Immersion microscope objective

[0001] This application claims priority of German Application No. 10 2004 051 357.0, filed Oct. 19, 2004, the complete disclosure of which is hereby incorporated by reference.

[0002] a) Field of the Invention

[0003] The invention is directed to an immersion microscope objective. Objectives of this kind are used, for example, in laser scanning microscopy (LSM), to observe the three-dimensional structure of biological objects, particularly tissue cultures and cell cultures. The cells are in a vessel with a solution. The vessel is closed by a thin coverslip. The side of the microscope objective facing the object is located on the other side of the vessel in an immersion liquid—at least the lens surface defining the objective on this side must be in the solution. In this connection, it is required that the refractive indices of the liquids on both sides of the coverslip are similar because only then can the layers of the specimen be imaged sharply at different depths. Since the living biological objects are generally in an aqueous solution, primarily water or glycerin are used as immersion liquids. Further, in order to be able to resolve the fine structure of the objects, immersion microscope objectives of the kind mentioned above need a high numerical aperture.

[0004] b) Description of the Related Art

[0005] Various immersion microscope objectives are known from the prior art. For example, U.S. Pat. No. 5,530,590 provides a microscope objective with three different variants, each with a numerical aperture of 1.15. This objective comprises three groups of lenses, the second lens group being displaceable relative to the two other groups along the optical axis in order to adapt the objective to the thickness of the coverslip for closing the sample, i.e., to compensate the spherical and chromatic aberrations which occur in objectives of this type having a high numerical aperture and which vary with this thickness.

[0006] It is the primary object of the invention to develop an immersion microscope objective which can be used for different immersion media. Moreover, the image contrast is improved over the prior art and the quality of the image contrast is consistently high as far as possible in different immersion media and within given coverslip thickness ranges and temperature ranges.

[0007] This object is met in an immersion microscope objective by providing solution adjusting means by which the objective can be adjusted to different immersion media.

[0008] Accordingly, the same objective can be adjusted to different solutions by means of the solution adjusting means—generally by varying the distances of the individual lenses or lens groups relative to one another. This ensures a uniform quality of the image contrast even with different solutions.

[0009] Further, in an advantageous development of the invention, temperature adjusting means are provided by which the objective can be adjusted and adapted to different temperatures of the respective immersion medium.

[0010] Further, in another development of the invention, coverslip adjusting means are provided by which the objective can be adjusted to different thicknesses of a coverslip which closes an object vessel.

[0011] In a preferred construction which makes it possible to adapt to different requirements in a simple manner and by which a uniformly good image contrast can be achieved, the immersion microscope objective comprises twelve lenses, some of which are combined in cemented lens groups. In order from the object plane along the optical axis, the objective comprises, first, a first lens group with a positive focal length comprising a first lens with a positive focal length and a second lens with a negative focal length, which two lenses are cemented together. Following at a distance A₁ from the first lens group between the lens surfaces along the optical axis is a meniscus-shaped third lens with lens surfaces whose centers of curvature lie on the object side and which has a positive focal length. Following the latter at a distance A₂ from the third lens is a second lens group with a positive focal length comprising a fourth lens with a positive focal length, a fifth lens with a negative focal length, and a sixth lens with a positive focal length. The fourth lens is cemented to the fifth lens and the latter is cemented to the sixth lens. Following at a distance A₃ from the second lens group is a third lens group with a negative focal length comprising a seventh lens with a negative focal length, an eighth lens with a positive focal length, and a ninth lens with a negative focal length. The seventh lens is cemented to the eighth lens and the latter is cemented to the ninth lens. This is followed next at a distance A₄ from the third lens group by a meniscus-shaped tenth lens with lens surfaces whose centers of curvature lie on the image side and which has a positive focal length. Finally, following the latter at a distance A₅ from the tenth lens is a fourth lens group with a negative focal length comprising a meniscus-shaped eleventh lens with a positive focal length and a biconcave twelfth lens which are cemented together. Following at a distance A₆ is a back shutter which closes the objective. The distances A₁, . . . , A₆ can be varied so that it is possible to adjust to the requirements of different solutions, solution temperatures and coverslip thicknesses.

[0012] The first lens and the twelfth lens are preferably made of the same material with a refractive index n_e<1.50 and an Abbe number ν_e>70 at a wavelength of λ_e=546.07 nm. The second lens is advantageously meniscus-shaped, and the centers of curvature of both lens surfaces lie on the object side. The first lens is inserted in the object-side concave curve of the second lens as a filling lens; the lens surface situated on the object side is preferably plane. This makes it possible to move very close to the coverslip so that only a thin film of immersion liquid is between the coverslip and the lens surface.

[0013] In order to adapt to different coverslip thicknesses, immersion media and temperatures and to compensate for various imaging errors that occur, the distances between the individual lenses or lens groups, A₁ to A₆, can be varied along the optical axis. However, since the variation of each individual distance contains many degrees of freedom independent from the others and an optical adjustment can be achieved, as a rule, only after much experimentation, a correction ring serving as solution adjusting means is provided at the objective barrel in a particularly preferred embodiment for varying and adapting the distances A₁ to A₆. The correction ring and the barrel are advantageously provided with solution marks which allow quick adjustment to the respective immersion media.

[0014] In another preferred construction, the correction ring also has the function of temperature adjusting means. For this purpose, the correction ring or the barrel is preferably provided with temperature marks; alternatively, both the correction ring and the barrel can also be provided with marks of this kind.

[0015] In another preferred construction, the correction ring also has the function of coverslip adjusting means. For this purpose, the correction ring or the barrel is preferably provided with coverslip thickness marks; alternatively, both the correction ring and the barrel can also be provided with marks of this kind

[0016] It is assumed by way of approximation first that the distances A₁ to A₆ are in a linear relationship and, further, that each of the distances is in a linear relationship with the temperature, the type of immersion medium, or the coverslip thickness. This approximation is valid for small variations in the coverslip thickness, the composition of the immersion media and slight changes in temperature.

[0017] For example, given two immersion media, water—designated by W—and glycerin—designated by G—the following equations apply for temperatures between T₀=23° C. and T_max=37° C. and for three coverslip thicknesses, taken by way of example, of D₀=0.15 mm, D₁=0.17 mm and D₂=0.19 mm:

with variations in the coverslip thicknesses

[0018] $\begin{array}{cc}\frac{A_{i,G/W,D_2,T}-A_{i,G/W,D_1,T}}{A_{i,G,D_1,T_0}-A_{i,W,D_1,T_0}}=\frac{A_{i,G/W,D_1,T}-A_{i,G/W,D_0,T}}{A_{i,G,D_1,T_0}-A_{i,W,D_1,T_0}}& \left(1\right)\end{array}$
between different air separations

[0019] $\begin{array}{cc}\frac{A_{i,G/W,D_2,T}-A_{i,G/W,D_1,T}}{A_{i,G,D_1,T_0}-A_{i,W,D_1,T_0}}=\frac{A_{j,G/W,D_2,T}-A_{j,G/W,D_1,T}}{A_{j,G,D_1,T_0}-A_{j,W,D_1,T_0}}& \left(2\right)\end{array}$
and with a change in temperature

[0020] $\begin{array}{cc}\frac{A_{i,G/W,D_1,T}-A_{i,G/W,D_1,T_0}}{A_{i,G,D_1,T_0}-A_{i,W,D_1,T_0}}=\frac{A_{j,G/W,D_1,T}-A_{j,G/W,D_1,T_0}}{A_{j,G,D_1,T_0}-A_{j,W,D_1,T_0}},& \left(3\right)\end{array}$
where i and j are natural numbers between 1 and 6 and G/W designates either water or glycerin, so that the distances in the numerators refer in each instance to one of the two solutions. T designates any temperature between T₀ and T_max. Of course, other coverslip thicknesses D can also be selected. In this way, the air separations between the individual lenses or lens groups can be changed to the various conditions with different immersion media, etc. and imaging errors such as spherical aberration and longitudinal chromatic aberrations in particular, but also coma, astigmatism, distortion and field curvature, can be corrected. Because of the linearity, the construction of the objective and its operation can be simplified considerably.

[0021] In this regard, the objective can be constructed in such a way that the distance between the object plane and the object-side surface of the tenth lens along the optical axis—with the thickness of the coverslip remaining the same—remains constant independent from the adjustment to the immersion medium that is used. Therefore, the tenth lens forms a fixed point in the objective.

[0022] An advantageous construction of the immersion microscope objective is given with the constructional data indicated in subclaim 14. The refractive indices refer to a wavelength of 546.07 nm. The Abbe numbers ν_e were calculated from the following equation:

[0023] $v_e=\frac{n_e-1}{n_{F^{\prime }}-n_{C^{\prime }}},$
where n_e is the refractive index at a wavelength of λ_e=546.07 nm, n_C′ is the refractive index at a wavelength of λ_C′=643.85 nm, and n_F′ is the refractive index at a wavelength of λ_F′=479.99 nm. At a distance A₆, the objective is first closed by a back shutter. The lens of a tube system with a focal length of 164.50 mm is located at a distance of 126.50 mm from this back shutter.

[0024] The objective, according to the invention, is suitable for different immersion media, particularly water and glycerin. When water is used the numerical aperture is 1.2, when glycerin is used the numerical aperture is 1.3. The imaging scale is at a magnification of approximately 63×, the field diameter is 20. The errors are best corrected for solution temperatures in the range from 23° C. to 37° C. and coverslip thicknesses between 0.15 mm and 0.19 mm.

[0025] The invention will be described more fully in the following with reference to an embodiment example.

[0034] FIG. 1 and FIG. 2 show possible constructions of the objective according to the constructional data in subclaim 7 which differ only with respect to the distances between the individual lenses or lens groups. The first lens group G1 which has a positive focal length is arranged at far left on the optical axis. It comprises a first lens L1 with a positive focal length and a second lens L2 with a negative focal length. The two lenses are cemented together. Following the first lens group G1 at a distance A₁ from the latter is a third lens L3 with a positive focal length and lens surfaces whose centers of curvature lie on the object side. A second lens group G2 with a positive focal length follows at a distance A₂ from the first lens group G1. The second lens group comprises a fourth, fifth and sixth lens L4, L5 and L6, respectively, having a positive focal length, a negative focal length and a positive focal length, respectively. The fourth lens L4 is cemented to the fifth lens L5, and the fifth lens L5 is cemented to the sixth lens L6. Following at a distance A₃ from the second lens group G2 is a third lens group G3 with a negative focal length. It comprises a seventh lens L7 with a negative focal length, an eighth lens L8 with a positive focal length, and a ninth lens L9 with a negative focal length. The seventh lens L7 is cemented to the eighth lens L8, and the latter is cemented to the ninth lens L9. Following at a distance A₄ from the third lens group G3 is a meniscus-shaped tenth lens L10 with a positive focal length and lens surfaces whose centers of curvature lie on the image side. At a distance A₅ from this lens, there follows a fourth lens group G4 with a negative focal length comprising a meniscus-shaped eleventh lens L11 with a positive focal length and a biconcave twelfth lens L12 which are cemented together. The centers of curvature of the lens surfaces of the eleventh lens L11 lie on the object side. Finally, following at a distance A₆ from the fourth lens group G4 is a back shutter which closes the objective and which is followed at a distance of 126.50 mm by the lens of a tube system—not shown in this drawing—with a focal length of 164.50 mm.

[0035] The two configurations shown in FIGS. 1 and 2 differ from one another only in the selected distances A₁ to A₆ which are determined depending upon coverslip thickness and immersion medium in accordance with the above equations (1) and (2).

[0036] When water is used as immersion medium, the following configurations are obtained, by way of example, at a temperature of 23° C.:

[0037]

	Coverslip thickness	0.15	0.17	0.19
	Working distance	0.215	0.197	0.178
	A1	0.624	0.659	0.693
	A2	1.687	1.553	1.419
	A3	1.267	1.167	1.067
	A4	0.071	0.270	0.470
	A5	4.796	4.770	4.745
	A6	2.504	2.529	2.555

[0038] All of the specified data are given in millimeters. FIG. 1 shows a configuration in which these distances are substantially realized. The working distance refers to the optical working distance between the coverslip and the vertex of the first lens surface.

[0039] When glycerin is used as immersion medium, the following configurations are obtained, by way of example, at a temperature of 23° C.:

[0040]

	Coverslip thickness	0.15	0.17	0.19
	Working distance	0.214	0.195	0.176
	A1	1.015	1.023	1.030
	A2	0.175	0.147	0.118
	A3	0.136	0.115	0.094
	A4	2.325	2.367	2.408
	A5	4.510	4.504	4.499
	A6	2.790	2.796	2.801

[0041] All of the specified data are given in millimeters. A configuration in which these distances are substantially realized is shown in FIG. 2.

[0042] Accordingly, the objective is suitable for different immersion media, in particular water and glycerin. When water is used, the numerical aperture is 1.2 and when glycerin is used the numerical aperture is 1.3. The imaging scale is −62.4 when water is used as immersion medium and −63.8 when glycerin is used as immersion medium. The field number in both cases is 20. The objective has very good transmission in the visible spectral region and an internal transmission of more than 72% at a wavelength of 365 nm, which is important particularly in applications in fluorescence microscopy.

[0043] FIGS. 3 to 6 show different imaging errors of the objective using water and glycerin, respectively, as immersion medium.

[0044] FIG. 3 shows an image error, namely the transverse aberration as a function of aperture, for the objective when water is used as immersion medium, specifically in the tangential section at two points in the image field, i.e., at the image center (a) and image edge (b). A scale division on the ordinate axis corresponds to 0.08 mm. The transverse aberrations are shown for three different wavelengths: the aberrations at λ_e=546.07 nm are shown by a solid line, the aberrations at λ_C′=643.85 nm are shown by a dashed line, and the aberrations at λ_F′=479.99 nm are shown by a dotted line. FIG. 4 shows the corresponding transverse aberrations of the objective when glycerin is used as immersion medium.

[0045] FIG. 5 shows other image errors of the objective according to the invention when water is used as immersion medium. The image height is shown in millimeters in the vertical axis. The scale divisions on the ordinate correspond, respectively, to √(1/4), √(2/4), √(3/4) and √(4/4) of the maximum field height of 10 mm. FIG. 5(a) shows the chromatic difference of magnification in per mil scaled to the respective image height. FIG. 5(b) shows the distortion of the microscope objective in percent. Finally, FIG. 5(c) shows the astigmatism of the objective in Rayleigh units (R.E.), specifically for the three wavelengths λe, λ_C′, and λ_F′ mentioned above. The solid line indicates the sagittal astigmatism and the dashed line shows the tangential astigmatism. FIG. 6 shows the corresponding errors when glycerin is used as immersion medium.

[0046] These imaging errors and other errors such as spherical aberration, coma and image field curvature are corrected at λ_e and corrected to the greatest extent at λ_C′ and λ_F′. Also, the longitudinal chromatic aberrations of the secondary spectrum—the deviation of the focus positions at λ_C′ and λ_F′ with reference to λ_e—were minimized to the extent that the difference in the focus positions between λ_e and λ_C′ lies within the area of the depth of focus—λ/NA², where NA is the numerical aperture. Since the focus positions coincide at wavelengths λ_C′ and λ_F′ and the image field is flattened, this is a semi-Apochromat Plan Neofluar objective.

[0047] Finally, FIG. 7 shows a correction ring 1 which runs around the barrel 2 of the objective and can be rotated around the latter relative to the barrel 2 so that, by rotating, it is possible to switch between the two immersion media, water and glycerin. In addition, two solution marks 3, 4 are arranged on the barrel 2. Solution mark 3 designates the adjustment to glycerin immersion medium which is illustrated by the lettering “Glyc” above this mark. Solution mark 4 designates the adjustment to the water immersion medium, which is illustrated by the letter “W” on the barrel 2 above this mark. The solution marks 3, 4 relate to a coverslip thickness of 0.17 mm. Coverslip thickness marks 5, 6 are provided to the left and right of the solution marks 3, 4. The coverslip thickness marks 5 designate the adjustment to a coverslip thickness of 0.19 mm for the selected immersion medium. Coverslip thickness marks 6 designate the adjustment to a coverslip thickness of 0.15 mm for the selected immersion medium. In the present example, this is the limiting case for which the objective satisfies the required conditions such as good image contrast and validity of the linear approximation described above, and the usable coverslip thickness range is illustrated once again by the lettering “0.19-0.15”. Of course, it is possible to adjust to other, intermediate coverslip thicknesses by visual interpolation. It is also possible to arrange additional marks, also for other solutions.

[0048] Two temperature marks 7, 8 are arranged on the ring to mark the limits of the temperature range for which the objective meets the required conditions. In this case also, intermediate adjustments are possible. Temperature mark 7 designates a temperature of 37° C., temperature mark 8 designates a temperature of 23° C. To facilitate operation, temperature mark 7 and temperature mark 8 can advantageously be maintained mnemonically in red and blue, respectively. By rotating the correction ring, the objective can be adapted to the conditions under which the microscope examination is to be carried out.

[0049] 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.

• [0050] A₁, . . . , A₆ distances between the lenses and lens groups
• [0051] G1, . . . , G4 lens groups
• [0052] L1, . . . , L12 lenses
• [0053] 1 correction ring
• [0054] 2 barrel
• [0055] 3, 4 solution mark
• [0056] 5, 6 coverslip thickness mark
• [0057] 7, 8 temperature mark