. The examples of the reporters that use these wavelengths are: two photons that become matches that use excitation of 980nm and emit in the visible region; and the nanocrystals that are optically excited in the UV, < 400nm, but emit in visible regions. For example, see, Bruchez et al., Science 281: 2013 (1998); Wright, W. H. et al., Procedures of SPIE - La International Society for Ultrasensitive Biochemical Diagnostics of Optical Engineering II Feb 10-Feb 12 97 v 2985 1997 San José, CA, USA All embodiments of the invention described above include system designs in which the regions of the sample illuminated by the array of the point are reflected by means of an optical system to the surface of a CCD detector. Additions Alternate systems use mechanical openings to provide the necessary confocal openings, with optical systems without image reflection, such as fiber optics or fiber groups to transfer light passed through the openings to the surface of the detector. In this way, the system designer can select the optimal spatial mappings between the excitation point arrangement and the detector surface, which allows the designer to select from a broader range of devices for the detector, or to use the detector more effectively. surface area of the device. For example, if the excitation point arrangement consists of a linear array of points, a linear array of apertures could be used to gather the light emitted from a sample that passes light to optical fibers with a fiber or group of fibers per aperture.

The outputs of multiple fibers or groups of fibers could be arranged, for example, in a two-dimensional square-shaped arrangement and project their image directly or by means of scattering optics to the surface of the detector. In this way the designer can provide a better match of the shape factor of a given CCD device, or fill the surface of a CCD with more points that can be used only with image optics.

Spectroscopy systems solved with time. The methods of the present invention can also be used to perform the MLSC resolved by time. As described above, in fluorescence microscopy resolved by time, the images of the sample are taken at pre-determined time intervals after the excitation illumination, which allows the observation of multiple fluorophores with different time-of-emission constants. . In the present invention, time resolved images can be obtained by reflecting images of a series of confocal CCD container openings on the sample at predetermined times behind the laser excitation point (s). The confocal containers are scanned on the sample by the same optical system that scans the laser point. The physical separation between the CCD vessels maps in the difference at the time of emission for the sample. The time separation is a function of the scanning speed of the sample.

Said system can operate as indicated below in an embodiment illustrated schematically in FIGURE 7. Specifically, the excitation light 11 provided by a laser 50, is passed through a dichroic filter 148 and reflected by the mirror 150 and a galvanometric scanning mirror 152 at the entrance of the pupil of objective 154 of a microscope. At t = 10, the laser spot excites a first region of sample 156 in which fluorophore Fl (158) and F2 (160) are present. Fl has a constant emission time in such a way that the fluorescence emitted from Fl is practically coincident with the excitation. Moreover, the light 161 of Fl retracts the path of the laser beam, passes through a dichroic filter 148 and is reflected in a confocal CCD vessel Bl (162). At t = 1, the scanning mirror has reflected and a second region of the sample receives the excitation illumination 164. Also at t = l, F2 that was excited in the first region of the sample at t = 10 emits. Since the scanning mirror a reflected between t = 10 and t = l, the light path followed by the emission F2 is not coincident with the path of the laser. As a result, the fluorescence of F2 follows a path that is misplaced by a relative amount previously determined to the fluorescence of Fl. The light of F2 could collect a separate confocal container, B2 (168) in the CCD. It will be understood that additional containers can be defined in the CCD to obtain additional time intervals. The distance between the containers (at constant scanning speed) determines the time interval between each fluorescence measurement event.

In other embodiments, a multipoint excitation arrangement is used as described above for time resolved measurements. In this embodiment, the CCD is divided into a container matrix. In one dimension, the vessels provide positional information for the light emitted. In the other dimension, the vessels provide fluorescence measurements resolved by time. For example, a system for performing three time-resolved measurements for each ten laser excitation points would comprise a 10x3 array of CCD vessels.

The dimensions of each container can vary independently to optimize the detection of particular fluorophores.

In other embodiments, time resolved spectroscopy is solved using multiple confocal openings implemented with optical fiber 184 operatively woven to the light detection devices 186. The space between the confocal openings determines the time intervals for the fluorescence measurement events. As in the incorporations that include CCD containers, the individual tiny confocal openings are scanned over a particular region of the sample at various times after the excitation of said region. As an alternative to tiny apertures, this incorporation can be done using optical fibers. The center of each fiber acts as a tiny hole, and the light transmitted through fibers is delivered in multiple PMTs. As before, the space between the illuminated end of the optical fibers determines the time interval between each measurement of fluorescence. This embodiment is illustrated schematically in FIGURE 8. The excitation light 11, provided by the laser 50 is passed through a dicrotic filter 170, reflected out of the scanning mirror 172 through repeating lenses 174 toward a target lens. 176 and focused on the sample 178. The emission light is reflected by a mirror 180, passes through focusing lenses 182 and then through the multi-orifice confocal apertures 184 toward PMT detectors 186.

In even greater incorporations, fluorescence spectroscopy resolved by time is performed one after the other with spectroscopy of the wavelength of the light emitted in each time interval.

In certain embodiments this is done using a series of tiny openings that are reflected on the sample as described above at different times behind a single laser excitation point.

The emission light that passes through each aperture then passes through the scattering optics - such as in a concave diffraction grating or a prism - which establishes the velocity of the light emitted at its component wavelengths, which from each time interval are reflected by means of scattering optics to a rectangular region separated from a CCD, in which the long axis of the rectangle is parallel to the axis the one that scatters the emitted light.

Each tiny aperture is mapped to a rectangular region separately to the CCD. The individual rectangles are subdivided on their long axes into the spectral vessels and the scattered light of each aperture falls into said vessels at a wavelength-dependently.

In even larger embodiments, the invention provides a method for performing multipoint fluorescence spectroscopy time-resolved one after the other with wavelength spectroscopy of the fluorescence emission. This system works in the same way as the multipoint system with a confocal slot architecture, as described above. However, in order to carry out the measures resolved by time, the system has a series of slot openings that are reflected on a sample at intervals of time behind the excitation points. Light passing through each aperture is dispersed in its component wavelengths and reflected to spectral vessels arranged in rectangles to a CCD, in which the rectangles are oriented orthogonally to the confocal slot.

In certain incrustations, separate CCDs are used to reflect the light that comes through each confocal opening, moreover, a three-slot configuration would require three CCDs. In other embodiments, a single CCD is used to reflect light from the rest of the openings. The CCD in this embodiment would be configured to provide the matrix of rectangles; each opening is reflected to a particular row (or column) of rectangles on the CCD. Thus, a CCD provides the information in the position, the resolution by time and the spectrum of each event of fluorescence emission.

Claims (8)

1. Claims 1. A confocal scanning system comprising: (a) means for generating and scanning on a sample a plurality of excitation light points, by which the emission light is released by said sample in response to the excitation by each of the mentioned points; (b) one or more detection devices positioned so that the emission light is reflected simultaneously in them.
2. 2. A confocal scanning system of claim 1 in which 3-100 points are generated.
3. 3. A confocal scanning system of claim 1 in which 5-25 points are generated.
4. 4. A confocal scanning system of claim 1 wherein said emission light comprises components of multiple wavelengths.
5. 5. The confocal scanning system of claim 4 wherein each of said component wavelengths is reflected in a separate detection device by one or more dichroic filters.
6. 6. The confocal scanning system of claim 1 wherein said sample comprises a blood sample contained within a capillary.
7. 7. The confocal scanning system of claim 6 wherein said capillary is translated orthogonal to the scanning of the excitation light.
8. 8. The confocal scanning system of claim 6 wherein the points of the excitation light are scanned in a direction parallel to the axis of said capillary. ^^ jffc ^ á The confocal scanning system of claim 6 wherein the points of the excitation light are scanned in a direction perpendicular to the axis of said capillary. The confocal scanning system of claim 1 wherein said one or more detection devices are CCD coupled charging devices. The confocal scanning system of claim 10 in which each CCD is separated into multiple containers so that each of the containers gathers a portion of the emission light resulting from the excitation of said sample by one of the mentioned points. The confocal scanning system of claim 11 wherein the dimensions of each of said containers are selected such that only the emission light from within a previously determined focal depth of said sample is collected. The confocal scanning system of claim 4 further comprising means for dispersing emission light at its component wavelengths. The scanning system of claim 13 wherein said means for scattering emission light at its component wavelengths is a dichroic filter. The confocal scanning system of claim 13 wherein said means for dispersing the emission light at its component wavelengths is a prism. The confocal scanning system of claim 13 wherein said means for scattering emission light in its component wavelengths is a network. The confocal scanning system of claim 13 in which a detection device is employed. The confocal scanning system of claim 17 wherein said detection device is a CCD. The confocal scanning system of claim 1 further comprising a microscope objective. The confocal scanning system of claim 19 wherein said excitation light and said emission light passes through said microscope. The confocal scanning system of claim 1 for further understanding optical fibers for transferring emission light to the one or more detection devices. The confocal scanning system of claim 13 of greater understanding with an aperture corresponding to each emission light spot so that said light within a previously determined focal depth of said sample passes through the detection device. The confocal scanning system of claim 11 of greater understanding with a slot opening corresponding to each emission light spot so that it within a previously determined focal depth of said sample passes through the detection device. A confocal scanning system comprising; (a) means for generating and scanning a sample on the axis a plurality of excitation light points; whereby the emission light is released by said sample in response to the excitation of said points; (b) a spatial filter comprised of a rectangular aperture, said spatial filter positioned such that said emission light is reflected therein; (c) means for dispersing the emission light passing through said spatial filter at its component wavelengths; and (d) a CCD, which is positioned in such a way that the component wavelengths are reflected in it; wherein said CCDs divide into rectangular pixel regions orthogonal to said spatial filter, such that each rectangular region receives a portion of emission light resulting from the excitation of said sample by one of said points; wherein each rectangular region is subdivided along its long axis into at least two spectral vessels, so that each spectral vessel meets different component wavelengths of said portion; and wherein the dimensions of each spectral vessel are selected such that said portion comprises the emission light of a previously determined focal depth of said sample. system for confocal scanning resolved by time comprising: (a) means for generating and scanning in a sample along an axis one or more points limited by the refraction of excitation light; in which the emission light is released by said sample in response to the excitation of said points; (b) a plurality of confocal openings, each of which is positioned so that the emission light resulting from the excitation of said sample by one of said points is reflected in this by means of said scanning means in a predetermined time following the excitation by said point; (c) means for detecting light operatively coupled to said confocal openings. 26. The system of claim 25, wherein said means for detecting light is a CCD, and wherein said confocal openings comprise pixel containers in said CCD. 27. A confocal scanning system comprising: - an excitation energy delivery system for directing energy to a first region of a sample; - an optical detection system for measuring the fluorescence of the sample induced by energy; - a spatial filter arranged optically between the sample and the optical detection system to limit the fluorescence to at least a second region of the sample; and - a mechanism coupling the energy delivery system and the spatial filter to the sample so that the first and second regions sequentially scan the sample. 28. The system of claim 27 in which the mechanism scans the first and second regions at a speed with a distance therein included so that the fluorescence is measured at a predetermined time after the energy. 29. The system of claim 27 further comprising a plurality of spatial filters comprising tiny apertures aligned so that the fluorescence is measured at a plurality of times after energy. The system of claim 27 in which the energy comprises laser energy.