Patent Application
20060214106
1. Field of the Invention
The invention relates to methods and arrangements for microscopy, in particular for fluorescence microscopy, laser scanning microscopy, fluorescence correlation microscopy and scanning near-field microscopy, primarily for examination of biological samples, preparations and their components. It also includes methods for the screening of substances (High Throughput Screening) based on fluorescence detection and methods for flow cytometry. Thus, simultaneous examination in real time of probes with multiple fluorophores becomes possible through simultaneous illumination of a probe having several illumination wavelengths, with overlapping fluorescence spectra, even in spatial structures of thick probes. An achromatic main beam splitter for Laser Scanning Microscopy is realized, which enables efficient excitation and detection in confocal imaging with or without extension of the depth of sharpness and integration of the same in Laser Scanning Microscopes. Multiple-point illumination is also explicitly included.
2. Description of Related Art
A classical field of application of light microscopes in the examination of biological preparations is fluorescence microscopy. See for example: Pawley, “Handbook of Biological Confocal Microscopy” Plenum Press 1995. In accordance with the classic application, certain dyes are used for specific marking of cell parts.
In fluorescence microscopy, the fluorescence light from an excitation beam is split with suitable dichroitic beam splitters in combination with block filters and is observed separately. Imaging of individual differently stained cell parts is thus possible. However, in principle, several parts of the preparations can be stained at the same time with different specifically absorbed dyes (multiple fluorescence). To differentiate between the fluorescence signals emitted by the individual dyes, special dichroic beam splitters are used.
The state of the art is explained in the following example on the basis of a confocal Laser Scanning Microscope (LSM) shown in
An LSM 10 comprises essentially 4 modules: light source 12, scan module 14, detection unit 16 and microscope 18. These modules are described in greater detail in the following description. In addition, reference is made to U.S. Pat. No. 6,167,173, which is incorporated by reference herein.
For specific excitation of the different dyes in a preparation, lasers 21-24 with different wavelengths are used in the LSM. The selection of the excitation wavelengths depends on the absorption characteristics of the dyes to be investigated. The excitation beam is generated in the light source module 12. Thereby different types of lasers are used (for example, glass laser: argon, argon krypton, solid state laser: TiSa laser, diodes). Furthermore, in a light source module, the selection of the wavelengths and the tuning of the intensity of the required excitation wavelengths is done, for instance, by using acoustooptical crystals 28,30. Thereafter, the laser beam reaches into the scan module 14 passing through a fiber 26 or a suitable mirror arrangement.
The laser beam generated in the light source is focused in diffraction limited manner by means of the objective 31, passing through the scanner 14, the scanning optical system 33 and the tube lens 35, onto the preparation 37. The scanner performs point-like rastering of the probe in x-y direction. The pixel dwell time during the scanning of the probe lies mostly in the range of less than a microsecond to a few seconds.
In the confocal detection (descanned detection) 16 of the fluorescence light, the light, which is emitted from the focal plane (specimen 37 and the planes above and below it, reaches, passing through the scanner, to a dichroitic beam splitter (HFT) 41. The latter separates the fluorescence light from the excitation light. Thereafter the fluorescence light is focused on a diaphragm (confocal diaphragm/pinhole) 43, which lies exactly on the plane conjugate to the focus plane. As a result, the portions of the fluorescence light outside the focus are suppressed. By varying the diaphragm size, the optical resolution of the microscope can be adjusted. Behind the diaphragm, there is another dichroic block filter EF1-EF4, which suppresses the excitation beam once again. After passing through the block filter EF1-EF4, the fluorescence is measured respectively by means of a point detector PMT1-PMT4.
In an application with multiphoton absorption, the excitation of the dye fluorescence takes place in a small volume in which the excitation intensity is especially high. This region is only insignificantly larger than the detected region in the applications with a confocal arrangement. Use of a confocal diaphragm is thus not necessary and the detection can take place directly after the objective (non-descanned detection) 49. In another arrangement for the detection of dye fluorescence excited with multiphoton absorption, descanned detection still does take place, however, in that case, the pupil of the objective is imaged in the detection unit (non-confocal descanned detection) 49.
Of a three-dimensionally illuminated image, only the plane (optical cross section) is reproduced by the confocal detection arrangement, which is located at the focal plane of the objective 31. Through the image data of the several optical cross sections in the x-y plane at different depths z of the probe, a computer-aided three-dimensional image of the probe can be recreated.
Therefore, the LSM 10 is suitable for examination of thick preparations. The excitation wavelengths are determined by the dye used according to its specific absorption characteristics. Dichroic filters tuned to the emission characteristics of the dye ensure that only the fluorescence light emitted by the respective dye is measured by the point detector.
In biomedical applications, in the present techniques, several different cell regions are marked simultaneously with different dyes (multifluorescence).
The individual dyes can be detected separately with state of the art technology on the basis of different absorption or emission characteristics (spectra).
For separate detection, additional splitting of the fluorescence light from several dyes takes place with secondary beam splitters DBS1-DBS3 and separate detection of the individual dye emissions in different point detectors PMT1-PMT4.
Flow cytometers are used in the examination and classification of cells and other particles. For that, the cells are dissolved in a liquid and are pumped by a capillary. To examine the cells, a laser beam is focused onto the capillary from a side. The cells are stained with different colors or fluorescent biomolecules.
What are measured are the excited fluorescence light and the backscattered excitation beam. The separation of the fluorescence signal of the probe from the excitation beam takes place by means of dichroic beam splitters (HFT see
The size of the cells can be determined from the backscattered signal. Based on the spectral characteristics of the fluorescence of the individual cells, different cells can be separated and/or sorted out or counted separately. The sorting out of the cells takes place by means of an electrostatic field in different capillaries. The result, that is, for instance, the number of the cells with stain A compared to cells with stain B, is frequently presented as histograms.
The flow rate is characteristically several 10-100 cm/s. Therefore, high-sensitivity detection is necessary. To limit the detection volume, the confocal detection takes place according to the state of the art.
In a Laser Scanning Microscope (LSM), according to the state of the art, the entire circular pupil is illuminated, due to which the main dye splitter (HFT) 41 is embodied as a dichroic splitter. This means that, for different excitation and detection wavelengths, different main dichroic splitters are required, which, mounted on a filter wheel, can then normally be swiveled in and out into the path of the beam.
Another alternative, known for a long time, for modifying the imaging characteristics of the microscope is to use special pupil illuminations. One possibility is to use an annular pupil illumination, which leads in particular to an extended depth of sharpness (C. J. R. Sheppard: The use of lenses with annular aperture in scanning optical microscopy, Optik 48 (1977) 329-334). Thereby, in combination with radial polarization, it also leads to increased resolution (C. J. R. Sheppard and A. Choudhury: Annular pupils, radial polarization, and superresolution, Appl. Opt. 43 (2004) 4322-4327). Other illuminations aimed at higher resolution are proposed in (T. Wilson and S. J. Hewlett: Superresolution in Confocal Microscopy, Op. Lett. 16(1991) 1062-1064). In confocal microscopy, illumination and detection take place in general with the same objective. Because of the heavy losses especially in detection with a highly limited pupil (for example one with an annular shape), use of such an objective aperture is out of the question at least in fluorescence microscopy. Besides that, some of the apertures described in T. Wilson et al have different limitations, such as, for example, extremely low excitation efficiency and asymmetrical Point Spread Function (PSF), which renders them in general not usable in confocal microscopy.
U.S. Pat. No. 6,809,324 B1, incorporated by reference herein in its entirety, describes an aperture division, in which the effective NA of the excitation is very low. The resolution of the excitation beam path is thus essentially worse than the resolution of the detection beam path. In general, the arrangement is used for suppressing the stray light (such as described in the Abstract and claim 1), whereby the probe is illuminated through a central part of the pupil with reduced NA (as described in claim 2). The same is true for the arrangement described in DE 299 13 707 U1.
In U.S. Pat. No. 6,785,302, incorporated by reference herein in its entirety, the exciting laser beam has a significantly smaller cross section than the fluorescence beam exiting from the objective (particularly as described in the Abstract and claim 1).
U.S. Pat. No. 6,888,148 describes an arrangement with a structured beam splitter for a line scanner and a far-field microscope. In both cases the non-point-like excitation of the probe takes place.
As further examples, the known use of a Nipkow disk represents a possible embodiment of multipoint scanners working parallel, as disclosed in U.S. Pat. No. 6,028,306 or U.S. Pat. No. 4,927,254 or U.S. Pat. No. 5,428,475. Besides that, the mentioned U.S. patent document describes a parallel scanning Laser Scanning Microscope with a multiaperture diaphragm plate, before which a corresponding microlens array is arranged, so that in the end, a multipoint source is generated. Other embodiments are related to fluorescence correlation spectroscopy (FCS), as described, for example, in U.S. Patent Application No. 2005/0271549 A1.
The invention enables achromatic separation of the excitation and detection beam paths in a single-point or multipoint scanning LSM, that is, with point-like excitation and detection. In this way, the resolution of the excitation beam path remains almost the same or identical to the resolution in the detection beam path, whereby, surprisingly, it turns out that a nearly identical optical resolution of the microscope, as in the classical LSM with filled pupil, can be achieved by generating special spatially coherent illumination patterns.
Another advantage of the invention is that it enables special operation modes in which the depth of the sharpness of the microscope is higher compared to the classical single-point or multipoint scanning microscope.
Another advantage of the invention is the accomplishment of the automatic regulation of the illumination patterns in the pupil, depending on the operation mode (for example, small focus volume or enlarged focus volume).
This is achieved in that excitation light with a spatially coherent illumination pattern is formed at the location of a pupil of the microscope. In the pupil, an element is arranged, which reflects the illumination pattern in the direction of the probe. The remaining areas of the element are preferably transparent, so that the detection light, which fills the remaining area of the pupil, can pass in the direction of the detection. A possible reversal involves a transparent area for the transmission of the illumination beam to the probe and a remaining reflecting area for the transfer of the detection beam in the direction of a detection unit. The element for the separation of the beam can be rigid or flexible, with an adjustable or a replaceable structure.
With the present invention, an achromatic beam splitter for a Laser Scanning Microscope is realized. In this way, compared to the state of the art, the splitter does not modify, or modifies only slightly, the characteristics of the image. To achieve the desired result, the main dichroic beam splitter is configured in the plane of the pupil (or near it) in such a way that it exhibits broad-band reflective function only in a certain region.
Light from the light source L arrives through a beam former SF to the beam splitter HFT and is reflected by it in the direction of the probe PR. The surface of the beam splitter is a vaporized coat reflector in the black area 1 so that the excitation is reflected in the direction of probe PR. The splitter is transparent in the remaining area 2.
With the excitation beam former SF, which is realized, for example, with a combination of phase masks PM and amplitude masks AM1, AM2 and classical optical system L1, L2 in the state of the art (for example, with diffractive or holographic elements (Such as described in “Diffractive optics improve product design,” Photonics Spectra, Laurin Publishing Co., Inc. September 1995)), the profile of the beam from the light source is influenced in such a manner that at the splitter HFT, it largely assumes the form of the structure of the splitter (area 1). With AM1, the amplitude of the beam profile can be influenced in the pupil. With the help of AM2, an additional spatial filter for the suppression of, for example, interfering diffraction orders that are generated by the PM or AM1, can be achieved. To that end, AM2 preferably takes the form of the illumination pattern at the location of the probe. For that, AM1 is arranged in or near a pupil and AM2 is arranged at an intermediate image. With that, a high efficiency is ensured in the excitation beam path. In the exemplary variants shown in
The fluorescence light generated in the probe arrives at the detector through the area 2 of the HFT. That part of the fluorescence light is lost, which is incident on the area 1. The detector is preferably embodied as a confocal detector, as in the state of the art. Further, devices for the suppression of the excitation light (for example emission filters) are arranged before the detector and after the HFT. In addition to that, the detection can take place simultaneously in several channels. This is achieved by arranging secondary dichroic splitters and more detectors as in
Case A in
In the arrangement according to the invention as in
A confocal PSF in an arrangement according to
If extended depth of sharpness is desired and the losses on the detection side do not play a role, an additional ring diaphragm RB (approximately of the same size as in the HFT) can be placed before the pinhole optics (see
One can achieve improved lateral resolution by using a laser beam with radial polarization (Shepard et al as cited above). The corresponding optics can be provided in the beam former part in
In order to take the fact into account that different objectives have different pupil radii, different main dichroic splitters can be mounted on a filter wheel and can be swiveled in according to the objective. In addition to that, dichroic HFT can also be mounted on the filter wheel in order to enable operation of the LSM according to the state of the art. Use of different ring apertures becomes naturally unnecessary, if zoom optics are provided between the HFT and the objective for adjusting the beam to different objectives.
An alternative solution follows from the following considerations. In the case of a limited number of objectives with different apertures, one can generate a common HFT, as shown in
For example, for s1=0.6 and s2=0.4, one obtains V=6% for ring size 0.02 times (1−ε) of the respective ring radius. The advantage in the arrangement realized in this manner lies in the exact justification of the HFT, which is possible with a stationary arrangement in the beam path.
Case B in
With plate width of b=0.02a, for instance, one obtains losses <3%. When the same HFT is used for an objective with aperture 0.4a, the losses increase to 16%. With linear adaptation of the plate width according to the distance r from the center so that b/r=const. (see
Shown are the linear (left) and the logarithmic (right) diagrams of the lateral PSF for the confocal case according to the state of the art confocal laser scanning microscope (CLSM) and with the HFT according to the invention (crosswise, landing plates are assumed infinitely thin in this case). The normalized lateral coordinate is v=2π/λNA r. λexc=488 nm, λfluo=520 nm.
Near the optical axis, the axial distribution of the PSF is identical to the confocal case according to the state of the art. There are visible but minor differences only far from the axis beyond v>2. The image characteristics are therefore actually practically identical to those in CLSM in this case.
Another embodiment uses a freely programmable HFT, as it can be realized for example with a Spatial Light Modulator (SLM). A possible embodiment is shown in
In addition to the free programming of the HFT, such an arrangement has the advantage of reduced losses, because the losses of 1−ε2 as described above occur only in the polarization direction of the fluorescence beam. The total losses are thus reduced to half.
Light from the light source L arrives, passing through the beam former SF, to a first polarization beam splitter PBS 1 and is reflected by it due to the corresponding selected polarization of the light source (for example linear) in the direction of the probe PR. On the path to the probe, it passes through an SLM. The latter lies in or near a pupil of the microscope. The SLM can be so regulated that the pixels in the black area (1) [see enlarged view of SLM] rotate the polarization of the excitation beam. The excitation beam from these areas thus passes through the second polarization beam splitter PBS 2. The remaining pixels (area 2) do not lead to any change in the polarization. Therefore, these parts of the excitation light are reflected in the direction of beam dump. Instead of a beam dump, a detector can also be arranged for monitoring of the power of the excitation beam.
If the polarization of the light source is not set in such a manner that it is completely reflected in PBS 1, these parts of the beam also reach the beam dump passing through the mirror M1/M2 and PBS 2.
The fluorescence light generated in the probe is split in PBS 2 into two polarized parts transversal to each other. One part is reflected in PBS 2 and reaches the detector after passing through M2/M1 and reflection at PBS 1. The part passing through PBS 2 arrives at the PBS 1 passing through the SLM (area 2), combines there with the reflected part and also reaches the detector. Only the part of the fluorescence light incident on the area (1) is lost.
With the help of the arrangements as in
Thereby the following, in part mutually dependent, measured quantities, serve as the automatic control parameters:
1.) The confocality of the microscope, which can take place, for example, through the measurement of the confocal signal on DE3. Either test probes (like fluorescent beads or thin fluorescence layers) or actual probes that are to be examined, can be used as probes. Use of test probes makes sense if the probe to be actually examined cannot be exposed to the dose of the illumination during the regulation. The regulation is manipulated in a way that preferably the signal with the amplitude as high as possible is measured at DE 3.
2.) The efficiency in the excitation beam path can be measured with a far-field detector at DE1. Thereby the signal should preferably be large as much as possible.
3.) The STREHL ratio of the excitation is measured with a confocal detector DE 2. Thereby the signal should preferably be as large as possible.
4.) From the position of the mirrors of the DMD or the pixel in the SLM, the area/efficiency of the beam splitter can be determined (ratio of the areas 1 to 2 in
The corresponding individual sizes can also be optimized. For example, both the efficiency as well as the resolution can be maximized by optimizing the measured variable I). In yet another variant, by optimizing the ratio of 1) to 2), the resolution and efficiency can have relative weights in the regulation.
For that, the splitter T is swiveled optionally into the beam path so that a part of the excitation light (for example 5%) is reflected on DE1 and DE2. The light as such is measured with the detector DE1, with DE2 the excitation-side point image and with the actual detector DE3, the detection-side point image through DMD.
In addition to that, the measurement 1) can take place with a fluorescence generating medium (F). For that purpose, it is swiveled into the beam path between the HFT and the scanner SC. For example, plane-parallel fluorescence optical cells (dye preferably matching to the stain used in the probe) or plane-parallel fluorescing glasses (for example erbium doped glasses) can be used. Preferably, F is so designed that no excitation light reaches in the direction of the probe. After the optimization of the microscope, that is, for the actual measurement of the probe, the splitter T and the probe F are swiveled out again for the actual measurement of the probe.
The HFT (comprising a DMD, SLM or different mirror masks as in FIGS. 2/6/7) and the beam former SF serve as the manipulating parameters for the regulation. In the beam former, the phase and amplitude are set at PM and AM. In the HFT, the patterns of the areas 1 or 2 are varied.
As the boundary parameters, the input parameters serve the purpose here, such as the type of the probe PR, the objective O, the light source and the operation mode B used by the user (for example, high or low depth of sharpness, high efficiency in the excitation, high efficiency in the detection). Preferably, these boundary parameters themselves automatically induce the corresponding optimization of the measured variables.
Surprisingly, the adjustment of the geometry with the regulator circuit of the splitter according to the invention can also be applied in the arrangements according to U.S. Pat. No. 6,888,148. Other similar arrangements of HFT, which are based on modifications of the previously mentioned arrangements, can be realized. Besides the ring-shaped arrangements, this is of relevance in particular when more landing plates are used (case A), for example, under 45, degrees with respect to the landing plate in the case A, use of landing plates with uneven thickness (for example, in the case A, landing plates that become thicker at the borders) landing plates with partial gaps (A and B) and combinations of these cases.
Several possible examples are shown in
The landing plates can cross each other in a star-like manner, with an even or odd number of plates. The plates can also be arranged in spider-like form, with a common center in the optical axis or outside the optical axis. Such arrangements of the plates can be combined with ring arrangements, and instead of the rings, spiral-shaped structures can also be provided or there can be several eccentrically arranged small rings or one—or several—triangles or other geometric figures, and other amplitude or phase patterns are also possible and can be adjusted, whereby the optimization can take place through the manipulation as described above.
In a Nipkow or a multipoint scanning microscopy system, the main dichroic beam splitter is substituted according to the invention by a patterned beam splitter and is arranged in or near a pupil.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2005 009 188.1 | Mar 2005 | DE | national |
