Title: Microscope
Abstract: The invention relates to a microscope comprising a microscope housing (18), an optics system (16) consisting of at least one lens system that contains at least one respective lens (48) and is positioned at one end of a passage (19) of the microscope housing (18), at least one observation device, in particular an ocular, located at the other end of the passage (19), an illumination device, whose light forms at least one illumination beam (44), originating from a plane of incidence (45) that vertically intersects the passage (19), said beam traversing the lens system and striking an object carrier (36) at a predetermined angle (β). According to the invention, the illumination beam or beams (44) originating from the plane of incidence (45) is/are provided by an optical device, whose cross-section lying in the vicinity of the passage (19) is substantially smaller than the cross-section of said passage (19).
Patent Number: 6,987,609 Issued on 01/17/2006 to Tischer, et al.
| Inventors:
|
Tischer; Christian (Heidelberg, DE);
Florin; Ernst-Ludwig (Gaiberg, DE)
|
| Assignee:
|
Europaisches Laboratorium fur Mole kularbiologie (EMBL) (Heidelberg, DE)
|
| Appl. No.:
|
488769 |
| Filed:
|
September 4, 2002 |
| PCT Filed:
|
September 4, 2002
|
| PCT NO:
|
PCT/EP02/09901
|
| 371 Date:
|
March 5, 2004
|
| 102(e) Date:
|
March 5, 2004
|
| PCT PUB.NO.:
|
WO03/023483 |
| PCT PUB. Date:
|
March 20, 2003 |
Foreign Application Priority Data
| Sep 05, 2001[DE] | 101 43 481 |
| Current U.S. Class: |
359/385 |
| Current Intern'l Class: |
G02B 21/06 (20060101) |
| Field of Search: |
359/368,385,387,388,389,390
|
References Cited [Referenced By]
U.S. Patent Documents
| 4783159 | Nov., 1988 | Takagi et al.
| |
| 5126877 | Jun., 1992 | Biber.
| |
| 5493443 | Feb., 1996 | Simon et al.
| |
| 5675145 | Oct., 1997 | Toda et al.
| |
| 5859727 | Jan., 1999 | Tsuchiya.
| |
| 6313944 | Nov., 2001 | Kawahito.
| |
| 6751018 | Jun., 2004 | Kawano et al.
| |
| 6819484 | Nov., 2004 | Aono et al.
| |
| Foreign Patent Documents |
| 42 43 144 | Jun., 1994 | DE.
| |
| 40 28 605 | Sep., 1997 | DE.
| |
| 198 42 153 | Mar., 2000 | DE.
| |
| 1 109 046 | Jun., 2001 | EP.
| |
| 10 096862 | Apr., 1998 | JP.
| |
| WO 00 5087/8 | Aug., 2000 | WO.
| |
Other References
Patent Abstracts of Japan, vol. 1998, No. 9, Jul. 31, 1998.
Tokunaga M., et al., "Single Molecule Imaging of Fluorophores and Enzymatic Reactions
Achieved by Objective-Type Total Internal Reflection Fluorescence Microscopy",
Biochemical and Biophysical Research Communications, vol. 235, 1997, pp. 47-53.
|
Primary Examiner: Robinson; Mark A.
Attorney, Agent or Firm: Rothwell Figg Ernst & Manbeck
Claims
What is claimed is:
1. A TIR microscope (
10) comprising
a microscope housing (
18),
an optics system (
16) at one end of a passage (
19) in said microscope
housing (
18), said optics system (
16) comprising at least one lens
system which includes at least one lens (
48), said optics system (
16)
determining an optical axis (
54) and an aperture of said microscope (
10),
at least one observation device (
14,
22), in particular an eyepiece,
at another end of said passage (
19),
an illumination device (
24) whose illumination light forms at least one
illumination beam (
44) which passes through said lens system, said at least
one illumination beam (
44) being actually or virtually focused in the focal
plane (
46) facing said at least one observation device (
14,
22)
of said at least one lens system (
48) through which said illumination beam
(
44) passes and striking a microscope slide (
36) at a predetermined
angle (
13) to the optical axis (
54) which is greater than zero, said
angle being in the range of a total reflection angle when said illumination light
strikes an interface between an object to be observed and a microscope slide, characterized
in that said illumination beam, after emanating from a plane of incidence (
45)
which perpendicularly intersects said passage (
19) between a front lens
(
48) located closest to the object to be detected of the optics system (
16)
and said observation device (
14,
22), is focused in the focal plane
(
46) facing said at least one observation device (
14,
22)
of said optics system (
16), and between said plane of incidence and said
lens system substantially extends parallel to and at a distance from the optical
axis, said focused illumination beam (
44) being supplied in the plane of
incidence (
45) by an optical device which is located in the region of said
optics system, which has a cross section in the area of said aperture which is
much smaller than the cross section of said aperture and which is displaceable
in the radial direction with respect to the optical axis (
54).
2. The microscope (
10) according to claim 1, characterized in that the
cross section of said optical device providing said at least one illumination beam
(
44) emanating from said plane of incidence (
45), said cross section
being situated in the area of said aperture, is at most half as large as the cross
section of said aperture.
3. The microscope (
10) according to claim 1, characterized in that the
cross section of said optical device providing said at least one illumination beam
(
44) emanating from said plane of incidence (
45), said cross section
being situated in the area of said aperture, amounts to at most 10% of the cross
section of said aperture.
4. The microscope (
10) according to claim 1, characterized in that the
cross section of said optical device providing said at least one illumination beam
(
44) emanating from said plane of incidence (
45), said cross section
being situated in the area of said aperture, amounts to at most 1% of the cross
section of said aperture.
5. The microscope (
10) according to claim 4, characterized by at least
one detection device in the area of said aperture.
6. The microscope (
10) according to claim 1, characterized in that said
optical device providing said at least one illumination beam (
44) emanating
from said plane of incidence (
45) is situated outside of the optical axis (
54).
7. The microscope (
10) according to claim 1, characterized in that the
divergence angle (a) of said at least one illumination beam (
44) emanating
from said plane of incidence (
45) is variable.
8. The microscope (
10) according to claim 7, characterized in that an
adjustable aperture diaphragm (
60) is arranged in the beam path of said
at least one illumination beam (
44).
9. The microscope (
10) according to claim 7, characterized in that the
focal depth of an optical device (
52) focusing said illumination beam (
44)
in said focal plane (
46) facing said at least one observation device (
14,
22) is variable.
10. The microscope (
10) according to claim 1, characterized in that said
at least one illumination beam (
44) is essentially coherent.
11. The microscope (
10) according to claim 10, characterized in that at
least two illumination beams (
44) cause interference in the area of an object (
38).
12. The microscope (
10) according to claim 1, characterized in that said
at least one illumination beam (
44) is incoherent.
13. The microscope (
10) according to claim 1, characterized in that the
fraction of said at least one illumination beam (
44) reflected back in the
area of said object (
38), i.e., the reflected beam (
56), is absorbed.
14. The microscope (
10) according to claim 13, characterized by at least
one absorber (
58) for said reflected beam (
56) in the area of said
passage (
19).
15. The microscope (
10) according to claim 1, characterized in that the
fraction of said at least one illumination beam (
44) that is reflected back
in the area of said object (
38), i.e. the reflected beam (
56), is detected.
16. The microscope (
10) according to claim 1, characterized by at least
one light source in the area of said aperture.
17. The microscope (
10) according to claim 1, characterized by at least
one deflecting unit (
50,
66) in the area of said aperture.
18. The microscope (
10) according to claim 17, characterized in that said
at least one illumination beam (
44) is coupled into said aperture by said
at least one deflecting unit (
50).
19. The microscope (
10) according to claim 17, characterized in that said
reflected beam (
56) is output out of said aperture by said deflecting unit (
66).
20. The microscope (
10) according to claim 17, characterized in that said
at least one deflecting unit (
50,
66) includes a prism (
150,
166).
21. The microscope (
10) according to claim 20, characterized in that said
prism (
650,
666) is connected to a lightguide (
678,
682).
22. The microscope (
10) according to claim 17, characterized in that said
at least one deflecting unit (
50,
66) includes a mirror (
350,
366).
23. The microscope (
10) according to claim 17, characterized in that said
at least one deflecting unit (
50,
66) includes a curved mirror (
450,
466).
24. The microscope (
10) according to claim 17, characterized in that said
at least one deflecting unit (
50,
66) includes a curved lightguide
(
778,
782).
25. The microscope (
10) according to claim 17 characterized in that a
deflecting unit (
576) into which at least two deflecting units (
50,
66) are integrated is provided.
26. The microscope (
10) according to claim 25, characterized in that said
deflecting unit (
576) is designed to be approximately ring-shaped and is
arranged concentrically with the optical axis (
54).
27. The microscope (
10) according to claim 17, characterized by a rotational
device for said illumination beam (
44).
28. The microscope (
10) according to claim 17, characterized by an aperture
diaphragm (
62) in said passage (
19) on the side of said plane of
incidence (
45) facing said at least one observation device (
14,
22),
for removing said at least one deflecting unit from the observation beam path.
29. The microscope (
10) according to claim 17, characterized by a filter
arrangement (
272) facing, with respect to said plane of incidence (
45),
the side of said at least one observation device (
14,
22).
30. An adapter (
40) for a microscope according to claim 1, characterized
in that said adapter (
40) can be inserted into said passage (
19)
in said microscope housing (
18) between said optics system (
16) and
said observation device (
14,
22) and includes at least one optical
device which provides said illumination beam (
44).
31. The adapter (
240) according to claim 30, characterized in that said
adapter (
240) can be inserted between said optics system (
16) and
an optics system connection of said microscope housing (
18).
32. The adapter (
40) according to claim 30, characterized in that said
adapter (
40) can be inserted into a receptacle (
32) of said microscope
housing (
18) which is provided for other optical elements, in particular
those used for DIC microscopy.
33. The adapter (
40) according to claim 30, characterized in that at least
one optical element is integrated into said adapter (
40).
34. An optics system (
816,
916,
1016,
1116) for a microscope
(
10) according to claim 1, characterized in that said optics system (
816,
916,
1016,
1116) includes said at least one optical device
which provides said illumination beam (
44).
35. The optics system (
816,
916,
1016,
1116) according
to claim 34, characterized in that at least one optical element is integrated into
said optics system (
816,
916,
1016,
1116).
Description
CROSS REFERENCE TO RELATED APPLICATION
This application is a 35 USC § 371 National Phase Entry Application from
PCT/EP02/09901, filed Sep. 4, 2002, and designating the U.S.
BACKGROUND OF THE INVENTION
This invention relates to a microscope. More particularly, this invention relates
to a microscope which includes a housing, an optics system having at least one
lens system which includes at least one lens, at least one observation device and
an illumination device producing at least one illumination beam that strikes a
microscope slide at a predetermined angle.
Such a microscope is known (
Biochemical and Biophysical Research Communications,
235, 47-53). It is used primarily for the microscopy technique known by the
acronym TIRM (=total internal reflection microscopy). In this technique, the illumination
beam is totally reflected at an interface formed between the microscope slide and
the object (the refractive index of the microscope slide is greater than the refractive
index of the object), wherein the illumination beam traverses the microscope slide,
so that an evanescent light field originating from the point of reflection penetrates
into the object, with the intensity declining exponentially. This light field is
used for strictly locally delimited illumination of areas of the object near the
microscope slide. These areas may then be examined in the usual way through the
optics system and the observation device, e.g., an ocular or a camera.
In one of two possible TIRM configurations, the object is on the side of the
microscope
slide facing the microscope with a corresponding guide for the illumination beam
in the manner of a back-lighting configuration (see, for example,
Nature,
vol. 374, pp. 555-559 or
Topics in Fluorescence Spectroscopy, vol. 3, ed.
by J. Lakowicz, Plenum Press, New York, 1992, p. 314 ff.). The alternative reflected
light arrangement, in which the object is situated on the side of the microscope
slide facing away from the microscope, is used in the related art cited in the
preamble. The illumination light here emanates from a laser outside of the passage
in the microscope and is directed via a mirror system at a dichroic beam splitter
mirror in the passage and then follows the path of the observation beam in parallel
with the optical axis through the optics system (objective) to the microscope slide
and the object. The dichroic mirror interferes with microscopic observation of
the object because it passes through the entire cross section of the passage and
thus weakens the light beam observable through the ocular in a ratio that depends
on the wavelength.
SUMMARY OF THE INVENTION
The object of this invention is to provide a microscope of the type defined in
the preamble so that it causes the least possible impairment in microscopy possibilities,
in particular with regard to the possible wavelengths of the illumination light
and/or the observation light.
This object is achieved by the fact that said at least one illumination beam
emanating from said plane of incidence is provided by an optical device whose cross
section in the area of said passage is much smaller than the Gross section of said passage.
The position of the plane of incidence may be selected as desired in the passage
between the front lens of the optics system (objective) and the observation device,
with lateral coupling into the front lens also being possible. Due to the fact
that the illumination light is concentrated on at least one illumination beam,
locally limited beam guidance elements may be used accordingly, which cause only
minor impairment in the observation beam path of the microscope (usually outside
of the optical axis). This fine illumination beam may be passed through the lens
system traversed by the beam (optionally the front lens) of the optics system without
requiring any additional measures. In particular, for coupling of the illumination
light it is possible to omit the use of any beam splitting equipment extending
through the passage.
A light source for the illumination light might be, for example, a laser which
emits an illumination beam bundle of parallel individual beams with an essentially
spot-shaped beam cross section. However, it is preferred that in the focal plane
facing the observation equipment hereinafter also referred to as the back focal
plane, of the lens system traversed by the beam of the optics system said at least
one illumination beam is virtually or actually focused at the point. This achieves
the result that the illumination beam emitted from the point in the focal plane
as a divergent beam bundle is formed by a beam bundle of individual beams running
in parallel after passing through the lens system, all of the individual beams
meeting the condition of total reflection equally.
The illumination beam could in principle also pass through the lens system of
the optics system at an inclination to the optical axis. However, it is preferable
for the at least one illumination beam to run essentially parallel to the optical
axis through the lens system traversed by the beam of the optics system. The illumination
beam here may fall outside of the optical axis as well as along the optical axis
through the lens system traversed by the beam of the optics system. The greater
the distance from the optical axis in the radial direction where the illumination
beam strikes the lens system of the optics system, the greater is the angle of
reflection of the illumination beam at the interface between the microscope slide
and the object. To meet the condition of total reflection, one would therefore
preferably select an arrangement in which the illumination beam runs near the edge
of the aperture diaphragm of the optics system through the lens system traversed
by the beam of the optics system. A special application is obtained when the illumination
beam traverses through the lens system traversed by the beam of the optics system
at the center of the passage along the optical axis. In this case, the illumination
beam strikes the interface between the microscope slide and the object essentially
at a right angle, so that it is reflected back into the microscope along the same
path. In this way it is possible to implement a microscope having a reflected light
configuration in which the light reflected back from the object can be observed
without having to use beam splitter equipment which would interfere with the observation
light, because only a small area at the center of the field of vision of the microscope
is included with the device providing the illumination beam
In an especially preferred embodiment, the at least one illumination beam is
displaceable
in the radial direction with respect to the optical axis. The lateral displacement
can be implemented through technically simple means, in particular through deflector
mirrors, deflector prisms or the like, all of which are displaceable in the radial
direction (with respect to the optical axis). A displacement in the radial direction
leads directly to a corresponding change in the angle of reflection at the interface
between the microscope slide and the object. The total reflection angle, which
varies from one case to the next (depending on the refractive indices of the microscope
slide and the object) can be adjusted in any desired manner. In addition, the depth
of penetration of the light field into the object behind the interface on which
the total reflection occurs depends on the angle of incidence of the light beam.
The depth of the illuminated object volume can thus be altered by radial displacement
of the illumination beam. In addition, the depth of the illuminated volume can
also be varied by choosing illumination light of a different wavelength. Since
the inventive microscope allows the use of illumination light of any wavelength,
a continuous variation in the illuminated object volume is also possible in this
way. A targeted change in the illuminated object volume and observation of the
intensity of the light emitted by this volume can be utilized for example, to determine
the size of an object in the area of this object volume, because the depth of penetration
of the illumination light can be calculated from its wavelength and the total reflection
angle set (see in this regard, for example,
Topics in Fluorescence Spectroscopy,
vol. 3, Plenum Press, New York, 1992, pp. 289 ff.).
Furthermore, it is proposed that the angle of divergence of the at least
one illumination beam emitted from the plane of incidence shall be variable. A
change in the angle of divergence results in a corresponding change in the cross
section of the illumination beam emitted from the lens system and thus also a change
in the size of the illuminated area in the object.
The angle of divergence can be varied according to the invention by adjusting
an aperture diaphragm in the beam path of the illumination beam or by varying the
focal depth of the optical unit focusing the illumination beam in the back focal plane.
If an as uniform as possible illumination of the object is required, then preferably
an incoherent illumination beam is used. However, if an increased positional resolution
and/or structured illumination is desired, then preferably one or more essentially
coherent illumination beams is used, with the possibility of interference in the
area of the object.
If the object to be observed is in the area of the optical axis, as is generally
the case, the totally reflected component of the illumination beam is then reflected
back into the optics system, at least when the microscope slide is arranged perpendicular
to the optical axis. Normally, with TIR microscopy, fluorescent light is observed
emanating from the illuminated part of the object and containing information about
the object. In order not to interfere with microscopic observation of the object,
the portion of the illumination beam reflected back, i.e., the reflected beam,
is absorbed according to this invention, preferably through an absorber or a filter
directly downstream from the lens system traversed by the beam. Alternatively or
additionally, the reflected beam may also be detected, whether for adjustment of
the arrangement, in particular for adjusting the total reflection angle, and/or
for absorption measurements on the object. A detection device may be provided in
the area of the passage for this purpose. Through the observation of absorption
processes at the interface between the microscope slide and the object as a function
of the wavelength of the illumination beam used, additional information about the
object can also be obtained from the reflected beam. It is important in particular
here that light of any wavelength can be observed, as is the case according to
this invention.
Greater freedom in the configuration and design of the detection device are
obtained when the reflected beam is guided out of the microscope beam path by means
of a deflecting unit in the area of the passage. A corresponding deflecting unit
may also be provided for coupling of the illumination beam into the microscope.
Here again, the advantage of a greater freedom in the arrangement and design of
the light source and the beam guidance is achieved by means of corresponding optical
systems of the illumination device. Nonetheless it may be advantageous in certain
situations, e.g., when not enough room is available for external light sources,
to place a light source with a spot-shaped cross section if possible, e.g., a laser
diode, directly in the passage in the area of the plane of incidence.
In a first embodiment of this invention, the deflecting unit includes a prism.
Deflection is then accomplished by total reflection on one of the prism faces.
The prism causes very little interference with the observation field of the microscope.
It can be held and mounted in a simple manner. A movable mount for adjusting the
beam path, in particular for adaptation of the reflection angle by displacement
in the radial direction, can be implemented with little structural complexity.
Thus, for example, it is possible to use a rod prism having a deflection prism
face in the passage with linear movement guidance and mounting outside of the passage.
Prisms are available in different shapes and designs, depending on the desired
specifications. A 90° prism is used in the basic version. The prism may be
rounded so as to interfere with the field of observation as little as possible.
Instead of the 90° configuration, other angles may also be selected. Coupling
an optical lightguide to a deflection prism offers special advantages with regard
to flexibility in introducing the illumination beam.
In cases in which dispersion of the illumination beam is to be prevented or the
wavelength of the illumination beam is varied, then instead of the prism, a deflecting
mirror may also be used. In general, a planar deflecting mirror will be used. However,
it is also conceivable to use a curved mirror, which can be useful in focusing
the beam in the back focal plane, so that corresponding lens elements may be omitted.
In the inventive configuration, at least one illumination beam is used, and a
plurality of illumination beams may also be used, depending on the particular requirements.
In such a case, a deflecting unit in which a corresponding plurality of deflecting
units is structurally integrated is also used to advantage. In this case, the deflecting
unit is preferably designed to be approximately ring-shaped and is arranged so
that it is concentric with the optical axis. Suitably inclined reflection surfaces
(mirror surfaces and/or prism surfaces) are provided on this ring in addition,
it is also possible to introduce the illumination beam into the beam path of the
microscope by using a curved optical lightguide. In this case the deflecting unit
is formed by the curved optical lightguide itself.
The configuration according to this invention, comprising individual illumination
beams, in particular a single illumination beam, makes it possible to illuminate
the object with an illumination beam coming from a single direction. In the case
of designs having a complex three-dimensional structure, such as cells, different
images of the object may be obtained, depending on the angle of incidence of the
illumination beam. This provides additional information regarding the object. Therefore
in such a case, it may be advantageous if, as proposed according to this invention,
the at least one deflecting unit or the light source (if a light source is situated
in the passage) is able to rotate about the optical axis. To detect or absorb the
reflected beam, it is also expedient to rotate the detection device and/or the
absorption device about the optical axis, if necessary.
Rotation of the deflecting unit and thus rotation of the illumination beam
striking the specimen may be performed between measurements, as mentioned above,
or even during a measurement in order to obtain uniform illumination of the object.
Since the illumination light is coupled into the microscope only in the area
of the optics system via the at least one deflecting unit according to this invention
and if necessary is also output again there, the light that serves to illuminate
the object and is not part of the reflected beam and nevertheless is reflected
and/or scattered back into the microscope can be removed easily from the observation
beam path, namely by a filter arrangement on the side of the at least one deflecting
unit that is remote from the object.
The restriction on the beam guidance of the illumination beam according to this
invention as well as that of the reflected beam to the optics system area permits
the use of an adapter, which is situated between the optics system and the observation
device and has at least the one deflecting unit or the one light source, if necessary.
With the help of the adapter, it is readily possible to couple the light source
of the illumination device, wherein the light source (in the case when it is not
situated in the passage) may be independent of the adapter or may also be integrated
into the adapter. The adapter can optionally be removed again or replaced by other
adapter devices, which allows versatile use of the microscope. Existing microscopes
can be upgraded or modified by replacing adapter inserts that are traditionally
present, in particular so-called DIC (differential interference contrast) sliders
which have optical components that are used for DIC microscopy, with an adapter
according to this invention. Another simple possibility of modifying existing microscopes
is provided by an adapter situated between the optics system and the optics system
connection of the microscope housing. Ideally such an adapter has a connection
on its side facing the optics system similar to the optics system connection of
the microscope housing into which the optics system normally fits. In addition,
it is advantageous if the adapter has a similar connection on its side facing the
microscope housing like the connection of the optics system facing the microscope
housing. In this case, the adapter can easily be inserted into the passage between
the optics system and the microscope housing without having to modify an existing
microscope, because in a microscope, the length of the passage between the observation
device and the connection of the microscope housing on the optics system end can
usually be varied.
It is even possible to integrate the deflecting unit into a suitably adapted
optics
system. The optical components can thus be optimally coordinated. Furthermore,
no other modification of the microscope is necessary, because one need only replace
a traditional optics system with an optics system according to this invention.
In addition to the deflecting unit, the light source and/or the detector unit
for the reflected beam may also be integrated into the adapter box and/or the optics
system according to this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
This invention is explained below on the basis of several exemplary embodiments
with reference to the drawing, showing:
FIG. 1 a roughly schematic sectional diagram of a microscope having a reflected
light configuration and an adapter, the adapter being indicated with dotted lines;
FIG. 2 a detailed view (arrow A) of the configuration in FIG. 1 with an embodiment
of an adapter designed according to this invention and an external light source
(not shown);
FIG. 3 a view according to FIG. 2 of a modified adapter with an internal light source;
FIG. 4 a sectional view like those in FIGS. 2 and 3 of another embodiment of
the adapter together with the optics system, omitting the other microscope components
and including a prism as the deflecting unit;
FIG. 5 a section of the configuration according to FIG. 4 according to line
V—V in FIG. 4;
FIG. 6 a section like that in FIG. 4 through another embodiment of the adapter,
omitting a part of the adapter that is remote from the object and having a mirror
as the deflecting unit;
FIG. 7 a view like that in FIG. 6 shows another embodiment of the adapter with
a curved mirror as the deflecting unit;
FIG. 8 a greatly simplified perspective schematic view of a deflecting unit
of concentrically arranged deflecting elements arranged in a ring together with
the lens system, represented by a single lens, and the microscope slide with the
object and the beam path indicated;
FIG. 9 a view like that in FIG. 6 with a prismatic deflecting element and an
optical lightguide connected;
FIG. 10 a view like that in FIG. 9 with a deflecting element formed by a curved
section of an optical lightguide,
FIG. 11 a sectional view with a deflecting unit in the form of a rod prism integrated
into the optics system;
FIG. 12 a configuration like that in FIG. 11 with a deflecting unit in the form
of a double prism integrated into the optics system;
FIG. 13 a view like that in FIG. 11 with an optical lightguide connected to
the rod prism, and
FIG. 14 a configuration like that in FIG. 13 with a curved section of optical
lightguide as the deflecting unit.
DETAILED DESCRIPTION
The schematic sectional drawing according to FIG. 1 shows a traditional microscope
in an inverted configuration (Carl Zeiss Axiovert 100, 135, 135M, Carl Zeiss Jena).
The main parts of the microscope, which is labeled as
10 in general can
be seen here, namely a visual observation part
12 with an ocular
14
at one end of the beam path and an optics system (objective)
16 on the other
end and a plurality of optical components inserted in between within a passage
19 of a microscope housing
18. The beam path of a photographic observation
part with a camera
22, the outline of which is shown here, can be coupled
into the beam path.
Furthermore, the beam path of an illumination part
24 can be
coupled into the visual beam path (via a partially transparent mirror
26
beneath an optics system revolver head
28 which carries the optics system
16 with the axis of rotation
30 of the revolver).
With the traditional microscope
10, an adapter receptacle
32 (indicated
with dotted lines in FIG. 1) is provided, serving to accommodate adapters (DIC
sliders), in particular a Wollaston prism for observation of objects in differential
interference contrast.
In FIG. 1, a microscope slide (omitted in FIG. 1) for the object to be observed
with the microscope is connected to the optics system
16 at the top. In
the arrangement shown here, the object is illuminated by the illumination part
from the observer's side. If transillumination of the object is desired, then an
illumination part (not shown in FIG. 1) for a corresponding illumination of the
sample (from above in FIG. 1) may be arranged on a mount
34 which protrudes
upward from the microscope
10 and is shown in a cutaway view in FIG. 1.
The area of the revolver head
28 indicated by the circular area A in FIG.
1 is shown in FIG. 2 together with the microscope slide
36 and the object
38 on its top side (side of the microscope slide
36 formed by a glass
slide facing away from the optics system
16).
An adapter
40 according to this invention is inserted instead of a traditional
adapter into the receptacle
32. This adapter
40 can thus be used
without further modification of the known microscope
10. It is used to illuminate
the object
38 according to the essentially known principle of TIR (total
internal reflection) microscopy.
In this microscopy technique light, coming from the microscope slide side is
totally
reflected at an interface
42 formed between the microscope slide
36
and the object
38 (refractive index of the object lower than the refractive
index of the microscope slide). A so-called evanescent illumination field extends
from the total reflection point into the object, declining exponentially with the
distance from the interface. This yields a type of illumination which is very sharply
delimited locally in the axial direction. To obtain the total reflection angle,
an optics system having a sufficiently high numeric aperture NA is used. For example,
in the case of a glass-water interface, an optics system with a high numeric aperture
(NA>1.33) must be used to obtain the total reflection angle.
The depth of penetration of this field depends on the particular reflection angle
β (which continues to obey the total reflection condition). Accordingly,
by varying the reflection angle β the depth of illumination can also be varied.
According to this invention, an illumination beam
44 having a greatly
limited cross section is used. The limitation is such that it essentially forms
a spot in the back focal plane
46 of the optics system
16. This refers
to the focal plane which corresponds to the lens system of the optics system
16
through which the illumination beam
44 passes (indicated by a single front
lens
48 in FIG. 2). Therefore, depending on the design of the optics system
16, other lenses or lens systems of the optics system
16 may also
be situated downstream from the coupling point (prism
50) of the illumination
beam
44 (in the direction of the ocular).
When using a precision laser beam as the illumination beam
44, it can
be passed through the lens system on the front (represented by the front lens
48)
without using focusing lenses, although a weak divergence of the beam emitted from
the front lens is unavoidable, because the partial beams which are still parallel
in front of the front lens then converge in the form of rays toward the focal point
F. Accordingly, the reflection angles of these partial beams differ slightly from
one another. In many cases, this may be acceptable.
Theoretically absolute parallelism of the partial beams after passing
through the front lens
48 of the optics system is obtained when the illumination
beam is focused into the back focal plane
46 (on the ocular end) with the
help of corresponding optical elements (indicated by a lens
52 in FIG. 2).
Then after the beam passes through the front lens
48, this necessarily results
in parallel partial beams of the illumination beam
44 with a joint reflection
angle β corresponding to the distance A of the partial beam
44 from
the optical axis
54 of the optics system
6. The degree of divergence
of the illumination beam
44 after passing through the front lens
48
is determined by the divergence angle α of the illumination beam
44,
which is focused in the back focal plane
46.
With the help of the prism
50 mentioned above, the illumination beam
coming from an illumination source (not shown; left of the revolver head
28
in FIG. 2) is deflected at a right angle so that it runs parallel to the optical
axis
54 and at a distance a from it. The cross section of the prism
50
in the passage
19 is significantly smaller than the total cross section
of the passage
19. For this reason, it is not necessary to use a beam splitter
such as a dichroic mirror which allows transmission of the observation light coming
from the object for coupling of the illumination beam because only a small portion
of the observation field of the microscope is covered by the prism
50. The
distance a may be varied according to this invention in an especially simple manner
by shifting the prism
50 either in the direction parallel to the optical
axis
54 or, as depicted here, in the radial direction (double arrow B).
The reflection angle β of the illumination beam
44 changes with the
distance a, and thus after exceeding the total reflection angle, the depth of penetration
into the object also changes. In order for the focus of the illumination beam
44
to still be in the focal plane
46 after displacement of the prism
50,
the position of the optical components provided in the beam path (represented by
the lens
52) must accordingly be adjusted. If, as shown in FIG. 2 the illumination
beam is deflected at a right angle, it is particularly advantageous to mount the
deflecting unit and the optical elements which permit focusing on a shared mount
because only this mount need be displaceable in the radial direction.
In the arrangement shown in FIG. 2 with the interface
42 at a right angle
to the optical axis
54 between the microscope slide
36 and the object
38, the illumination beam again passes through the front lens
48
into the optics system
16 (symmetrical with the illumination beam
44)
after being reflected on the interface
42 (now called the reflected beam
56). In order for the reflected beam
56 not to interfere with microscopic
observation of the object
38, it is absorbed by an absorber
58 in
the embodiment according to FIG. 2, said absorber being situated here on the other
side of the optical axis
54 at a location corresponding to the location
of the prism
50. To interfere with microscopic observation as little as
possible, the absorber
58 may be moved radially outward as much as possible;
as indicated by the double arrow B′.
The aperture diaphragm
60 in front of the lens
52 should also be
mentioned, because the aperture diaphragm limits the beam cross section of the
illumination beam
44 and thus via the divergence angle α determines
the degree of divergence of the illumination beam
44 after the beam passage
through the front lens
48. Another possibility of varying the divergence
angle α consists of varying the focal depth of the lens
52, in which
case then the position of the lens
52 (see double arrow D) and optionally
the joint position of lens
52 and prism
50 must then be readjusted
in the axial direction (see double arrow E) so that the illumination beam is still
focused in the back focal plane
46.
In case of need, the adapter
40 can be removed from the receptacle
32
again and optionally replaced by a conventional adapter. Furthermore, the adapter
may also easily be used in combination with any other optics systems of the revolver
head
28, because a receptacle
32 is usually assigned to each optics system.
With regard to the optical structure, it should also be added that an additional
aperture diaphragm
62 (indicated beneath the prism
50 in FIG. 2)
may also be used in the passage
19 between the coupling point of the illumination
beam (prism
50) and the observation device. This aperture diaphragm removes
the prism
50 from the observation beam path and thereby prevents any interfering
asymmetrical diffraction images (image distortion) of the object due to the prism
50 introduced into the illumination beam path. The slightly reduced numeric
aperture and resolution are then acceptable.
Another variation in the type of illumination of the object
38 can
be achieved if the prism
50 together with the optical components (lens
52)
connected in front is shifted in parallel with the optical axis
54. If the
focus then moves out of the back focal plane
46, the result is a certain
divergence of the beam striking the interface
42 and a change in the degree
of divergence.
FIG. 3 shows another embodiment (labeled as
140) of the adapter
40
according to FIG. 2. Accordingly, the other components, inasmuch as they correspond
to components of the adapter
40, are provided with the same reference numbers,
but each has the number
100 added to it.
In contrast with the adapter
40 having the external light source, the
light
source
164 with the adapter
140 is integrated into the adapter
140.
The lens
152 and the prism
150 are connected thereto. After passing
through the front lens
148 of the optics system
116 and after total
reflection at the interface
142, the illumination beam
144 and/or
the reflected beam
156 is deflected by 90° through another prism
166
(diametrically opposite the prism
150 together with the light source
164)
and is captured in a detector
170 after passing through a lens
168.
With the detector
170, the optical arrangement can now be adjusted in a
targeted manner; in particular, the total reflection angle can be adjusted as a
function of the position of the prism
150 and a desired range after exceeding
the total reflection angle can be set. On the other hand, under some circumstances
local changes in refractive index, absorption processes or the like can be detected
in the area of the interface
142. Variation of the wavelength of the light
of the light source
164 is also conceivable here.
The radial adjustability of the prisms
150,
166 is indicated by
the double arrows B and B′; the mobility of the prisms
150 and
166
together with the light source
164 and the lens
152 and/or the detector
170 and the front Jens
168 in parallel with the optical axis
154
is indicated by the double arrows E and E′; the adjustment mobility of the
lenses
152,
168 is indicated by the double arrows D, D′.
In principle, it is also conceivable to design the adapter
140 so that
it can optionally be rotated about the optical axis
154. This offers the
advantage that in the case of objects having anisotropic optical properties, the
incident beam direction of the observation beam
144 may optionally be varied.
Uniform illumination of the object can also be achieved optionally by rotation
during the measurement.
FIGS. 4 and 5 show another embodiment of the inventive adapter, now labeled
as
240, shown here in a side view and a sectional view. Its components,
which correspond in function to those in FIG. 3, are labeled with the same reference
numbers, each increased by
100.
Here again, the prisms
250,
266 with the lenses
252,
268
connected in front are provided for coupling of the illumination beam
244
and/or for output of the reflected beam
256. In contrast with the embodiment
according to FIG. 3, the reflected beam
256 is guided out of the adapter
240 and sent to a detection device (not shown here). The light source (also
not shown here) is outside of the adapter
240 (in accordance with FIG. 2).
The adjustment options are the same here as those in the embodiment according to
FIG. 3, which is indicated by the corresponding double arrows D,D′, B, B′
and E, E′.
The adapter
240 is situated between the optics system
216 and the
revolver head
228. On its side facing the optics system, it has a connection
290, which corresponds to the optics system connection
292 of the
revolver head
228 into which the optics system
216 is normally inserted.
On its side facing the revolver head
228, the adapter
240 has a connection
286, which corresponds to the connection
288 on the ocular end of
the optics system
216, which is normally fitted into the optics system connection
292 of the revolver head
228. In this way, the adapter
240
can easily be integrated into an existing microscope without having to modify the microscope.
The two prisms
250,
266 have rounded reflective surfaces so as
to interfere with microscopic observation as little as possible (see FIG. 5). In
addition, a filter wheel
272 with an axis of rotation
274 in the
observation beam path is situated in the adapter
240 on the side of the
prisms
250,
266 facing away from the object
238. Thus, for
example, the portions of the illumination beam
244 which are not output
as reflected beam
256 out of the adapter
240 (in particular diffusely
scattered fractions), can be filtered out of the observation beam path. This is
advantageous in fluorescence measurements in particular.
The embodiment of the inventive adapter shown in FIG. 6 is labeled as
340.
It differs from the embodiment according to FIGS. 4 and 5 only in that the prisms
250,
266 have been replaced by planar mirrors
350,
366.
The total reflection angle can be varied in the same way when using a prism due
to the displacement of the mirror
350 in a direction perpendicular to the
optical axis
354 (while retaining the three-dimensional orientation).
In the embodiment labeled as
440 of the adapter according to FIG. 7, the
two planar mirrors
350,
366 according to FIG. 6 have been replaced
by curved mirrors, in particular concave mirrors, also called concentrating reflectors
450,
466. Under some circumstances, coupling and focusing of the
illumination beam
440 can be accomplished according to the exemplary embodiments
described above without requiring other optical elements such as a front lens.
Beam guidance of the reflected beam
456 through the mirror
466 is
accomplished symmetrically here.
In a highly simplified, perspective, schematic view, FIG. 8 shows a deflecting
unit
576 which is used for coupling of a plurality of illumination beams
544,
544′,
544",
544′" and for output
of the corresponding reflected beams
556,
556′,
556",
556′".
The deflecting unit
576 consists of a concentric arrangement of deflecting
elements held together in the shape of a ring, depicted in FIG. 8 as trapezoidal
mirrors
550,
550′,
550′,
550",
566,
566′,
566",
566′". An illuminating beam, e.g.,
labeled with the reference number
544, is reflected here on the particular
deflecting element, e.g.,
550, so that it runs parallel to the optical axis
554 after reflection and is imaged by the optics system
548 on the
microscope slide
536 with the object
538. The illumination beam,
e.g.,
544 totally reflected at the interface between the microscope slide
536 and the object
538 strikes the deflecting element, e.g.,
566
as a reflected beam, e.g.,
556 after passing through the optics system
548
again in parallel with the optical axis
554, and is reflected again at the
deflecting element, so that it is output out of the beam path of the microscope.
With the arrangement depicted in FIG. 8, the object
538 can be illuminated
simultaneously from several directions, with each of the illumination beams
544—
544′"
satisfying the total reflection condition in the same way.
FIGS. 9 and 10 show additional embodiments of the adapter, which was already
shown in FIGS. 2-7 and is depicted here in a side view. The components of this
adapter, labeled with reference numbers
640 and
740, the function
of which corresponds to that of the adapter shown in FIG. 4, are labeled with the
same reference numbers but in this case they are increased by the number
400
or
500 in each case.
In the embodiments of the adapter
640 and
740 shown in FIGS. 9
and
10, the illumination light is introduced into the adapters
640 and/or
740
via a lightguide
678 or
778, e.g., a glass fiber. An adapter optics
680 or
780, e.g., composed of microlenses, is connected to the lightguide
678 or
778 to focus the illumination beam
644 or
744
coming from the lightguide
678 or
778 in the back focal plane
646
or
746.
In the embodiment depicted in FIG. 9, the lightguide
678 is introduced
into the adapter
640 at a right angle to the optical axis
654, so
that it must be deflected in a direction parallel to the optical axis
654
by a deflecting unit, which is shown in FIG. 9 as a prism
650. On the other
hand, the lightguide
778 in the exemplary embodiment depicted in FIG. 10
is curved, so that the focused illumination beam
744 leaving the adapter
optics
780 is already parallel to the optical axis
754.
In both exemplary embodiments, the reflected beam
656 or
756 can
be output with the help of the optical elements which correspond to those used
for coupling. This is implemented in FIG. 9 by the prism
666 and the lightguide
682, which is connected by the adapter optics
684 to the prism
666.
In FIG. 10 this is done through the lightguide
782 and the adapter optics
784 attached to the end thereof, and with respect to the optical axis
754,
said optics are also mounted symmetrically with the adapter optics
780 used
for coupling the illumination beam
744.
FIGS. 11-14 show embodiments of the present invention in which the deflecting
units, which are used for coupling of the illumination beam and/or for output of
the reflected beam are integrated into a special optics system (objective)
816,
916,
1016,
1116. Components whose functions correspond to
those in the preceding figures are labeled with the same reference numbers in FIGS.
11-14, but each of the numbers has been increased by 100. In comparison with the
embodiments in which the deflecting units are integrated into an adapter, the embodiments
depicted in FIGS. 11-14 offer the advantage that the optical components required
for the deflecting units can be adapted to the lens system of the optics system
816,
916,
1016,
1116, so that the quality of the optical
imaging can be optimized on the whole. In addition, such special optics systems
can also be used for microscopes which do not have an adapter receptacle, as depicted
in FIG. 2.
FIG. 11 shows the illumination beam
844 coupled via a rod prism
850
into the beam path of the microscope. Likewise, the reflected beam
856 is
guided out of the optics system
816 via another rod prism
866. Both
of the prisms
850,
866 are situated here in the area of the optics
system so that they are displaceable in the radial direction for adjusting the
reflection angle in the beam path of the microscope (represented by the double
arrows B, B′ in FIG. 11). An alternative arrangement of the rod prism
850
is shown with dotted lines in FIG. 11. In this arrangement, the rod prism
850′
for coupling of the illumination beam
844 is situated between the illuminated
front lens of the optics system and its focal plane
846 on the ocular end.
The illumination beam
844 in this case is focused virtually on the focal
plane
846 on the ocular end of the lens system through which the beam passes.
This arrangement allows a particularly space-saving design of a special optics
system with an integrated deflecting unit.
In the exemplary embodiment illustrated in FIG. 12, in contrast with FIG. 11,
a double prism
950 is used for coupling of the illumination beam
944,
so that the illumination beam
940 can be deflected by 180°.
In the exemplary embodiments illustrated in FIGS. 13 and 14, lightguides
1078,
1082.
1178 are used to couple and/or output the illumination light
coming from a light source (not shown) in the area of the optics system into the
beam path of the microscope. In the arrangement depicted in FIG. 13, which is similar
to that in FIG. 9, an adapter optics
1080 mounted on the lightguide
1078
and a prism
1050 which is connected to the adapter optics
1080 are
used for coupling of the illumination beam
1040. The reflected beam
1056
is output through a prism
1066, which is situated symmetrically with respect
to the optical axis
1054, and an adapter optics
1084 which is connected
thereto and has a lightguide
1082 connected to it. In contrast with this,
FIG. 14 shows an embodiment in which a curved lightguide
1178 is connected
to the adapter optics
1180 aligned in parallel with the optical axis
1154
to couple the illumination beam
1144 into the beam path of the microscope
in parallel with the optical axis.
FIGS. 1-14 illustrate various embodiments of a microscope which can be used
in particular for TIR microscopy. The microscope is operated in a reflected light
arrangement in which the light which is used to illuminate the object passes through
at least a portion of the optics system before striking the object to be observed.
The illumination beam is coupled thr
