a) Field of the Invention
The invention is directed to a method in microscopy, particularly fluorescence microscopy, laser scanning microscopy, fluorescence correlation microscopy and scanning nearfield microscopy, for examining primarily biological specimens, preparations and associated components. This includes methods for screening active ingredients (high throughput screening) based on fluorescence detection. Simultaneous examinations of specimens with multiple fluorophores in real time by means of simultaneous illumination of the specimen at a plurality of points on the specimen are therefore possible with overlapping fluorescence spectra even in three-dimensional structures of thick specimens.
b) Description of the Prior Art
A typical area of application of light microscopy for the examination of biological preparations is fluorescence microscopy (Pawley, “Handbook of Biological Confocal Microscopy”; Plenum Press 1995). For this purpose, determined dyes are used for specific labeling of cell parts.
The irradiated photons having a determined energy excite the dye molecules, through the absorption of a photon, from the ground state to an excited state. This excitation is usually referred to as single-photon absorption (
The fluorescent light is split off from the excitation radiation by suitable dichroic beam splitters in combination with blocking filters and is observed separately. This makes it possible to show individual cell parts that are dyed with different dyes. In principle, however, multiple parts of a preparation can also be dyed simultaneously with different dyes which bind in a specific manner (multiple fluorescence). Special dichroic beam splitters are used again to distinguish the fluorescence signals emitted by the individual dyes.
In addition to excitation of dye molecules with a high-energy photon (single-photon absorption), excitation with a plurality of lower-energy photons is also possible (
The prior art will be explained more fully in the following by way of example with reference to a confocal laser scanning microscope (LSM) (
An LSM is essentially composed of four modules: light source, scan module, detection unit, and microscope. These modules are described more fully in the following. In addition, reference is had to DE19702753A1.
Lasers with different wavelengths are used in an LSM for specific excitation of different dyes in a preparation. The choice of excitation wavelength is governed by the absorption characteristics of the dyes to be examined. The excitation radiation is generated in the light source module. Various lasers (argon, argon/krypton, Ti:Sapphire lasers) are used for this purpose. Further, the selection of wavelengths and the adjustment of the intensity of the required excitation wavelength is carried out in the light source module, e.g., using an acousto-optic crystal. The laser radiation subsequently reaches the scan module via a fiber or a suitable mirror arrangement.
The laser radiation generated in the light source is focused in the preparation in a diffraction-limited manner by the objective through the scanner, scan optics and tube lens. The focus is moved in two dimensions in x-y direction over the specimen. The pixel dwell times when scanning over the specimen are mostly in the range of less than one microsecond to several seconds.
In confocal detection (descanned detection) of fluorescent light, the light emitted from the focal plane (specimen) and from the planes located above and below the latter reaches a dichroic beam splitter (MDB) via the scanner. This dichroic beam splitter separates the fluorescent light from the excitation light. The fluorescent light is subsequently focused on a diaphragm (confocal diaphragm/pinhole) located precisely in a plane conjugate to the focal plane. In this way, fluorescent light components outside of the focus are suppressed. The optical resolution of the microscope can be adjusted by varying the size of the diaphragm. Another dichroic blocking filter (EF) which again suppresses the excitation radiation is located behind the diaphragm. After passing the blocking filter, the fluorescent light is measured by a point detector (PMT).
When using multiphoton absorption, the excitation of the dye fluorescence is carried out in a small volume in which the excitation intensity is particularly high. This area is only negligibly larger than the detected area when using a confocal arrangement. Accordingly, a confocal diaphragm can be dispensed with and detection can be carried out directly following the objective (nondescanned detection).
In another arrangement for detecting a dye fluorescence excited by multiphoton absorption, descanned detection is carried out again, but this time the pupil of the objective is imaged in the detection unit (nonconfocal descanned detection).
From a three-dimensionally illuminated image, only the plane (optical section) coinciding with the focal plane of the objective is reproduced by both detection arrangements in connection with corresponding single-photon absorption or multiphoton absorption. By recording a plurality of optical sections in the x-y plane at different depths z of the specimen, a three-dimensional image of the specimen can be generated subsequently in computer-assisted manner.
Accordingly, the LSM is suitable for the examination of thick preparations. The excitation wavelengths are determined by the utilized dye with its specific absorption characteristics. Dichroic filters adapted to the emission characteristics of the dye ensure that only the fluorescent light emitted by the respective dye will be measured by the point detector.
Currently, in biomedical applications, a number of different cell regions are labeled simultaneously by different dyes (multifluorescence). In the prior art, the individual dyes can be detected separately based on different absorption characteristics or emission characteristics (spectra). For separate detection, an additional splitting of the fluorescent light of a plurality of dyes is carried out with the secondary beam splitters (DBS) and a separate detection of the individual dye emissions is carried out in various point detectors (PMT x).
Flow cytometers are used for examining and classifying cells and other particles. For this purpose, the cells are dissolved in a liquid and are pumped through a capillary. In order to examine the cells, a laser beam is focused in the capillary from the side. The cells are dyed with different dyes or fluorescing biomolecules. The excited fluorescent light and the backscattered excitation light are measured. The separation of the fluorescence signal of the specimen from the excitation light is carried out by means of dichroic beam splitters (MDB, see
The size of the cells can be determined from the backscattered signal. Different cells can be separated and/or sorted or counted separately by means of the spectral characteristics of the fluorescence of individual cells. The sorting of the cells is carried out with an electrostatic field in different capillaries. The results, that is, for example, the quantity of cells with dye A in comparison to cells with dye B, are often displayed in histograms. The through-flow rate is typically about 10-100 cm/s. Therefore, a highly sensitive detection is necessary. According to the prior art, a confocal detection is carried out in order to limit the detection volume.
According to the prior art, line scanners, as they are called, are also used instead of point scanners (Corle, Kino, “Confocal Scanning Optical Microscopy and Related Imaging Systems”, Academic Press 1996). The basic construction essentially corresponds to that of an LSM according to
In another arrangement for real time microscopy according to the prior art, the entire field to be examined is illuminated by an expanded light source. However, only special point patterns of the total field to be scanned are uncovered by a rapidly rotating disk. These methods are mostly known in technical literature as Nipkow disk methods (Corle, Kino, “Confocal Scanning, Optical Microscopy and Related Imaging Systems”, Academic Press 1996).
In another method according to the prior art, known as structured illumination (see
where I(x, angle) describes the intensity at the respective pixel in the corresponding phase image.
It is simplest to carry out the recording of three or more phase images sequentially. In this connection, it is assumed that the specimen is not moved during the measurement of the images. The section images or section stacks which are calculated from the phase images in this way can be displayed subsequently on a standard PC and monitor by means of 3-D evaluating software. The spatial resolution along the optical axis depends on the wavelength of the light, the numerical aperture of the objective and the modulation frequency.
For a detailed description of the calculation algorithm, reference is had to T. Wilson, et al., “Method of obtaining sectioning by using structured light in a conventional microscope”, Optics Letters 22 (24), 1997.
Arrangements for screening dyes, for example, in so-called chip readers are similar in optical construction to laser scanning microscopes. However, they scan an appreciably larger image field for the investigation of macroscopic specimens, for example, screening of active ingredients on a biochip. The edge length of the scan fields amounts to about 10 mm. These scan fields can be achieved, e.g., by increasing the scan angle of the galvoscanner, by arranging the specimen in an intermediate image of the microscope arrangement or by a special objective arrangement (macroobjective) which images the intermediate image on the specimen in magnified manner.
According to the prior art, the separation of the excitation light from the light emitted by the specimen is carried out by spectral separation using Stokes shift by restricting the numerical aperture of the optics used for specimen illumination and detection or by splitting into different polarization directions.
Special dichroic beam splitters are used for the spectral separation of the excitation light from the light emitted by the specimen. As is shown in
The separation of excitation light from emitted light by restricting the numerical aperture of the specimen illumination optics (4 in
Problems of the Prior Art
In all of the methods according to the prior art, it is disadvantageous that the separation of the excitation light from the light emitted by the specimen is carried out in a wavelength-dependent manner, i.e., can not be adjusted in a flexible manner, or with a limited efficiency of typically 70% to 90%, depending on the required spectral characteristics and the quantity of illumination lines.
The primary object of the invention is to overcome the stated problems of the prior art. The invention describes methods and arrangements by which the excitation light can be separated from the light radiation (e.g., fluorescence or luminescence) excited and/or backscattered in the specimen in a particularly advantageous manner with high efficiency. In contrast to the prior art, the separation is adjustable in a flexible manner as regards spectrum without the movement of mechanical components and is therefore particularly suitable especially for use in multifluorescence microscopy, i.e., for simultaneous excitation of different dyes. In addition, the suppression of interference light is improved by at least an order of magnitude. Accordingly, fast switching between several excitation wavelengths or spectral detection wavelength ranges—so-called multitracking, as described in EP977069 A2—can be realized in a particularly simple manner. Further, it is possible to separate the light scattered by the specimen in the direction of the detector from the light reflected on a direct path and to measure it separately. In addition, a measurement of the polarization direction of the light coming from the specimen can be carried out. A further advantage consists in that fluctuations in laser output caused by unstable coupling into a glass fiber can be prevented by regulation, so that the output can be kept constant at the location of the specimen.
Unlike the arrangements for separating the excitation beam path from the detection beam path according to the prior art, optical resolution is not impaired by the arrangements according to the invention. Further, the illumination distribution can be manipulated at the site of specimen interaction. This makes it possible to scan so-called regions of interest (ROI) in real time. In addition, illumination methods known from widefield microscopy such as oblique illumination can be realized.
The solution according to the invention can be used in image-generating microscope systems as well as in analytic microscope systems. The microscope systems are image-generating systems such as laser scanning microscopes for three-dimensional examination of biological preparations with an optical resolution of up to 200 nm, nearfield scanning microscopes for high-resolution examination of surfaces with a resolution of up to 10 nm, fluorescence correlation microscopes for quantitative determination of molecular concentrations and for measuring molecular diffusions. Also included are methods based on fluorescence detection for screening dyes and methods for flow cytometry.
In all of the systems mentioned above, fluorescent dyes are used for specific labeling of the preparations. The above-stated objective is achieved by methods and arrangements according to the independent patent claims. Preferred further developments are indicated in the dependent claims.
The quantity of dye signatures that may be used simultaneously, i.e., the quantity of characteristics, for example, of cells that can be investigated simultaneously, can be increased by means of the methods according to the invention. When the spectral signatures of the individual dyes overlap extensively or are very close to one another, the detected wavelength range or numerical aperture must be limited, according to the prior art, for separate detection of the fluorescence signals of individual dyes. This reduces the sensitivity of detection, i.e., increases the noise of the detectors, because greater amplification must be used. This is avoided by the methods and arrangements according to the invention.
In the drawings:
a is a representational drawing showing single photon absorption;
b is a representational drawing showing multiphoton absorption;
a is a graphical representation of the spectral separation of the excitation light from the light emitted by the specimen by the use of special diochroic beam splitters;
b and 3c are cross-sectional representations of a splitter for separating excitation light from emitted light;
Various arrangements by which the light radiation (hereinafter, detection light) excited and/or backscattered in the specimen can be separated from the excitation light in a particularly efficient manner will be described more fully in the following. The arrangements are accordingly suitable in particular for fast multitracking with a flexible separation of the excitation radiation from the detection light that is adapted with respect to spectrum. In the following context, light that is emitted by the specimen is light which is preferably radiated from the specimen in a large solid angle. This light is not polarized (unpolarized). This refers in particular to fluorescent light and luminescent light excited in the specimen and backscattered light.
1. Principle of Operation of the Arrangement for Variable Separation of Excitation Light from Detection Light
The construction of the arrangement for separating the excitation light from the detection light is shown schematically in
The SLM is traversed in areas a and b (see
The principle of operation will be explained in the following with reference to
By means of the arrangement, light radiation entering through the input 2 can accordingly be spatially separated in the different outputs 1, 5 and 4 independent from the polarization state due to their different spectral composition and can accordingly be further processed optically separately.
A rotation of the polarization by angles other than 90 degrees in the detection beam path for fluorescence measurement is possible, but is less advisable because the components of the fluorescent light then reach the coupling ports 1 and 5 also and are therefore not detected by a detector.
The principle of operation in the excitation beam path is effected in a corresponding manner and will be described with reference to
The spatial separation of the spectral components through L2 and D2 is canceled again subsequently and the light travels to P2. P2 separates the components, according to polarization, into the output 3 and output 2, respectively. In the example above, the polarization for wavelength λ2 is rotated by exactly 90° through the SLM. Therefore, all light of this wavelength is directed into output 3 through P2. In contrast, the polarization for wavelength λ1 is only rotated by an angle not equal to 90°. Therefore, the light output is split in the two outputs 2 and 3. The splitting ratio is given by the adjusted angle of rotation of the polarization at the SLM. A rotation of the polarization by angles not equal to 90 degrees in the excitation beam path is suitable for attenuating the excitation light, since the ratio of output in the coupling ports 2 and 3 can be adjusted continuously corresponding to the following equation: P2/P3=tan (angle of rotation).
Accordingly, by means of the arrangement, light radiation entering through input 1 can be spatially separated in the different outputs 2, 3 and 6 independent from the degree of polarization due to its different spectral composition and can accordingly be separately further processed optically.
2. Laser Scanning Microscope
The arrangement according to the invention of an MDB (main color splitter) for separating illumination light and specimen light for a laser scanning microscope (LSM) in the xz-plane is shown schematically in
The linearly polarized excitation light coupled in the direction of output 2 (see
The light emitted by the specimen is collected through optics O (e.g., a microscope objective) and is imaged together with the tube lens TL in an intermediate image plane ZB of the microscope arrangement. It travels from the latter location via the scanners SX/SY and the relay optics RL to the input 2 of the MDB again. Since the light emitted by the specimen is usually unpolarized, it is divided at the beam splitter P2 (see also
Subsequently, the light of the specimen which travels through output 4 of the MDB by means of imaging optics (PO) is focused through a confocal diaphragm (PH), so that detection light which is outside of the focus is suppressed. In nonconfocal detection, the diaphragm can be omitted. A detector (DEI) which detects the light radiation excited in the specimen is located behind the confocal diaphragm. When recording fluorescence or luminescence, an emission filter (dichroic filter) F can be swiveled in for additional suppression of the excitation light backscattered from the specimen or for limiting the spectral detection area.
When the polarization of the emitted light of the specimen is to be detected (e.g., when determining fluorescence anisotropy), this can be carried out by detectors DE1 and DE2. The respective polarization can be composed from two portions or components which are polarized perpendicular to one another. The two portions which are polarized perpendicular to one another are measured separately by DE1 and DE2, e.g., by switching on a half-wave plate, not shown, between P1 and P3. The respective polarization can then be calculated by taking the ratio of the signals of detectors DE1 and DE2. Another method consists in controlling the corresponding SLM pixels in such a way that the signal is equal in both detectors DE1 and DE2. The corresponding polarization direction of the fluorescent light can then be determined from the control characteristic of the SLM pixel. A spectrally resolved measurement of fluorescence anisotropy is made possible in this way in that the pixels of the SLM are adapted sequentially for the individual colors.
The backscattered or reflected excitation light of the specimen which travels through the output 5 of the MDB (one portion travels in the direction of the light source LQ and is masked out) can also be focused through a confocal diaphragm (PH) by means of imaging optics (PO), so that detection light which lies outside of the focus is suppressed. A detector which detects excitation light that is backscattered from the specimen is located behind the confocal diaphragm.
Many different types of light sources from a broad spectral range are currently used in fluorescence microscopy. Due to a lower transmission of the SLM in certain wavelength ranges, e.g., in UV light sources (less than 400 nm) or with multiphoton excitation (greater than 800 nm), the coupling of the light sources through the SLM is not desirable. These light sources (LQ2) can be combined correspondingly with the light sources LQ1 passing through the SLM by a conventional beam splitter MDB2 between the output 2 and the first scanner, e.g., SX. In these light sources, detection is usually carried out in the wavelength range between 400 and 800 nm, that is, for example, through the SLM or by detectors according to the prior art.
The light emitted by the specimen which strikes output 4 of the MDB I can also be split by dichroic beam splitters NFT according to the prior art onto different confocal detectors (e.g., DE1 and DE2).
By means of this arrangement, a spectrally resolved measurement of the polarization characteristics can be carried out in a particularly advantageous manner corresponding to the method described above.
Outputs can also be exchanged in a corresponding manner in all of the arrangements according to the invention.
The MDB shown in
In principle, the function of the scanner shown herein can also be taken over by a corresponding scan table (object scanner).
The invention is used in a particularly advantageous manner in a widefield microscope, a fluorescence microscope, a point-scanning laser scanning microscope or a line-scanning laser scanning microscope, a flow cytometer, a parallel confocal microscope with a pinhole array in the illumination beam path and/or detection beam path or in a microscope arrangement for forming a periodic structure on an object with a phase displacement of the structure and the recording of phase-shifted images of the object.
While the foregoing description and drawings represent the present invention, it will be obvious to those skilled in the art that various changes may be made therein without departing from the true spirit and scope of the present invention.
Number | Date | Country | Kind |
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102 41 472 | Sep 2002 | DE | national |
This application is a divisional application of Ser. No. 10/654,702, filed Sep. 4, 2003 now U.S. Pat. No. 7,298,551, which claims priority of German Application No. 102 41 472.6, filed Sep. 4, 2002, the complete disclosure of which are hereby incorporated by reference.
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Number | Date | Country | |
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Parent | 10654702 | Sep 2003 | US |
Child | 11833761 | US |