The invention relates to a confocal laser scanning electron microscope with an illuminating configuration, which provides an illuminating beam for illuminating a probe region, with a scanning configuration, which guides the illuminating beam over the probe while scanning, and with a detector configuration, which via the scanning configuration images the illuminated probe region by means of a confocal aperture on to at least one detector unit.
Confocal laser scanning electron microscopes of the type initially mentioned are known in the state of the art, for example, let us cite the German patent DE 197 02 753 A1 in reference thereto. Most recently, components and technical systems from microscopy, specifically from confocal imaging laser scanning microscopes, have been ever more frequently applied to spectroscopic imaging techniques. In this manner, it is possible to survey the spectroscopic characteristics of a selected probe region without destroying or touching the probed area. Confocal optic microscopy thereby makes it possible to selectively detect optical signals, which are generated within a diffractionally limited confocal volume whose magnitude lies in the realm of micrometers. Laser scanning microscopes with scanning laser beams and/or with probe feed units can generate high focal resolution for two or three dimensional representations of the probe under examination. Owing to this characteristic, confocal laser scanning microscopy has nearly asserted itself as the standard for fluorescent probes in the field of biomedical technology.
Based on the highly exponential power of the chemical contents, confocal Raman microscopy is very attractive, particularly with regards to its application. The patent EP 0 542 962 B1 describes a setup for confocal Raman microscopy in which the appropriate selection of a focally resolving surface detector is used to produce the condition of confocality.
A problem in Raman spectroscopy consists in that the levels of signal intensity are frequently lower by several orders of magnitude as compared to classical fluorescence spectroscopy.
In practice, the integration time per measuring point frequently exceeds 1 minute. The periods of measurement resulting from that often span many hours or days which naturally sets narrow limits on the capabilities of confocal Raman spectroscopy for recording two or three dimensional microscopic images with high dot density. A reduction in the integration time would theoretically be conceivable by increasing laser output performance, however, this quickly leads to the destruction of the probe.
One is faced with similar problems in the area on non-linear optical microscopy, which, for example, offers attractive contrasting methods with the second harmonic generation but whose practical application however, is highly limited as well due to the low intensity of the signals and to the long measurement periods associated therewith.
A confocal microscope setup is known from the U.S. Pat. No. 6,134,002 in which a spectral analyzer is used as the detector. The analyzer admits the radiation through an entrance slit, whereby the linear slit region corresponds to a line region on the probe. A dotted image is scanned on the probe. The radiation to be detected is decoupled to the analyzer via a beam splitter acting as the primary color splitter, said beam splitter being located either between two scan mirrors of a scanning unit or being arranged in front of the scanning unit as seen in the direction toward the probe. In the first-named variant, the dotted image on the probe scanned by the two scan mirrors is only descanned in one spatial direction so that the spectral analyzer is impinged by radiation scanned along one line. In the second variant, the radiation is completely descanned by the scan mirrors and thereby comes to a rest state and can therefore be once more expanded with a cylindrical optical system toward a pinhole. The construction known from the U.S. Pat. No. 6,134,002 achieves an acceleration in image recording speed by shortening the periods of spectral analysis, which is why it forcibly depends on a spectral analyzer with a slit-shaped entrance region serving as a detector, thus it is highly limited with regards to a plurality of possible detectors. The initially mentioned problems associated with high laser performance are also a given factor in the design according to the U.S. Pat. No. 6,134,002.
The task set forth by the invention is therefore to expand upon a laser scanning microscope in such a manner that it can also record spectroscopic signals that are low in intensity within the shortest possible amount of time.
This task is resolved, in accordance with the invention, with a confocal laser scanning microscope of the type initially mentioned, in which, the illuminating configuration of the scanning configuration provides a line-shaped illuminating beam, the scanning configuration guides the line-shaped illuminating beam over the probe while scanning and the confocal aperture is designed as a slotted aperture or as a slotted region in the detector unit acting as a confocal aperture.
The present invention is dedicated to the problems described by combining line-shaped illumination of the probe with confocal detection by means of a slotted aperture or of a region acting as a slotted aperture.
In contrast to point scanners, as used in the U.S. Pat. No. 6,134,002, a line-shaped region is illuminated on the probe and confocally formed on a detector that is at least line-shaped.
As compared to a conventional confocal point laser scanning microscope with the same image recording time, with the same surface imaged in the sample, with the same visual field and with the same laser performance per pixel, a signal to noise ratio is produced that is improved by a factor of √{square root over (n)}, if n is defined as the number of pixels in the detector line. For this, a value ranging from 500 to 2,000 is typical. The prerequisite for this is that the line illuminating the probe exhibits the n-fold power of a laser focus of a comparable confocal point scanner.
If one does not wish to improve the detection speed or the signal to noise ratio with the laser scanning microscope in accordance with the invention, as an alternative and by comparison to a confocal point scanner with the same image recording time and the same signal to noise ratio, one can lower the radiation to which the probe is exposed by a factor of n, if the punctually produced beaming power realized for a confocal point scanner is now distributed throughout the illuminating line.
As compared to a confocal point scanner, the line sensing laser scanning microscope thus makes it possible, by a factor of n, to more quickly form low intensity signals for sensitive probe substances with the same signal to noise ratio and the same probe exposure, to form an improved signal to noise ratio, by a factor of √{square root over (N)}, with the same recording time, or with the same recording time, with the same signal to noise ratio and with a probe exposure that is lower by a factor of n.
Depending on the desired resolution, one will want to illuminate a line varying in width on the probe and detect it confocally. Illumination that is variable is therefore preferred. This can basically be achieved as early as the generation of line-shaped illumination. However, since the generation of linear illumination in a laser scanning microscope is purposefully accomplished in an illumination module, the expenditures for setting changes can run relatively high, if beams from different beam sources are collimated in the illumination module. It is therefore preferable, to use an optical zoom system disposing to this end of a zoom function for varying the width of the line of an already generated linear illuminating beam and said zoom system preferably located in a region of the beam path in which the illuminating radiation and the radiation to be detected are guided through the same optical elements, that is to say, are not yet separated.
In the case of laser scanning microscopes, the scanned image region can be selected in the scanner by the proper control for adjusting the zoom function, but only when combined with a galvanometer scanner in the case of individual point scanning. Here, in the present case of parallel scanning, that is to say, simultaneous scanning of several points by laser scanning microscopes, a zoom function based on changing the position of the scanning configuration cannot be implemented since the points individually scanned on the line stand in an established geometric relation to one another. The variable magnification attained by the optical zoom system makes it possible to change the magnification setting of the scanned field for such multiple spot scanners that operate in parallel, in which a zoom function that is based on intervention at the level of the scanning arrangement is not possible due to the fixed geometric interrelation of the points projected in parallel over the probe. The known approach of controlling spot to spot scanning in confocal scanning electron microscopes in such a manner that an image field is scanned in the desired and adjustable magnification is just as impossible in such parallel scanning systems as it is in systems that operate with resonance scanners, that is to say, in rotating mirrors driven by resonance vibrations, since the maximum deflection available there cannot be adjusted.
A possible position for the optical zoom system is to directly place it in front of the scanner unit (seen in the direction of the probe). A preferably motor-driven optical zoom system makes it possible, by an adaptation of the zoom factor it produces, to continuously vary the diagonal visual field within a specific range of adjustment. Especially preferred is an optical zoom system which is adjustable in three optical degrees of freedom so that important parameters do not vary, such as focusing position, pupil position and imaging dimensions, when the width of the line is changed.
An objective achieves its maximum resolution in the case when the entrance pupil is fully illuminated. It is therefore purposeful to provide the appropriate means to ensure that the optical zoom system always fully illuminates the entrance pupil of the objective, regardless of the setting on the optical zoom system. As a consequence, another purposeful embodiment of the invention provides for the arrangement of an element acting as an aperture in the exit pupil of the optical zoom system, said element not being larger than the smallest exit pupil size, which occurs when the optical zoom system is in operation. As a result of this, the size of the entrance pupil is independent from the selected setting on the optical zoom system.
Said size is purposefully equal or smaller than the size of the objective's entrance pupil.
During operation of the optical zoom system, the exit pupil can become very small when magnification is set to less than 1.0. If one wishes to avoid this very small exit pupil size as the lower value limit for the design, then it is purposeful to connect a telescope in front of the optical zoom system which shall effect the corresponding pupil dilation. Purposefully, this telescope shall only be activated during beam sweep when the optical zoom system operates in the scaled down mode. In this context, the concepts of “magnify” and “scale down” here relate to the image of the probe.
The activation of this telescope ensures that the exit pupil of the zoom, which is provided at a magnification of 1.0, can be established as the lower limit for the design without causing the exit pupil to become so small during scaled down mode of the optical zoom system that the objective's pupil might possibly become underfilled. Based in the interchangeability of the objective, it is purposeful to design the element operating as an aperture as being interchangeable if one intentionally wishes to underfill the objective's pupil, that is to say, not to fully illuminate. In that case, for example, an adjustable iris diaphragm or a mechanism with different interchangeable apertures would come under consideration such as, for example, a focal wheel with different pinhole apertures.
In an especially compactly built form of embodiment, the element acting as a lens aperture is realized by the scanning unit; for example, the limited dilatation of the scanner mirrors can act as a lens aperture.
In an especially preferred form of embodiment of the invention, an optical zoom system is used which exhibits an exit side pupil in which an aperture is provided. In practice, this aperture can also be delimited by the surface of a mirror in the scanning unit. Based on the action of this exit side aperture of the optical zoom system, a fixed pupil size is always formed on the scan configuration or on the objective of the confocal laser scanning microscope, regardless of the setting selected for zoom magnification. The aperture advantageously prevents the incidence of unwanted stray light in the realm of the scanning configuration.
If one also wishes to set the zoom factor at less than one, it is advantageous to preposition the cylindrical telescope so as to fill the pupil. Preferably, this setting can be automatically activated, for example, in the form of a pivoting device. The exit side pupil, or the aforementioned aperture of the zoom objective, for example, is thereby prevented from being insufficiently illuminated. Regardless of any change in setting on the optical zoom system, it is thereby always ensured at the site of the objective pupil that there will always be an illuminated line adjustable in size so that probe regions adjustable in size can be analyzed.
Upon activating the cylindrical telescope, an image brightness jump unavoidably occurs, since on the one hand, the cylindrical telescope is absorbing radiation and on the other hand, since above all, the intensity of the radiation is distributed over a longer line. In order to compensate for this effect in terms of the viewer, it is preferably provided in another form of embodiment, when the cylindrical telescope is switched in the beam path, that a control unit compensates for the reduction in image brightness caused by the telescope by an adjustment of the magnification factor of the detector configuration or by an adjustment in the scanning speed of the scanning device. Purposefully, this change carried out by the control unit is displayed for the user, for example, by the corresponding toggling of automatic controllers in a user program.
The inventors recognized that the problems associated with the axially varying position (in the direction of illumination) of the entrance pupil of the microscope objective could surprisingly be resolved by the appropriate design of the optical zoom system. The optical zoom system is advantageously designed in such manner that the imaging length (the distance between the entrance pupil and the exit pupil on the optical zoom system) can be varied so as to equilibrate the fluctuations in the axial positioning of the entrance pupil on the microscope's objective. The optical zoom system in accordance with the invention therefore achieves a double function in that, on the one hand, the scanning field parameters can be adjusted by varying the magnification, and on the other hand, the transmittance length can be adjusted in such a manner that an axially varying pupil position on the microscope's objective can be compensated for.
Furthermore, it is therefore purposeful that the optical zoom system is controlled by a control unit to be adjustable in such a manner that in a first mode of operation, a variable imaging length is produced. In order to adapt the optical zoom system to an activated objective, such as to a pivoting objective, it is purposeful to maintain magnification at a constant in this mode of operation.
Once the setting for the position of the pupil is in place, another mode of operation can be advantageously realized in which the magnification is set by guidance of the control unit so as to implement a zoom function without varying the imaging length. By virtue of the action of the optical zoom system in this mode of operation, the scanned field can be adjusted in terms of its size. If one synchronously uses a controllable double axis scanning unit, then in addition to and depending on the adjustment change in zoom magnification, a random region can be selected within the maximum permissible scanning field as a so-called “region of interest”, whereby this “region of interest” need not be symmetrically located relative to the optical axis. During detection beam sweep, this displacement factor as well as the zoom magnification in the direction of the detector are once more cleared so that the observation of specific regions in a probe is possible. In addition to this, images from different “regions of interest” can be acquired and subsequently recomposed into an especially highly resolved image.
An especially purposeful mode of construction of the optical zoom system uses four optical groups to implement variable pupil imaging. For the sake of manufacturing, it is favorable to provide the four optical groups, as seen in the direction of illumination, with positive refracting power, with negative refracting power as well as twice with positive refracting power. Purposefully, at least three of the optical groups are individually and independently adjustable by means of drives, and the displacement occurs in such a manner that the focus from infinite to infinite remains intact and depending on the mode of operation, the magnification or imaging length (pupil position) is adjusted. It can also be advantageous to design the last group, as seen in the direction of illumination, as one unit together with a scanning objective that is standard to a confocal scanning electron microscope, said scanning objective being positioned in front of the scanner unit. Each group is preferably comprised of at least one lens. In order to achieve the best possible characteristics in terms of available spectral range as well as possible apertures/field angles, the groups preferably have self-correcting capabilities in terms of image defects/imaging errors.
The mentioned selection of a “region of interest” either exclusively by way of the zoom function realized by the zoom objective, or also in addition to that, by way of an asymmetrical scanning mode of operation in the possible scanned field can further be improved by the use of an element that rotates the beam path. If, for example, an Abbe König prism is inserted into the pupil of the illuminating beam path, then the scanned, zoomed scan field can be rotated. In the detection beam path (mode), this rotation is once again cleared by the prism. Such an Abbe König prism can be obtained, for example, from LINOS Photonics, Germany and is known in the state of the art. For the mentioned design, it is rotatably arranged in the beam path, in proximity of the pupil since the beam cones converge at their narrowest here, and therefore an especially small prism can be used. Depending on the rotational angle, it introduces a rotation around the double angle of the image field.
Linear illumination can be produced in many ways. Especially advantageous however, is the use of at least one aspheric mirror. The basic principle involved in beam formation in the illuminating device is the case when by means of an aspheric mirror, a redistribution of spectral energy is achieved at least in one sectional plane and a inhomogeneous, specifically Gaussian profile distribution is transformed in such a manner that a largely homogeneous distribution of spectral energy predominates in the sectional plane. If the mirror is aspherically designed in two cross sectional directions, homogenization is obtained in two sectional planes, or therefore, an homogenized field is obtained. By the use of an aspheric mirror, a large spectral bandwidth can be covered by the illuminating radiation while simultaneously maintaining homogeneous illumination. It was thereby recognized that the reflecting aspheric mirror, which is more pronouncedly curved in one sectional plane in the region of the point of incidence of the source beam than in regions removed from the point of incidence, is suited for avoiding codependency on the wave length during focusing and distribution of spectral energy, whereby the concept of variable curvature of the aspheric mirror simultaneously opens up a great multitude of spectral energy distribution patterns. With said illuminating device, Gaussian beam bundles can be exemplarily reshaped in such a manner that in over 80% of the illuminated region, the intensity does not fall below 80% of the maximum value. This is a substantially homogeneous distribution of the type quite relevant here.
A variant with biaxial aspheric curvature can be applied in an especially advantageous manner for homogenization on an intermediate image plane. In the case of multiple point scanning microscopes, homogeneous illumination of an intermediate image in front of the element that generates the point cluster (e.g. Nipkow disc) makes it possible to evenly illuminate the probe with a beam intensity that is essentially uniform for the region. The converting unit also makes a full illumination of the objective's pupil possible so that an especially good image (highly resolved) is achieved because when the pupil is homogeneously filled, this permits the optical resolution to be fully exploited.
A form of embodiment that is especially simple in execution is a mirror that is designed to be wedge-shaped with a rounded off dome. Such a mirror can be simply manufactured from a quadrangular prism and it yields a focal line with homogeneous distribution of spectral energy.
In a variant that can quite easily be mathematically described, the mirror is defined by a conic constant such as the rounded off radius of the dome and it satisfies the equation y2/[c+(c2−(1+Q)y2) 1/2] for the (x,y,z) coordinates relative to the z-coordinate, wherein “c” is the rounded off radius of the dome and “Q” is the conic constant.
For the column-shaped illumination, it is desirable to not only homogeneously distribute the radiation along a longitudinally extended line, but rather to possibly match the width of the line to the diameter of the entrance pupil of the subsequent optical system. In order to achieve this, the aspheric mirror must also effect an expansion of the beam perpendicular to the direction of the line. This is especially easy to achieve with the above-mentioned variant of the wedge-shaped mirror with a rounded off dome in that the mirror surface or at least the dome along the dome's longitudinal axis is spherically or aspherically curved.
The aspheric mirror with the rounded off dome is then curved in two dimensions, whereby in a first sectional direction (perpendicular to the longitudinal axis) a cone with a rounded off point can be provided, and in the second sectional direction (along the dome), a parabolic, spheric or aspheric curvature can be provided. The latter curvature then sets the width of the illuminated field, whereas the aspheric form, perpendicular to the longitudinal axis, effects the expansion along the field and based on the aspheric quality, it determines the distribution of spectral energy. Along the field, a largely homogeneous spectral energy distribution is thereby achieved.
An additional mirror, spherically or parabolically curved along the dome can be mathematically simply expressed as follows:
f(x,y)=√{square root over ((a(y)−rx)2−x2)}−rx, wherein rx is the radius of curvature along the dome, that is to say, in the second sectional direction mentioned above.
In order to effect adaptative image matching in the case of the mirror curved in two directions (e.g. aspheric in the first sectional direction, spheric in the second) for full illumination of an intermediate image or of the entrance pupil to the immediately consecutive optical system, it is purposeful to arrange after the mirror a converging optical system, e.g. in the form of a concentrating reflecting mirror.
Normally, to this end, a cylindrical or toric concentrating reflecting mirror is used for generating a rectangular field. For other field forms, the shape of the mirror can vary, for example, one can also use the mentioned aspheric mirror for this second mirror in order to achieve a combination of homogenizing the light filled pupil in a first direction (by one of the aspherical mirrors) and the intermediate image in the remaining direction (by the other aspherical mirror). By the use of the additional aspherical mirror, image defect/imaging error compensation can also be effected. The second aspherical mirror can also be provided in addition to the concentrating reflecting mirror.
For the form of embodiment of the aspheric mirror with spherical curvature in the second sectional plane, it is therefore preferable that the concentrating reflecting mirror exhibits a curvature radius equal to rx+2·d in the x-direction, whereby “d” is the distance between the aspheric mirror and the concentrating reflecting mirror. The radius of curvature rx of the aspheric mirror in the second sectional plane then directly scales the height of the illuminated rectangular field or of the profile of the illuminating beam.
Of course, for homogenous pupil illumination, a mirror can be used that is aspheric in both sectional directions. In the case of a rotationally symmetrical aspheric mirror, this would then create a homogeneously illuminated circular field; otherwise one would obtain an elliptical field.
When the pupil is illuminated in such a manner, one is then able to select and use individual regions in the scanning process, e.g. by means of Nipkow discs, slotted apertures or such similar.
For illuminating the aspheric mirror, it is advantageous to set up the axis of symmetry of the mirror at an angle of between 4° and 20° to the incident axis of the source beam, which has, for example, a Gaussian-shaped profile, since a compact layout can then be obtained. The subsequently arranged concentrating reflecting mirror, which can, for example, be designed as cylindrical or toric, gathers the radiant energy distributed around by the aspherical mirror and compensates for disseminated wave aberrations during propagation. In simple cases where such wave aberrations are of no consequence, then a spherical lens can be used in place of the concentrating reflecting mirror.
Preferably provided in the laser scanning microscope is a decentralized zoom function produced by means of a second optional, independently acting scanning unit, that is to say, with crop function.
Confocal imaging can be produced by a slotted aperture in the laser scanning microscope. Preferred is a slotted aperture which can be continuously adjusted in terms of its aperture width so that a random airy diameter can be created on the detector. The continuous adjustment can, for example, be achieved by slotted apertures realized by means of solid state articulation technology. As an alternative, a slotted aperture unit can also be used with several interchangeable slotted apertures of different aperture width.
For example, fixed slit diaphragms, e.g. structured chrome masks, of different widths can be arranged on a slider/shutter.
In a solution that is especially simple to realize, the detector unit itself acts as a slotted aperture. To this end, for example, a detector line with pixels in a row can be used. It is also possible that the detector unit is comprised of a focally resolving surface irradiation sensor, crosswise to the direction of the slot, said sensor being arranged in the confocal plane, whereby the surface irradiation sensor acts as a confocal slotted aperture in selecting a subregion on the surface. In this manner, the effect of a variation in the slotted aperture diameter can be achieved at the level of the sensor by a corresponding selection of the region of interest, for example, with a CCD [charge coupled device] array or a CMOS [complementary metal oxide semiconductor] detector array.
In the laser scanning microscope, of course, various spectral channels can be used for illumination as well as for detection. In terms of this, an especially large variation is possible when the scanning configuration separates the projected radiant illumination from the irradiation returned by the probe region by means of a primary color splitter, wherein the color splitter can be produced as a strip mirror in accordance with the German patent DE 102 57 237 A1, whose revealed contents are explicitly integrated here.
Such a strip mirror acts as a spectrally independent primary color splitter. It lies in a pupil plane of the scanning configuration in which radiant illumination reflected at the level of the probe is formed in the shape of a line, that is to say, as coherent radiant illumination. In contrast to this, the incoherent signal radiation emitted that is to be detected fills the entire plane of the pupil and is only insubstantially weakened by the narrow strip mirror. Thus, the concept of the “color splitter” also covers splitter systems that act non-spectrally. An homogenous neutral splitter (e.g. 50/50, 70/30, 80/20 or such similar) or a dichroic splitter can also be used instead of the spectrally independent color splitter described.
To make an application based selection possible, the primary color splitter is preferably provided with mechanical means that allow for a simple change, for example, with a corresponding splitter disc containing individual, interchangeable splitters.
A dichroic primary color splitter is especially advantageous in the case when coherent, that is to say, when oriented radiation is to be detected such as, for example, Stoke's or anti-Stoke's Raman spectroscopy, coherent Raman processes of the higher order, general parametric non-linear optical processes such as second harmonic generation, third harmonic generation, sum frequency generation, two photon absorption and multiple photon absorption or fluorescence. Several of these processes from non-linear optical spectroscopy require the use of two or several laser beams that are co-linearly superimposed. To this end, the described unification of illuminating beams from several lasers proves to be especially advantageous. Basically, the dichroic beam splitters widely used in fluorescence microscopy can be applied. It is also advantageous for Raman spectroscopy to use holographic notch splitters or filters in front of the detectors to suppress Rayleigh scattering.
In the case of detection with several separate spectral channels, the emitted signal radiation is separated into spectral segments by means of a secondary color splitter, whereby each secondary color splitter provides an additional spectral channel. The individual spectral segments are then focused on the strip-shaped region, conjugate with the object plane, by means of round and/or cylindrical optical systems. This slotted aperture region exhibits partial confocality and after being spectrally filtered, for example by an emission filter, with the help of an optical group (e.g., by means of cylindrical lenses) is imaged on a suitable detector with focal resolving power in the direction of the slot, e.g. a CCD line camera or an optical multichannel analyzer.
As an alternative or additionally, a spectrometer can be used as a detector, which spectrally divides the linear radiation crosswise to the line and directs it to a surface irradiation detector. An entrance slit in the spectrometer can serve as a confocal aperture for this. If one wishes to analyze processes with timing resolution, it is purposeful to use a streak camera as the detector unit that temporally divides the linear radiation crosswise to the line and directs it to a surface irradiation detector.
The positionally resolved signal from the detector then contains the space coordinate in the one coordinate, and the time and wave length coordinate in the other, which reflects the temporal development or the spectral composition of the radiant signal emitted by the individual pixels along the space coordinate.
For the detection of linear or non-linear Raman signals and based on a polarization dependent illumination or detection, one can analyze the symmetry of the molecular vibrations to be detected and/or suppress the non-Raman resonant background fractions. It is therefore preferable for such applications, that at least one polarizer be used in the illuminating configuration and that at least one polarization analyzer be used in the detector configuration.
In the mentioned form of embodiment with several spectral channels, which are each respectively comprised of one detector unit, an independent slotted aperture can be used in each spectral channel. For the purpose of simplifying the construction, a common slotted aperture can also be optionally positioned in front of all of the spectral channels.
For simply aligning the position of the confocal slot-shaped element (e.g. of the slotted aperture or of the detector), one can design this element to be correspondingly sliding. Simpler in terms of construction however, is to provide a correcting device in the illuminating configuration and/or in the detector configuration that has at least one plane parallel transparent plate, which is supported in a holding fixture in the path of the beam and based on said fixture, which can be driven into a tilting and/or pivoting motion around at least one axis so that by virtue of the change in the tilt position of the plate, a certain parallel shift of the beams can be set in the beam path.
The correcting device has the advantage of making a simple compensation or correction possible of the defects/errors arising for imaging in the optical configuration, more specifically, the following can be simply corrected: ambient temperature or system temperature, position of the interchangeable or movable elements in the configuration, errors in color due to the wave length or wave length ranges of the radiation used. Depending on the requirement, a uniaxial tilting or pivoting motion can hereby suffice. If one wishes to provide a biaxial parallel shift, either a biaxial tilting or pivotable plate can be provided, or two different uniaxial tilting or pivotable plates can be provided. Essential for the invention is that the plane parallel plate with the holding fixture can be tilted in the path of the beam in a defined and known manner. For a biaxial positional change, any combination of tilting and pivoting motion is valid. A combination of a tilting motion with a pivoting motion is hereby relatively simple to mechanically produce and surprisingly, it does not yield any disadvantage along the optical axis in spite of the shift occurring for the plane parallel plate during the pivoting motion.
The correction effected by the device can be initiated manually by the user, for example, during adjustment in the manufacturing plant. Especially preferred however, is an expanded form of embodiment with a setting/correcting unit which is comprised of at least one operating parameter of the optical configuration and which sets the tilting position as dependent on the value of the operating parameter. The tilting position can exemplarily be stored to memory in calibrating tables. It is also possible, to optimize a correction, permanently, regularly or on demand, by setting the tilting position via active control loops. For such a design, it is preferred to provide a feedback control system that uses the tilting position of the plate as a correcting variable so as to balance the depicted effects on the imaging optical configuration. In this manner, eventual errors of temperature or long term drift errors present in the optical configuration can be simply corrected.
Since it is known that the parallel shift of the plane parallel plate depends on the refractive index of the transparent plate material, it is possible in the case of polychromatic radiation, that chromatic cross-aberrations may develop in the beam path of the optical configuration owing to a wavelength related parallel shift due to dispersion of the plate material. By constructing the plane parallel plate out of one or several graduated plates, one can compensate for such chromatic cross-aberrations originating from the plane parallel plate.
The correcting device can also be applied for use in countering varying chromatic cross-aberrations of the optical image that are due to operating conditions. For example, if an optical configuration is suited to operate with different wave lengths, then it is possible that chromatic cross-aberrations arise that are associated with wave lengths and are therefore dependent on operating conditions. The correcting device can then bring the plane parallel plate into another tilting position, depending on the wave length range currently in use in the optical configuration and on the therewith associated chromatic cross-aberrations, so that in the end effect, in spite of operation with different wave length ranges, the optical image remains unchanged in the configuration. Also for said correction, an appropriate setting/correcting unit can be used, as previously mentioned, which could also exhibit a feedback control system.
The requirements in precision or sensitivity with which the drive is displaced over the holding fixture, as well as the accessible parallel shift range and the thickness of the plane parallel plate can be programmed in.
As previously mentioned, the correcting device reduces the number of required movable optical elements in the optical imaging configuration. This advantage is especially significant when the confocal microscope exhibits exchangeable beam splitters with which an adaptation to various applications ensues, that is to say, a change in the projected wave lengths or selected wave lengths. The correcting device corrects the errors originating from the variable optical elements without interfering with the optical image. Furthermore, the correcting device can also be positioned between the confocal aperture and the detector and can thus aptly parallel shift the beam path (image) between the aperture and the detector.
Compensation for deviations perpendicular to the slotted aperture as well as compensation for deviations parallel to the slotted aperture can be adjusted via a corresponding setting for the tilting position of the plane parallel transparent plate.
In the first case, it must be checked whether the light originating from the probe falls exactly on the slotted aperture, and is not off center above or below the slotted aperture. In the second case, it must be checked whether the light originating from the probe correctly hits the line detector and that there is no pixel misalignment between the images of two detection channels in the system, which exemplarily each have their own line detector. In this manner, the confocal microscope can achieve an image registration that is accurate down to the subpixel while configured with multiple channels.
Furthermore, the correcting device is additionally advantageous in the confocal microscope in the sense that from now on, a narrow detector line can be used without requiring any movement from the slotted aperture or the detector. It is then altogether avoided, in the case of misalignment (caused by tilting and wedge errors in interchangeable elements) after adjusting the slotted aperture to increase resolution, that light flux is unnecessarily lost and thereby causing the signal to noise ratio to drop.
Since the tilting or wedge errors of individually switchable optical elements are likely to be repeated, the tilting position of the transparent plane parallel plate can be selected in a simple manner. Upon changing a switchable optical element, only one specific drive of the plane parallel transparent plate is needed in order to set the new tilting position required for the desired configuration of the microscope. Therefore, an expanded form of embodiment of the microscope is preferred, in which interchangeable or positionally adjustable elements are provided in the path of the beam and in which the setting/correcting unit interprets a configuration of interchangeable or positionally adjustable elements in the form of operating parameters and sets the tilting position based on the value of operating parameters.
An example for such parameters, in which not only the maladjustment of the optical image is corrected in terms of the slotted aperture, but also the chromatic cross-aberrations, advantageously provides for the presence of radiation in different wave lengths in the beam path of the microscope, whereby the setting/correcting unit sets the tilting position based on the length of the waves. One or several plane parallel plates is/are then arranged in front of each detection channel and the tilting position of the plane parallel plate is set by the setting/correcting unit depending on the length of the waves or on the wave length range in the actual channel.
An especially comfortable user operation is obtained when a control loop is provided which maximizes the radiant intensity in the detector unit, and/or which minimizes the image shift/misalignment in that the tilting position of the plane parallel plate is used as a correcting variable. With this, long term effects or temperature changes which bring about maladjustments can be rectified at any time without needing a service technician.
In another expanded form of embodiment, the microscope in accordance with the invention provides the probe with wide field illumination and that said probe is imaged by scanning of the dotted spot or dot group spot.
Henceforth, the invention advantageously uses wide field illumination in combination with scanning detection. With this surprisingly simple measure, there is no need for a separate detector. One simultaneously obtains an additional plethora of advantages.
For wide field illumination, radiation sources can be used that are already present in laser scanning microscopes for normal optical observation. A switch-over mechanism is no longer required. On the whole, the resulting construction is simplified. Preferably, the wide field illumination source is achieved by passing light illumination of the probe. As an alternative and in addition, wide field incident light illumination is also possible, for example, to conduct epifluorescent measurements or optical reflectance measurements. Both modes as well can be realized simultaneously (incident light illumination and passing light illumination).
The depth discriminating capability of the confocal detector configuration thereby permits a transmittance measurement that is resolved for depth.
By using wide field illumination sources usually already in place that conventionally have very broad bands as compared to the excitational illuminating sources provided for scanning, white light transmittent illumination operations can be performed, which were not possible in this manner in the conventional laser scanning microscopes due to the requirements of confocal imaging or if so, then only under enormous expenditures on the light source side. By analogy, the same is true for wide field incident light illumination.
Based on scanning of a probe with wide field illumination by means of the scanned detectors, the spectral analysis capability of the detector configuration present in the laser scanning microscope can also be used during transmittance operations thus leading to improved characterization of the probe.
The wide field illumination can be operated independent of the scanned spot-shaped illumination. Of course, the control unit can also initiate simultaneous operation in that the probe is then simultaneously analyzed in the transmittance operation mode as well as in the conventional fluorescent operation mode.
Exemplarily, the control unit can aptly select different spectral channels so that in some of the spectral channels, fluorescent information on the probe is acquired, whereas in other spectral channels, transmittance information is acquired. The proper synthesis of said information, for example, in an overlay image yields an analysis of the probe that is superior to that derived from conventional systems. It is therefore preferred to have the control unit simultaneously control both the operation of spot illumination configuration and of the wide field illumination source so as to properly select the spectral channels for the spot detector configuration.
Another advantage of the approach in accordance with the invention lies therein that it is also possible to have simultaneous transmittent light scanning at several points now, which was not permitted by conventional detectors separately arranged under the probe due to the lack of suitable focalized resolution. The use of multiple point or point group scanners under transmittent light operation now opened by the invention reduces eventual problems associated with temporal fluctuations in wide field illumination, since said problems can be corrected by the proper extension of the integration time for multiple point or point group systems. It is therefore preferred that both wide field illumination and the scanned point or point group shaped illumination be simultaneously carried out. Under “point group”, we understand any configuration of multiple points, in particular, in the form of a line which the laser scanning microscope confocally illuminates and images. By virtue of this approach, the probes are exposed to lesser radiation stress and shorter measuring periods are advantageously realized which were not possible in the prior state of the art. It is therefore especially preferred that the spot detector configuration implement a confocal point group formation, for example, with at least one Nipkow disc and with at least one matrix detector. The spot detector configuration can also use a confocal slotted aperture with a line detector when the point group is composed of a line.
The use of wide field illumination finally opens up entirely new contrast media processes for transmittent light measurement. Now, all of the contrast media processes are possible as they are known in the state of the art for conventional optical light microscopy. In order to realize this, it is preferable that the wide field illumination source exhibits a condenser in which the contrast media are switchable. For example, one can realize the illumination of a dark field in that a suitable annular lens is arranged in the condenser.
Other additional contrast media methods are also conceivable if the scanning configuration exhibits a scan objective in which suitable contrast media can be switched at the level of the pupil. In combination with the introduction of contrast media in the condenser, then not only is dark field contrast possible, but rather phase contrast, VAREL contrast, polarization contrast or differential interference contrast as well.
An additional analysis of time resolved processes is possible in the laser scanning microscope with the aid of gated detector arrays and of known pump and probe technology.
Based on the partial confocality generated by the aperture slot, an improved separation of microsectional thickness is achieved as compared to the optical wide field illumination. In combination with high speed zoom focusing, the line scanner in accordance with the invention therefore also succeeds in producing a three dimensional reconstruction of expanded probes. High speed z-canning can be realized by moving the probe in the z-direction (e.g. by means of high speed mechanical drives or piezoelectric probe movement), by moving the objective in the z-direction (e.g. by means of high speed mechanical drives or piezoelectric objective movement), or by objective internal focusing or high speed adaptive optical systems.
The laser scanning microscope in accordance with the invention enables processes for the detection of weak spectroscopic signals in microscopic configurations. The fields of application cover the vast expanse from microspectroscopy and microanalytics to “real” two dimensional and three dimensional microscopic image rendition. A hereby preferred process covers Stoke's confocal or anti-Stoke's Raman microscopy. But basically, any spectroscopic method can be used with the microscope in accordance with the invention for microscopic contrasting—and preferably such methods with signals that are weak in intensity. Conceivable as such methods, for example, are: luminescence spectroscopy (fluorescence, in particular, fluorescence polarization measurements, chemoluminescence, bioluminescence, phosphorescence), infrared microscopy, circular dichroism (CD) spectroscopy, hyper-Raman spectroscopy, stimulated Raman spectroscopy, coherent Stoke's or anti-Stoke's Raman spectroscopy (CARS, CSRS as well as all coherent Raman processes of the higher order, so-called HORSES), general parametric non-linear optical processes such as second harmonic generation (SHG), third harmonic generation (THG), sum frequency generation (SFG), two photon absorption and multiple photon absorption or fluorescence.
Several of the above-mentioned methods of non-linear optical spectroscopy require two or several lasers whose beams are overlaid in a co-linear manner. The represented collimation of beams from several lasers proves to be especially advantageous for this.
Potential uses for the invention cover all of the methods in which high focal microscopic resolution is routinely combined with classical spectroscopy. The application of the invention is particularly advantageous when, on a routine basis, that is to say, without high temporal expenditures, two dimensional and three dimensional substance distributions are to be quasi followed in real time. A very promising field of application is therefore found in the chemical and pharmaceutical characterization and process control of active ingredient distribution in fibers, foils, lacquers/polyurethane paints, dispersions, suspensions, emulsions, plastics, tablets, etc. Particularly interesting hereby is the analysis of crystalline and amorphous solid matter (e.g. the analysis and distribution of imperfections in crystals). In addition to the microanalytical characterization of existing substances, the pursuit of chemical process sequences is also conceivable, for example, for transformations into microfluid reagents in the rearrangement of crystals and in solid state polymerization.
In the area of medical technology, especially the focally resolved, non-invasive determination of active substances with the aid of Raman spectroscopic methods is of particular interest. The use of Raman spectroscopy in the area of medical applications is frequently limited due to the required laser spectrum density output in the focus and because of the associated destruction of living human tissue. As compared to point scanners counterparts, the use of the line scanner in accordance with the invention makes it possible to conduct measurements on the probe in the same amount of recording time and with the same SNR while lowering probe exposure by a factor of n (n=500-2000). As a practical application, let us mention the determination of inhomogeneous distribution of pigments and antioxidants in the human eye and skin.
Another potential field of application for the described invention exists in high throughput Raman screening of microtiter plates (multiwell plates) in the area of pharmaceutical development of active ingredients. Frequently of special interest hereby are Raman spectroscopic studies on polymorphism, which not only require less expenditures equipment-wise than in x-ray structure analyses but which can also be conducted in probes with a supernatant solution.
In the following, the invention shall yet be more closely detailed with reference to the drawing as an example. Shown in the drawings are:
a, 20b and 21a as well as 22a, 22b different settings for the optical zoom system in
The beaming source module 2 generates illuminating radiation, which is suited for laser scanning microscopy, more specifically, radiation which can release fluorescence. Depending on the application, the beaming source module exhibits several sources of radiation to this end. In a represented form of embodiment, two lasers 6 and 7 are provided in the beaming source module 2, after which are connected on the load side a light valve 8 as well as an attenuator 9 and which couple their radiation into a fiber optical wave guide 11 via a coupling point 10. The light valve 8 acts as a beam deflector by which beam cut-out can be effected without having to switch off the operation of the very lasers in the laser unit 6 or 7. The light valve 8 is designed as an AOTF which deflects the laser beam, before coupling into the fiber optical wave guide 11, in the direction of a light trap, not represented here, for the purpose of cutting out the beam.
In the exemplary representation of
The radiation coupled into the fiber optical wave guide 11 is concentrated by means of optical collimation systems 12 and 13 sliding over beam uniting mirrors 14, 15 and is modified in terms of its beam profile in a beam forming unit.
The collimators 12, 13 ensure that the radiation conducted from the beaming source module 2 to the scanning module 3 is collimated into an infinite beam path. In each case, this is advantageously achieved by a single lens which, under the control of a (non represented) central control unit, has a focusing function by its displacement along the optical axis in that the distance between the collimator 12, 13 and the respective end of the fiber optical wave guide is modifiable.
The beam forming unit, which shall later be explained in more detail, generates a column-shaped beam from the rotationally symmetrical, Gaussian profiled laser beam, as it exists emergent from the beam uniting mirrors 14, 15, said column-shaped beam no longer being rotationally symmetrical in its profile but rather suited for generating an illuminated rectangular field.
This illuminating beam, also referred to in the following as column-shaped, serves as excitation radiation and is guided to a scanner 18 via a primary color splitter 17 and via an optical zoom system, yet to be described. The primary color splitter shall also be detailed later, but let it just be mentioned here, that it has the function of separating the excitation radiation from the irradiation returning from the probe that originated from the microscope module 4.
The scanner 18 deflects the column shaped beam into one or two axes, after which said beam passes through a scanning objective 19 as well as through a tube lens 20 and an objective 21 to be bundled into a focus 22, which lies in a preparation or in a probe 23. The optical image is hereby produced such that the probe 23 is illuminated in a focal line with excitation radiation. A biaxial deflection by the scanner 18 is optional; it can, as shall later be detailed, be used for the selection of a scanning region or ROI [region of interest] lying asymmetrically to the optical axis.
Fluorescent radiation excited in the linear focus 22 in such a manner arrives, via the objective 21, the tube lens 20 and the scanning objective 19, back at the scanner 18 so that in the reverse direction toward the scanner 18, a beam is to be found once more at rest. One therefore also speaks of it in such terms that the scanner 18 descans the fluorescent radiation. The probe is simultaneously illuminated and scanned in parallel at several points on the line. The line therefore represents a point group.
The primary color splitter 17 lets the fluorescent radiation lying in wave length ranges other than those of the excitation radiation pass so that it can be rerouted via the deflecting mirror 24 in the detector module 5 and can then be analyzed. The detector module 5 exhibits in the form of embodiment in
Each spectral channel comprises a slotted aperture 26 which produces a confocal or partially confocal image of the probe 23 and whose aperture size establishes the depth of focus with which the fluorescent radiation can be detected. The geometry of the slotted aperture 26 therefore determines the microsectional plane within the (thick) preparation from which fluorescent radiation is detected.
Arranged after the slotted aperture 26 is also a blocking filter 27, which blocks off undesirable excitation radiation arriving in the detector module 5. The column-shaped fanned-out beam separated off in such a manner, originating from a specific depth segment is then analyzed by an appropriate detector 28. The second spectral detection channel is also constructed in analogy to the depicted color channel, and also comprises a slotted aperture 26a, a blocking filter 27a as well as a detector 28a.
In addition to confocal scanning of an probe region illuminated with a focal line, the laser scanning microscope 1 represented in an optional mode of construction in
Transmitted fractions of this illumination are also scanned in the scanning process by means of the objective 21, the tube lens 20, the scan objective 19 and the scanner 18 and are spectrally analyzed by means of the primary color splitter 17 in the detector module 5. Detection via the scanner 18 effects the focal resolution in the form of probe sensing and at the same time, a wide field of illumination is made possible by the halogen lamp 29.
The same concept can also be applied to the evaluation of rereflected radiation and epifluorescent radiation in that the illuminating radiation is coupled into the telescopic barrel of the microscope module 4 via a mercury vapor lamp 34 with an optical lamp system 35 on a beam splitter 36. This radiation then arrives on the probe 23 via the objective 21. The illumination here is also produced without the participation of the scanner 18. In contrast, detection process then takes place in turn via the optical scanning system 19 and the scanner 18 in the detector module 5. For this expanded form of embodiment, the detector module 5 therefore has a double function. On the one hand, it serves as a detector for scanned projected excitation radiation, whereby the scanner 18 serves both for the projection of the excitation radiation as well as for the descanning of the detecting radiation. On the other hand, the detector module 5 acts as a focally resolving detector when no further structured radiation is being projected on to the probe, namely either in the form of wide field illumination from below or via the objective 21.
But the scanner 18 also has a double action as well since it achieves focal resolution by point group or point formation scanning of the probe not only for projected point group or point formation excitation radiation, but rather also for wide field illumination.
Beyond this, the laser scanning microscope 1 in
For wide field illumination, a field aperture is preferably provided between the optical lamp system 30 and the condenser 31 in order to adjust the illuminated field. Furthermore, the aperture diaphragm in the condenser 31 is switchable. It lies in a position conjugate with the planes of the pupils of the laser scanning microscope. In the case these pupil planes, we are dealing with the plane of the pupil in which the scanner 18 lies as well as with the plane in which the primary color splitter 17 is arranged. As an aperture diaphragm such as in the plane of the pupil, one can now integrate different kinds of optical elements so as to be able to use known contrast methods from classical microscopy, such as for example, dark field, phase contrast, VAREL contrast or differential interference contrast. Suitable aperture diaphragms or elements to be introduced in the plane of the pupil are detailed, for example, in the publication “Microscopy from the very beginning”, Carl Zeiss Microscopy, D-07740 Jena, 1997, pages 18-23. The revealed contents of this company publication are hereby explicitly integrated in reference to this. For such contrasting method interventions of course, this pupil plane is not the only one coming under consideration. There are also other pupil planes suitable to this end. For example, intervention could be implemented in the proximity of the primary color splitter 17 or by means of an optical relay system after the secondary color splitter 25 in one (or several) spectral channels of the detector beam path.
The use of a confocal slotted aperture in the detector module 5 serves only as an example. In principle, random configurations of multiple points such as point clusters or Nipkow disc concepts can be used for parallel scanning of point groups. However, what remains essential is that the detector 28 be focally resolving so as to enable the scanner to detect several probe points in parallel on a sweep run.
Based on this concept, the non-descanned detectors required to date in the state of the art in the microscope module 4 are no longer needed. Furthermore, owing to confocal detection, high focal resolution can be attained that would otherwise only be feasible with expensive matrix sensors in the case of non-descanned detection. Beyond this, temporal fluctuations in the projecting wide field illumination, e.g. of the halogen lamp 29 or of the mercury vapor lamp 34 among other things, can be phased out by proper integration in the focally resolving detector 28, 28a.
For this mode of operation of the laser scanning microscope 1, the primary color splitter 17 as well as the secondary color splitter 25 are naturally to be properly set. This also makes it possible, to simultaneously operate both types of illumination, that is to say, the wide field illumination from below and the illumination through the objective 21 when the color splitters are designed to be suitably dichroic. Also, random combinations with scanned illumination from the radiation source module 2 are possible. A corresponding overlaid graphic representation of the evaluated signals then offers image information that is outstanding when compared to conventional concepts.
The combination of a confocal line formation, that is to say, of a line scanner with multiple channel spectral detection makes it possible to acquire highly parallel data. An image recording rate of over 200 images per second can be achieved and an ability to render “real time” that has not been realized to date with laser scanning microscopes is a given. As an alternative, the laser scanning microscope 1 also enables highly sensitive detection of particularly weak signal intensities. Compared to a conventional confocal point scanning laser microscope, a signal to noise ratio is obtained that is improved by a factor of √{square root over (n)}, for the same image recording time, for the same surface imaged in the probe, for the same visual field and for the same laser performance per pixel and with “n” being designated as the number of pixels in the detector line. A value ranging from 500 to 2,000 is typical of this.
The beam source module 2 of the laser scanning microscope 1 fulfills the requirements necessary to this end, namely the illuminating line which is made available by the beam forming unit 16, which exhibits n-fold output, as well as the laser focus of a comparable confocal point scanner.
As an alternative, and by comparison to a confocal single point scanner, for the same image recording time and the same signal to noise ratio, the probe exposure, that is to say the quantity of radiation the probe shall be exposed to, can be lowered by a factor of “n” if the radiation output applied to date in a confocal point scanner is now limited to just being distributed on the line.
As compared to a confocal point scanner, the line sensing laser scanning microscope with the beam forming unit 16 therefore makes it possible, by a factor of n, to more quickly form low intensity signals from sensitive probe substances with the same signal to noise ratio and the same probe exposure, and to form a signal to noise ratio improved by a factor of √{square root over (n)} with the same recording time, or with the same recording time, with the same signal to noise ratio and with a probe exposure that is lower by a factor of n.
In
For reshaping the beam, an aspheric mirror 38.1 is used which expands the radiation. The expanded radiation is again made parallel by means of a concentrating reflector mirror 38.2. The aspheric mirror 38.1 is impinged by a source beam 38.3 from the beam source, said source beam exhibiting a rotationally symmetric Gaussian-shaped beam profile. In the section represented in
The action of the aspheric mirror 38.1 provided in
The aspherical curvature on the (z, y) plane effects the propagation of energy, as represented in
The asphericity detailed for the one sectional direction can naturally also be provided in the other sectional direction. One can therewith achieve a homogeneously illuminated elliptical or round shaped field; said round shaped field being achieved with a rotationally symmetrical aspheric mirror 38.1. As an alternative, one can omit the sphericity in the x-direction. The aspheric mirror 38.1 would then have the profile form of the sectional line 38.12 for each respective x-coordinate.
The reflecting mirror surface represented in
The convex or concave reflecting mirror surface 38.6 of the aspheric mirror 38.1 can be produced in a variety of manners. Thus, the profile corresponding to the sectional line 38.12 can be integrated into a cylinder that has a radius of curvature which corresponds to the radius of curvature rx of the reflecting mirror surface in the (z, x) plane. If one wishes to obtain a reflecting mirror surface 38.6 that is not curved in the (z, x) plane, that is to say, whose radius of curvature in this sectional plane can be assumed to be infinite, then the arrangement can be done with a quadrangular prism or a wedge which would then be rounded off in the region of the dome, corresponding to the curvature “c” dictated by the parabola 38.10.
In the case of rx-radii being smaller than 0, then replica techniques or duplicating techniques can be used to form the reflecting mirror surface 38.6 of the aspheric mirror 38.1.
For creating the shaped beam 38.5, a concentrating reflecting mirror 38.2 is positioned after the aspheric mirror 38.1, as shown in
In order to especially simplify the implementation of the setting for the height of the rectangular field to be illuminated, the radius rtx is selected as rtx+2 d for the toric mirror, wherein “d” designates the distance between the aspheric mirror 38.1 and the concentrating reflecting mirror 38.2 on the optical axis. One then obtains a beam expansion factor of rx/rx and therefore about 1+2d/rx.
Instead of the concentrating reflecting mirror 38.2, one can naturally also use a corresponding achromatic toric lens. Furthermore, at least one cylindrical mirror can be used for correcting the modified ray bundle diameter transverse to the homogenized direction, said cylindrical mirror having such dimensions that, together with the radius rx of the aspheric mirror 38.1 as well as with the radius rx of the concentrating reflecting mirror 38.2, it can purposefully change the focus and the ray bundle diameter transverse to the homogenized direction. This cylindrical mirror can be positioned in front of the aspheric mirror 38.1 or after the toric concentrating reflecting mirror 38.2. Its function can also be realized by at least one achromatic cylindrical lens.
The illuminating configuration with the aspheric mirror 38.1 can serve to evenly fill in a pupil between a tube lens and an objective. The optical resolution of the objective can be fully exploited by so doing. This variant is purposeful in a point scanning or in a line scanning microscope system (in the latter case, in addition to the axis in which the probe is being focused on or in).
As explained, the linearly transformed excitation radiation is directed to the primary color splitter 17. In a preferred form of embodiment, the latter is designed as a spectrally neutral splitter mirror in accordance with the German patent DE 10257237 A1, whose revealed contents are fully integrated here. The concept of “color splitter” also includes splitter systems that act in a non-spectral manner. Instead of the described spectrally independent color splitter, an homogenous neutral splitter (e.g. 50/50, 70/30, 80/20 or such similar) or a dichroic splitter can also be used. In order to make an application based selection possible, the primary color splitter is preferably equipped with mechanics that make it possible to implement a simple change, for example, by means of a corresponding splitter disc which contains individual, interchangeable splitters.
A dichroic primary color splitter is especially advantageous in the case when coherent, that is to say, when oriented radiation is to be detected such as, for example, reflection, Stoke's or anti-Stoke's Raman spectroscopy, coherent Raman processes of the higher order, general parametric non-linear optical processes, such as second harmonic generation, third harmonic generation, sum frequency generation, two photon absorption and multiple photon absorption or fluorescence. Several of these processes from non-linear optical spectroscopy require the use of two or more laser beams that are co-linearly superimposed. To this end, the described unification of beams from several lasers proves to be especially advantageous. Basically, the dichroic beam splitters widely used in fluorescence microscopy can be applied. It is also advantageous for Raman spectroscopy to use holographic notch splitters or filters in front of the detectors to suppress Rayleigh scattering.
It is advantageously shown in
The cylindrical telescope 37 working together with the optical zoom system 41 is also activatable by a motor and is arranged in front of the aspherical unit 38. It is selected in the form of embodiment represented in
If a zoom factor of less than 1.0 is desired, the cylindrical telescope 37 is automated to pivot into the optical path of the beam. This prevents the aperture diaphragm 42 from being incompletely illuminated when the zoom objective 41 setting is scaled down. The pivotable cylindrical telescope 37 thereby ensures that even with zoom factor settings of less than 1, that is to say, independent of any adjustment change in the optical zoom system 41, there will always be an illuminated line that is constant in length on the site of the objective's pupil. As compared to a simple visual field zoom, losses in the laser's output as expressed in illuminating beam power are avoided owing to this.
Since an image brightness jump cannot be avoided in the illumination line when the cylindrical telescope 37 is being pivoted, it is provided in the (non-represented) control unit, that the feed rate of the scanner 18 or the gain factor for the detectors in the detector module 5 is adapted accordingly when the cylindrical telescope 37 is activated so that the image brightness can be maintained at a constant.
The
Furthermore, in an exemplary variant it is purposeful to design group G1 as one unit together with the scanning objective following it; in this variant, the scanning objective is therefore positioned before the scanner in the direction of illumination (not shown).
Each group is comprised of at least one lens. To satisfy the requirements for the desired spectral ranges as well as for the targeted aperture/field angle, the groups are self-correcting, to the extent possible, in terms of imaging errors/image defects.
For the sake of visualization in
The entrance pupil is hereby set to the 0 position. The figures designated with “a” respectively show a sectional plane which is rotated by 90° as compared to the figures designated with “b”. Thus,
In
The zoom objective 41 can therefore be operated in two different modes of operation. On the one hand, it is possible to change the setting for magnification m [v] while maintaining a constant imaging length L. A change from the position drawn in
The concept of “magnification” once more relates to the action of the optical zoom system, that is to say, to the magnification of the image. An image magnification is then attained when the optical zoom system, in the direction of illumination, has indeed achieved a reduction of the image transmitted by the illumination source, that is to say, for example, when a focal line has been shortened. In contrast, in the direction counter to illumination, that is to say, in the direction of detection, an enlargement takes place.
Based on the previously mentioned action of the scanner 18 to descan and based on a repeat run through the optical zoom system 41, the selection of the region of interest (ROI) in the detection beam path is again cleared in the direction toward the detector. One can hereby make any selection for the desired region of interest (ROI) within the range offered by the scanning image SF. In addition, for different selections within the region of interest (ROI), one can acquire images and then compose them into an image that is highly resolved.
If one not only wishes to shift the selected region of interest by the measure of an offset OF relative to the optical axis, but also wishes to rotate said region, there is a purposeful form of embodiment which provides an Abbe König prism in a pupil of the beam path between the primary color splitter 17 and the probe 23, which obviously leads to the rotation in the image field. This image is also cleared again in the direction of the detector. Now, one can measure images with different offset spacings OF and with different angles of rotation and after that, they can be computed into a high resolution image, for example, by means of an algorithm, as described in the publication by M. Gustafsson, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination” in “Three-dimensional and multidimensional microscopy: Image acquisition processing VII”, Proceedings of SPIE, Vol. 3919 (2000), p 141-150.
In addition to the motor driven optical zoom system 41 as well as to the motor activated cylindrical telescope 37, there are also remote controlled adjusting elements provided in the detector module 5 of the laser scanning microscope in
Additionally provided for the sake of compensation is a correcting unit 40 which shall be described in the following based on
The CCD line 43 receives radiation via the color splitter 25 which then by way of the slotted aperture 26 that acts as a confocal aperture becomes incident on CCD line 43.
Together with a circular lens 44 arranged in front of the slotted aperture as well as with the equally prepositioned first cylindrical optical system 39 as well as with the postpositioned second cylindrical optical system, the slotted aperture 26 forms a pinhole objective of the detector configuration 5, whereby the pinhole is realized here by the slotted aperture 26.
The linear or line-shaped region of the probe 23 illuminated for the purpose of generating fluorescent excitation which is confocally formed is schematically represented in
A change in the color splitter 25 or in the blocking filter 27 unavoidably causes a certain tilt or wedge error during pivoting. The color splitter can cause an error between the probed region and the slotted aperture 26; the blocking filter 27 can cause an error between the slotted aperture 26 and the CCD line 43. To avoid the necessary readjustment of the position of the slotted aperture 26 or of the CCD line 43, the plane parallel plate 40 is arranged between the circular optical system 44 and the slotted aperture 26, that is to say, in the imaging beam path between the probes 23 and the CCD line 43 so that said plate can be brought into various rocking positions by the activation of a controller C. The plane parallel plate 40 is adjustably mounted in a holding fixture (not represented in
The plane parallel plate 40 causes a parallel offset which is drawn in
A change in the tilt position of the plane parallel plate 40 makes it possible to adjust the position of the probe line in such a manner vis a vis the slotted aperture 26 (as an alternative, this also applies to the position of the aperture vis a vis the CCD line 43 also acting as an aperture if the plate 40 is inserted after the slotted aperture 26) that, for a given set of conditions in the beam path subject to being modified when the color splitter 25 is changed, an optimal position will always result, that is to say, a biaxial centered position. This is visualized in
The offset dx leads to the result that the signal to noise ratio is unnecessarily compromised. If one wishes to improve the depth resolution in the confocal microscope by dimming the slotted aperture 26, that is to say, by reducing its latitude in the x-direction, it can happen, in the case of an offset dx, which is greater than half the height of the probe line 23, that there is no longer any incident radiation upon the CCD-line.
The offset dx then leads to the outcome that the depth resolution attainable with the laser scanning microscope becomes lesser than should be the case accomplished with said equipment. The same applies to the possible alternate or cumulative variant for adjusting the slotted aperture 26 and the CCD-line 23.
The optical adjustment of the probe spot image attained for the slotted aperture 26 by setting the tilting position of the plane parallel plate 40 has the effect that, seen in the x-direction, no surface area of the CCD-line 43 unnecessarily fails to be illuminated.
In contrast, the offset dy has the effect, in the y-direction, that the regional information detected by the CCD-line 43 does not correspond to the real conditions of emission and reflection in the probe 23. Artefacts can result in the image (or a substitute image). The setting of the tilting position of the plate 40 makes it possible to minimize the offset dy, preferably to even bring it to 0, so that the slotted aperture 26 lies in dead center on the CCD-line 43 and all pixels in the CCD-line 43 are correctly illuminated. This is especially important in the case when the laser scanning microscope is comprised of several detector channels which read out different color channels via the secondary color splitter 25. Since based on the individual adjustments of the settings, there would otherwise be different offsets of dy, this would lead to an error in the reconstituted image for such a multiple channel laser scanning microscope given the configuration of the individual color channels.
Depending on the wave length or the wave length range, which is evaluated in the detector module 5, the pinhole objective can display a chromatic cross-aberration that is variable. The same is true for elements prepositionally connected, for example, the color splitter 17, 25 or other optical systems configured on the optical axis OA. By setting the tilting position of the plate 40, this chromatic cross aberration can be compensated purposefully. To this end, the controller C guides the plate 40 into the tilt position, whereby each wave length range or each wave length is assigned its own tilt position for which the detector module 5 can be used.
If relatively broad band radiation is being conducted in the detector channel, then the plane parallel plate itself can generate a chromatic cross aberration if the dispersion of the transparent material of the plane parallel plate 40 is such that the wave length dependent offset for the emergent bundle of rays A is different from that of the incident bundle of rays E. The mode of construction for the plane parallel plate 40, comprised of two graduated plates 40a, 40b, represented in
The controller C sets the tilt position of the plate 40 based on input user specifications after evaluating the current configuration (in particular, also considering ambient temperature or device temperature or other external factors of influence) of the laser scanning microscope or does so continuously or at intermittent intervals within the process control cycles. The tilt position of the plate 40 is used as a correcting variable for control. In a calibrating step, the radiation intensity or the image offset on the CCD-line 43 can be analyzed as a controlled variable.
The drive 40.11 controlled by the controller C is represented in perspective in
A holding plate 40.14 is provided for tilting around the x-axis to which a pair of leaf springs 40.15 are screwed down which secure a frame 40.16 in which the plane parallel plate 40 is mounted. The leaf springs 40.15 establish the tilting axis. They press a roller 40.17 that is secured to a frame 40.16 on a cam disc 40.18, which is driven by a stepping motor 40.12, which is also seated on the holding plate 40.14. Depending on the position of the cam disc 40.18, the roller 40.17 and with it the frame 40.16 are thereby made to deflect in different positions whereby tilting of the plate 40 is achieved around the x-axis.
The holding plate 40.14 itself is the arm of a lever 40.19 which is rotatable around a swivel axis 40.20. The swivel axis 40.20 represents the axis for the movement of the plate 40 around the y-plane. The other arm 40.21 of the lever 40.19 carries on its end a roller 40.22 which rests against a cam disc 40.23, which is driven by the stepped motor 40.13. Similar to the manner in which the leaf springs 40.15 press the roller 40.17 against the cam disc 40.18, there is a spring element provided on the swivel axis 40.20 which presses the roller 40.22 against the cam disc 40.23.
By controlling the stepped motors 40.12, 40.13, the controller C, which is connected to the stepped motors by lines that are not represented here, can set the tilting or the pivoting position of the plane parallel plate 40 in the beam path of the detector configuration by motor power. Based on the incremental control of the stepped motors 40.12, 40.13 and in combination with the startup reference position assumed at the beginning of each operation, the controller C knows the actual position of the plate 40 at any point of the operation so that the position of the plate 40 can be used as a correcting variable in a regulating loop or it can be set depending on the stored input settings.
As an alternative to biaxial positioning, two adjustable uniaxial plates can also be connected one behind the other as exemplarily shown in
In addition to positioning by means of the correcting unit 40 or 40a, the slotted aperture 26, 26a itself can be made to change its setting. This specifically occurs to thereby match the width of the slotted aperture 26, 26a to the airy diameter on the detector. By changing the width setting of the slot on the slotted aperture 26 or 26a, the depth of field and thereby discrimination of sectional depth in the z-direction is set, that is to say, along the optical axis in the probe 23. An additional cross displacement movement serves for roughly adjusting the region outside of the adjustment range of the correcting unit 40 or 40a.
In
The forms of embodiment for the laser scanning microscope described up to this point provide spectrally discrete detector paths in the detector module 5. If one wishes to simultaneously analyze a wide band spectral range, then the conception systematically represented in
In a simplified form of embodiment, the slotted aperture 26 can be realized by an entrance slit of the spectrometer 49; the slotted aperture 26 and the inlet window 48 formed as an entrance slit then coincide.
In particular, in the case of the use of holographic Raman notch filters, it is also conceivable in Raman microscopy to use a simple monochromator instead of the two-way or three-way spectrometers.
Another possible mode of construction for a laser scanning microscope 1 is shown in
As a modified arrangement of the mode of construction in
The optical zoom system 41 corresponds, for example, to the mode of construction previously detailed, whereby the scanner 18 naturally becomes redundant due to the Nipkow disc 64. Nevertheless, said scanner can be provided if one wishes to undertake the selection of a region of interest (ROI) detailed in
An alternate approach with multiple spot scanning is shown in schematic representation in
As another form of embodiment coming under consideration is multiple point scanning, as described in the U.S. Pat. No. 6,028,306, whose revelation is fully integrated here in terms of this. Here as well, a detector 28 with positional resolving power is to be provided as in
The described invention represents a significant expansion of the application possibilities for high speed confocal laser scanning microscopes. The significance of such expanded development can be deduced from the standard literature on cell biology and from the processes described there on superfast cellular and subcellular processes' and from the applied methods of analysis with a multitude of dyes2.
See, for example:
The invention has an especially great significance for the following processes and developments:
Development of Organisms
The described invention is, among other things, suited for the analysis of developmental processes which are characterized foremost by dynamic processes ranging from tenths of seconds to hours in duration. Exemplary applications are described here, for example, at the level of cell groups and whole organisms:
This applies to the following points of emphasis in particular:
The last mentioned item in combination with the ones preceding it.
Transport Processes in Cells
The described invention is excellent in its suitability for the analysis of intracellular transport processes since the truly small motile structures involved here are to be represented, e.g. proteins with high speeds (usually in the range of hundredths of seconds). In order to capture the dynamics of complex transport processes, applications are also often used such as FRAP with ROI bleaching. Examples for such studies are described here, for example:
Zhang et al. describe in 2001 in Neuron, 31: 261-275 live cell imaging of GFP transfected nerve cells wherein the mobility of granules was analyzed based on a combination of bleaching and fluorescent imaging. To this end, the dynamics of the nerve cells set very high requirements for the imaging speed.
Molecular Interactions
The described invention is particularly well suited for the representation of molecular and other subcellular interactions. To this end, very small structures with high speeds (in the range of hundredths of seconds) must be represented. In order to resolve the spatial position necessary for the observation of molecular interactions, indirect techniques must also be applied such as, for example, FRET with ROI bleaching. Exemplary applications are, for example, described here:
The described invention is excellent and very well suited for the analysis of signal transmission processes that are usually extremely rapid. These predominantly neurophysiological processes set the highest demands on temporal resolution since the activities mediated by ions transpire within the range of hundredths to smaller than thousandths of seconds. Exemplary applications of analyses on the muscle and nervous systems are described here, for example:
Number | Date | Country | Kind |
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10 2004 034 977.0 | Jul 2004 | DE | national |