This application claims priority of German Application No. 103 55 523.4, filed Nov. 21, 2004, the complete disclosure of which is hereby incorporated by reference.
a) Field of the Invention
The invention is directed to a stereo microscope which is suitable particularly for fluorescence observation with incident light.
b) Description of the Related Art
The use of fluorescence contrast in stereo microscopy has become increasingly important in recent years because of new goals for applications in biology, medicine, science and technology. One example of such applications is the use of the GFP (Green Fluorescent Protein) fluorochrome in microbiology.
Known equipment for fluorescence microscopy with stereo microscopes is based on different principles:
a) Equipment for External Oblique Incident Light Fluorescence Excitation with Lightguides and Focusing Attachments
Equipment for external, oblique incident light fluorescence excitation with focusing attachments are made available by the present applicant in the Stemi 1000/2000, SV6/SV11/SV11 Apo stereo microscopes. They are described in the Carl Zeiss information sheet “F1 S Equipment—Fluorescence Contrast with Stereo Microscopes” (40-510/9.97). An incident light fluorescence adapter SZX-FLUV is known from Olympus.
An embodiment example for external, oblique incident light fluorescence excitation in a stereo microscope is shown in
This external, oblique incident light fluorescence excitation with lightguides and focusing attachments has the following advantages: the intensity of the excitation can be optimized by displacing the focusing head on the light-guiding illumination part and by adjusting the distance of the illumination part from the object; the intensity of the excitation is high (illumination apertures are larger than the observation aperture); and hardly any interference light can reach the observation channel from the excitation beam path because the illumination channel and observation channel are spatially separated from one another.
This design has the following disadvantages: the light spot impinging in the object plane is elliptic to some extent, depending on the angle of inclination of the illumination, the distance of the focusing attachment from the object and the adjustment of the focusing attachment, and therefore has a drop in intensity in the longitudinal axis of the illumination; the light spot has a fixed size even when the magnification is changed at the stereo microscope zoom, and the illuminated object field is always more or less greater than the object field detected by the microscope and higher objective magnifications therefore have less image brightness; excitation light can be reflected toward the observer with back illumination; the excitation filter and blocking filter with different changing locations lie far apart from one another and must therefore be changed individually in a time-consuming manner (so that modular construction is impossible); and the holder with the illumination must be readjusted and optimized when changing the objectives or when changing the height of the adjusting plane.
b) Equipment for External, Oblique Incident Light Fluorescence Excitation with Ring Lamps
Equipment of this kind is known from the NIKON Fluorescence Ring Illuminator C-FPS and the MEIJI Fluorescent Ring Light MA 305.
An embodiment example of external, oblique incident light fluorescence excitation with ring lamps in a stereo microscope is shown in
The external, oblique incident light fluorescence excitation with ring lamps has the following advantages: the light spot impinging in the object plane is circular; the illumination and light spot remain in the object plane when changing objectives or when changing the height of the adjusting plane; and hardly any interference light can reach the observation channel from the excitation beam path because the illumination channel and observation channel are spatially separated from one another.
It has the following disadvantages: the light spot has a fixed size even when the magnification is changed at the stereo microscope zoom, and the illuminated object field is always larger to some extent than the object field detected by the microscope and higher objective magnifications therefore have less image brightness; the light spot in the focused object plane is optimally illuminated only with certain objectives; the illumination intensity is low because the radiating direction of the ring lamp is more polydirectional; the excitation filter and blocking filter with different changing points lie far apart from one another and must therefore be changed individually in a time-consuming manner (here again, modular construction is not possible).
c) Coaxial Incident Light Fluorescence Excitation through both Channels of the Stereo Microscope
Equipment of this kind is the GFP Illuminator by Kramer Scientific Corporation, Elmsford, N.Y., USA, for the Carl Zeiss SV 6/SV stereo microscope, whose basic principle is described in U.S. Pat. No. 6,147,800.
An embodiment example of coaxial incident light fluorescence excitation through both channels of the stereo microscope is shown in
This kind of incident light fluorescence excitation through both channels of the stereo microscope has the following advantages: the light spot impinging in the object plane is circular and is illuminated and observed at the stereo angle; the size of the light spot is determined by the respective zoom magnification and objective magnification; the optimally illuminated light spot always lies in the focused object plane and, therefore, it is possible to change the objective and to refocus at the microscope without manipulating the illumination; and the excitation filter, dichroic mirror and blocking filter lie close to one another so that they can easily be constructed as modules for a slide or filter wheel, which makes it possible to change the fluorescence modules quickly on a slide or filter wheel that has been loaded.
It is disadvantageous that the illumination is carried out through the observation channel and the illumination intensity is therefore determined by the zoom aperture; it can only be optimized by adjusting the lamp collector. The transmission of the optics, which are actually designed for stereoscopic observation, in the zoom channels has a disadvantageous result for the intensity of the fluorescence excitation in the UV range, and the excitation light can cause stray light in the zoom channels which worsens the image contrast.
d) Coaxial Incident Light Fluorescence Excitation through One Channel of the Stereo Microscope
Such devices as the LEICA fluorescence module for MS-MZ, Olympus Fluorescence Illuminator P-FLA and the Nikon SZX-RFL Fluorescence Illuminator are known.
This type of incident light fluorescence excitation through one channel of the stereo microscope has the following advantages: the light spot impinging in the object plane is circular; the size of the light spot is determined by the respective zoom magnification and objective magnification; the optimally illuminated light spot always lies in the focused object plane and therefore makes it possible to change the objective and to refocus at the microscope without manipulating the illumination; and the excitation filter, dichroic mirror and blocking filter lie close together so that they can easily be constructed as modules for slides or filter wheels, which makes it possible to change the fluorescence modules quickly on a slide or filter wheel that has been loaded.
It is disadvantageous that the illumination is carried out on one side through the one observation channel at the stereo angle and that the illumination intensity is determined by the zoom aperture; it can only be optimized by adjusting the lamp collector. The transmission of the optics, which are actually designed for stereoscopic observation, in the zoom channels has a disadvantageous effect on for the intensity of the fluorescence excitation in the UV range, and the excitation light can cause stray light in the excitation channel and worsen the image contrast, so that the image is brighter in the excitation channel than in the pure observation channel. In the pure observation channel, arrangement of the blocking filter alone is sufficient for wavelength selection; when wavelength regions lie close together, the dichroic mirror matching the excitation is needed in addition to the blocking filter.
e) Integrated Incident Light (Fluorescence) Illumination through Two Illumination Channels Lying Outside of the Observation Channels of the Stereo Microscope
This kind of arrangement of illumination channels in a stereo microscope, preferably with two lightguides that are inclined somewhat relative to the optical axis is described in the German Laid Open Application DE 19822255 by the applicant.
An embodiment example of this illumination arrangement is shown in
This illumination arrangement has the following advantages: it is carried out with a minimal space requirement and the basic mechanical-optical construction is influenced only minimally; the maximum visible object field enables a bright, homogeneous and reflection-free illumination of the object regardless of the position and observation direction of the stereo microscope; the light spot impinging in the object plane is circular and always lies in the focused object plane and it is therefore possible to change the objective and to refocus at the microscope without manipulating the illumination; and the focusing optics of the lightguides can be also be constructed as zoom systems coupled to the observation zoom and the size of the light spot can accordingly be adapted to the respective zoom magnification and objective magnification.
It is disadvantageous that the excitation filter and blocking filter with different changing locations lie far apart from one another and must therefore be changed individually (modular construction is therefore impossible).
f) Integrated Coaxial Incident Light Fluorescence Excitation through a Third Channel which is Analogous to the Stereo Channels and which Lies Between the Normal Stereo Channels so as to be Offset with Respect to the Center
This arrangement of a third illumination channel which is analogous to the stereo channels and lie between the normal stereo channels so as to be offset with respect to the center is described in the protective application EP 1 010 030.
EP 1 010 030 describes a single-channel zoom body (magnification changer) and a two-channel (stereo) zoom body (likewise referred to as a magnification changer), each of which has another illumination beam path lying at least approximately parallel to the observation beam path.
This illumination arrangement has the following advantages: the light spot impinging in the object plane is circular, its size is determined by the respective zoom magnification and objective magnification, and the optimally illuminated light spot always lies in the focused object plane so that it is possible to change the objective and to refocus at the microscope without manipulating the illumination device. The zoom optics in the separate illumination channel are optimized for better transparency to UV and blue light. Any possible autofluorescence does not interfere with observation: stray light or worsened image contrast are prevented. The excitation light is coupled into the third illumination channel in a simple manner, the dichroic splitter mirror is replaced by a reflection element, and the excitation filter and blocking filter which lie close together can be constructed as a flat module for slides or filter wheels making it possible to change the fluorescence modules quickly on a slide or filter wheel that has been loaded or equipped.
It is disadvantageous that the illumination intensity which is increased by a better transparency of the zoom optics to UV and blue light is still limited by the illumination aperture (corresponding to the respective observation aperture).
It is the primary object of the invention to overcome the disadvantages of the prior art and to provide an incident light fluorescence excitation with a high illumination aperture for all observation zoom magnifications that is greater than the aperture of the observation zoom itself.
This object is met by an incident light fluorescence stereo microscope comprising two eyepieces, an objective with two observation beam paths and at least one illumination beam path. The illumination beam path preferably extends on the side opposite from the eyepieces. A reflecting component is provided which deflects the illumination beam path in such a way that it passes through the objective preferably parallel to the observation beam paths.
According to the invention, this illumination, coming from the back, is deflected by illumination optics vertically downward into an objective between, in front of, or after the channels of the observation zoom, wherein the optical axes of the deflected illumination beam path and of the channels of the observation zoom are approximately parallel in the space between the illumination deflecting element, the optics at the entrance of the observation zoom and the objective. In order to adapt the size of the illuminated image field to the scale ratios of the observation zoom with the objective, the illumination optics are constructed, according to the invention, as an illumination zoom—hereinafter referred to as light zoom. The movements of the light zoom and observation zoom are coupled with one another so as to advantageously correspond to the scale ratios. Accordingly, the light spot imaged by the light zoom is circular and always lies in the focused plane of the microscope objective.
According to a preferred embodiment form of the present invention, the fluorescence illumination and the stereo microscope observation channels are integrated in an incident light fluorescence stereo microscope body. The illumination according to the invention includes a light staircase, a light protection shutter, a light zoom and a stationary deflecting element. The illumination beam path is guided via the light staircase into the light zoom which images the light spot, corresponding to the respective magnifications adjusted in the observation channels, in the object plane by the deflecting element through the excitation filter and the objective. The objective, two blocking filters and the optics in the observation channels are associated with the stereoscopic observation beam path. The fluorescing object is imaged through the objective, two blocking filters and the optics in the observation channels for observation in the microscope tube. A suitable coupling between the light zoom and the observation zoom causes the scale ratios in the light zoom to be adapted corresponding to the scale ratios in the illumination zoom. Excitation filters and blocking filters are advantageously mounted in modules. The modules themselves are outfitted with various filter combinations or must be equipped individually and can therefore be exchanged, changed or switched quickly. The light protection shutter automatically covers the illumination beam path when exchanging, changing or switching the modules.
In this connection, it is advantageous when the incident illumination for fluorescence excitation is deflected coming from behind vertical to the optical axes of the observation beam paths by a deflecting element which has a cemented convex lens and which is arranged at the height of the front lenses of the observation zoom in front of the optical axes of the observation beam paths toward the objective in such a way that the illumination beam path is coaxial and parallel to the observation beam paths in the space between the front lens, observation zoom, deflecting element and objective.
In a preferred solution, the illumination beam path and the observation beam paths pass through the objective in a circle diameter containing the maximum effective (free) diameter of the observation systems and illumination system. The objective diameter is greater than this circle diameter containing the maximum effective (free) diameter of the observation systems and illumination system. The plane defined by the observation beam paths has a parallel offset relative to a meridian plane of the optical axis of the objective.
According to the invention, the illumination with a fluorescence light source is guided via the illumination beam path. At least one blocking filter for short-wave light beams is arranged in the observation beam path and an exciter filter for limiting the bandwidth of the fluorescence light is arranged in each illumination beam path. The blocking filter and exciter filter lie in a plane and are mounted in a common filter holder. A filter wheel having receptacles for three exchangeable filter holders with different combinations of blocking and exciter filters which can be switched into the channels of the observation beam paths and illumination beam path alternately by rotating the filter wheel is arranged in the space between the front lens, observation zoom, deflecting element and objective. The filter wheel itself can also be exchanged for another filter wheel. A filter carrier without a blocking filter and exciter filter or with attenuation filters instead of the blocking filter and a UV filter instead of the exciter filter is provided for normal illumination without fluorescence excitation.
According to the invention, the observation zoom and the illumination zoom are coupled with one another or also selectively adjusted by two drives, respectively, preferably spindle drives. As is described in DE 198 222 56, the disclosure of which is hereby adopted herein in its entirety by reference, the spindle drives for the observation zoom and for the light zoom are synchronously controlled in a stepwise manner by an electronic control unit working with application-specific software corresponding to the respective linear or optionally nonlinear paths in such a way that the illumination in the illumination beam path is correspondingly adapted to the size ratios and aperture ratios in the object plane in the coupled mode when changing the magnification in the observation beam path.
The solution, according to the invention, of an incident light fluorescence stereo microscope body is particularly advantageous for the user because the coaxial incident light fluorescence excitation is carried out with a high illumination aperture for all observation zoom magnifications and, because of the coupling of the light zoom and observation zoom, the size of the sharply defined circular image field, which is illuminated in a sufficiently homogeneous manner, is always adapted to the scale ratios of the observation zoom with objective and lies in the focused object plane. It is advantageous when the optics of the light zoom have a high light-transmitting value and are optimized for a sufficiently high transparency for UV and blue light so that a high illumination intensity can be achieved in the object plane. Another advantage consists in that the excitation filters and blocking filters arranged in the space between the illumination deflecting element, the optics at the entrance of the observation zoom and the objective which is suitable for fluorescence can be quickly changed, exchanged and covered.
In the drawings:
Preferred embodiment examples of the incident light fluorescence stereo microscope body, according to the invention, with integrated fluorescence illumination and stereo microscope observation channels are described more fully with reference to the drawings in FIGS. 7 to 14.
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In the illumination beam path, AL designates the measurement of the distance from the objective to the light outlet surface of the deflecting prism with lens, BL designates the measurement of the distance from the objective to the light outlet surface of the first zoom lens, CL designates the measurement of the distance from the objective to the light outlet surface of the second zoom lens, DL designates the measurement of the distance from the objective to the light outlet surface of the third zoom lens, and EL designates the measurement of the distance from the objective to the light outlet surface of the fourth zoom lens. The diagram shows the movement dynamics of the zoom systems in the observation beam path and illumination beam path as a function of microscope magnification.
A preferred embodiment example for the zoom system in the illumination beam path is shown in the following table:
Surface number 1 is assigned to the objective exit surface, surface numbers 2 to 5 are assigned to the first lens group (LG 1), surface numbers 6 and 7 are assigned to the second lens group (LG 2), surface numbers 8 and 9 are assigned to the third lens group (LG #), surface numbers 10 and 11 are assigned to the fourth lens group (LG 4) and surface numbers 12 and 13 are assigned to the fifth lens group (LG 5). During the zoom movement, the prism entrance surface (surface number 2) is the aperture diaphragm of the illumination beam path with zoom magnifications in the observation beam path of 4×-10×, the movable collecting lens (surface number 7) is the aperture diaphragm of the illumination beam path with zoom magnifications in the observation beam path of 0.8×-4×.
The surface numbers of a lens or lens component, the radius of curvature of the respective surface, the distance to the next surface, the index of refraction (ne) and the dispersion (Abbe number ve=(ne−1)/(nF′−nC′)) are listed in the columns of the table. An air gap is represented by an empty row in the material parameters and L1, L2, L3 and L4 designate the variable distances between the lenses or lens groups of the zoom system.
When the observation zoom is coupled with the light zoom, the distances L1, L2, L3 and L4 are specified in the following table depending on the zoom magnification in the observation beam path qZB, wherein the size of the observed and illuminated object field diameter Ø OFBA and the distances L1, L2, L3 and L4 are associated with the zoom magnification in the observation beam path qZB:
The zoom magnification in the observation beam path (qZ), the size of the object field diameter (Ø OFBA) observed and illuminated as a function of the zoom magnification, and the variable distances L1, L2, L3 and L4 between the lenses or lens groups are listed in the columns of the table.
The invention is not limited to the embodiment examples shown herein. Further developments in the art do not lead to a departure from the protective scope of the claims
While the foregoing description and drawings represent the present invention, it will be obvious to those skilled in the art that various changes may be made therein without departing from the true spirit and scope of the present invention.
Reference Numbers
Number | Date | Country | Kind |
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103 55 523.4 | Nov 2003 | DE | national |