EP3615977A1

EP3615977A1 - Microscope and microscope illumination method

Info

Publication number: EP3615977A1
Application number: EP18728319.7A
Authority: EP
Inventors: Benjamin DEISSLER; Arnold Müller-Rentz
Original assignee: Leica Microsystems CMS GmbH
Current assignee: Leica Microsystems CMS GmbH
Priority date: 2017-05-16
Filing date: 2018-05-16
Publication date: 2020-03-04
Also published as: WO2018210906A1; US20200201014A1; CN110622055B; CN110622055A; DE102017110638B3; CN115793223A

Abstract

The invention relates to microscope (10) for examining a sample in phase-contrast transmitted-light illumination and/or in fluorescence reflected-light illumination, comprising a phase-contrast transmitted-light illumination device (11) and a fluorescence reflected-light illumination device (12), wherein the phase-contrast transmitted-light illumination device (11) comprises a transmitted-light illumination source (101) and a transmitted-light illumination optical unit (103) with a ring stop (102, 201), wherein the ring stop (102, 201) has a light-opaque inner stop region (203), which is surrounded by an at least partly light-transmissive ring-shaped region (202), and wherein the fluorescence reflected-light illumination device (12) has a reflected-light illumination source (121) and a reflected-light illumination optical unit (122), and wherein the microscope (10) comprises an objective lens (105) with a phase ring (106), wherein the fluorescence reflected-light illumination beam path, produced by the fluorescence reflected-light illumination device (12), lies within the inner stop region (203) of the ring stop (102, 201) of the phase-contrast transmitted-light illumination device (11) with the cross section thereof after passing through the object plane (104) of the microscope (10), and to a corresponding microscope illumination method.

Description

Microscope and microscope illumination method

description

The present invention relates to a microscope and a

Microscope illumination method, in particular a microscope for examining a sample in phase-contrast transmitted light illumination and then or alternately or simultaneously in fluorescence incident illumination and a corresponding microscope illumination method.

State of the art

In cytodiagnostics and pathology, stained specimens are usually examined with a microscope in transmitted-light brightfield illumination. For diagnosis, the color of the microscopically examined sample is an important criterion. For example, with other microscopic examinations

Contrasting methods, such as phase contrast or Differential Interference Contrast (DIC), make the color of the sample less important. With such

Contrast methods are usually examined uncolored samples, which are in the transmitted light bright field microscopy as predominantly transparent. The contrasting methods then serve to visualize phase properties of the sample.

In phase contrast microscopy is a so-called phase ring in or on the microscope objective and a ring diaphragm in the condenser optics of Transmitted light illumination device installed. The ring diaphragm, also known as the light ring, limits the incidence of light on the sample to a certain angle of incidence range. The phase ring causes a phase shift of the incident light of 90 °. By diffraction of the light, for example, on cell structures, light passing through the object is deflected in such a way that, for the most part, it does not pass through the phase ring. However, the diffraction in the sample also causes a phase shift depending on the refractive index. The phase difference between diffracted object light and passing through the phase ring

Backlight causes interference in the image plane. By appropriate dimensioning of the phase ring can be represented in this way, the object, for example. Dark against a light background (positive phase contrast). An image in negative phase contrast is also possible.

Another known examination method is fluorescence microscopy. Here, the sample to be examined is illuminated by means of an incident-light illumination beam path which traverses a so-called excitation filter. The excitation light leads to fluorescent light in the fluorescently labeled object, wherein the emitted fluorescent light determines the resulting microscope image of the sample. The said microscopy methods are known per se for a long time. For further details reference is made to the existing state of the art.

The predominantly used in transmitted light microscopy in the past halogen lamp is becoming more and more by solid state light sources, eg.

Light-emitting diodes (hereafter LEDs), with their known advantages replaced. These advantages include higher light output with lower power consumption and longer life. For the

Transmitted light illumination is mainly used with white light LEDs. Such solid state light sources often exhibit luminescence upon excitation by an external light source. This is z. For example, in the case of LEDs, where a phosphor layer is used to produce certain spectral components (in particular White light LEDs, but also z. B. in the green spectral range). For microscopes that combine transmitted light illumination and fluorescence incident light illumination, the solid state light source used for transmitted light illumination can be used by the

Light source of fluorescence incident illumination are excited. A large part of the excitation light for the fluorescence excitation passes through the sample and from there via the transmitted light illumination axis up to the

By light illumination source. That generated there due to stimulation

Luminescent light becomes a disturbing background in the fluorescence image

perceived. This effect also occurs when the solid state light source of the transmitted light illumination is switched off.

In DE 10 2011 079 941 A1, this problem is treated alternately or simultaneously in transmitted light bright field illumination and epi-fluorescence illumination in connection with a microscope for examining a sample. To avoid said luminescent light, a matching filter is introduced into the transmitted light illumination axis, which is the luminescence

causing excitation light from the fluorescence incident illumination spectrally blocks. The adaptation filter can remain on the transmitted light illumination axis even when there is a change to transmitted-light bright-field illumination, since in this way the spectrum of, for example, a white-light LED used can be approximated to the spectrum of a halogen lamp. According to this document, however, it is useful when using a contrasting method such as phase contrast, the adaptation filter from the illumination beam path of the

Manual or motorized removal of transmitted light illumination so that a higher light intensity is available for the selected contrasting method. However, such a switchable adaptation filter is elaborately constructed, requires a larger space, is expensive to produce and also slow in the switching. The same disadvantages occur when using a locking device (eg.

Shutter), which is switchable on the transmitted light illumination axis. For example, it is proposed in DE 10 2011 079 942 Al that, when activating reflected-light fluorescence illumination, a switchable shutter is forcibly switched on or introduced on the transmitted light illumination axis in order to prevent excitation of the white light LED used as transmitted light bright field illumination source, in which case Activation of transmitted-light brightfield illumination of this shutter forced off or

is swung out.

The present invention is therefore based on the object to improve the examination of a sample with a microscope in phase-contrast transmitted light illumination and / or in fluorescence incident illumination, wherein the

Suppression of interfering luminescence switchable elements to be avoided. Disclosure of the invention

According to the invention, a microscope, the use of a ring diaphragm in such a microscope and a method for microscope illumination with the

Characteristics of the independent claims proposed. Advantageous embodiments are the subject of the dependent claims and the following description.

The invention is based on the finding that one in the

Transmitted light illumination optics of the phase contrast transmitted light illumination device located ring diaphragm for shielding the transmitted light illumination source, which is usually a solid-state light source, can be used before incident radiation of fluorescence Auflichtbeleuchtungseinrichtung. A microscope according to the invention for examining a sample in

Phase contrast transmitted light illumination and / or in fluorescence Incident light illumination has a phase contrast transmitted light illumination device and a fluorescence incident illumination device, wherein the phase contrast transmitted light illumination device is a transmitted light illumination source, in particular a solid state light source, in particular one or more LEDs, in particular one or more white light LEDs, and a

Transmitted light illumination optics, in particular a condenser optics, having a ring diaphragm, wherein the annular diaphragm (light ring) has an opaque inner diaphragm portion of at least partially

translucent substantially annular area is surrounded. The fluorescence incident illumination device has a

Incident light source and incident light illumination optics, in particular with a beam splitter, on. Furthermore, the microscope for the phase contrast transmitted light illumination is equipped with a lens with a phase ring. To avoid the above-described luminescence by stimulating the

Durchlichtbeleuchtungsquelle the microscope setup is selected such that the fluorescence Auflichtbeleuchtungsstrahlengang generated by the fluorescence Auflichtbeleuchtunglichtseinrichtung with its cross section after passing through the object plane of the microscope - even with an object located there - for the most part, but especially within the inner aperture of the ring diaphragm of the phase contrast Durchlichtbeleuchtungseinrichtung lies. In this way, there is a particular complete shading of Auflichtbeleuchtungsstrahlengangs after entering the phase contrast transmitted light illumination device before hitting the

By light illumination source.

The corresponding microscope setup can be achieved in various ways. Preferably, the transmitted light illumination optical system comprises a

Condenser optics or a condenser or consists of such

Condenser optics or such a condenser, in the rear focal plane, the annular aperture is arranged. Preferably, the annular aperture is fixed on the Transmitted light illumination axis arranged. It can then the other existing in the microscope optics, namely transmitted light illumination optics,

Incident light optics and lens, individually, in combination or all together adjusted so that the above-mentioned shielding occurs optimally. "Adjustments" of the optics or of the objective means that lenses located there are changed in their focal length and / or such lenses are displaced along the optical axis. Advantageously, the existing incident illumination optics, also as

Designated fluorescence axis, set such that at a passage of the fluorescence Auflichtbeleuchtungsstrahlengangs through the object plane - both in an object there and in the absence of an object - this with its cross-section in particular completely within the inner

Aperture area of the bezel comes to rest. The epi-illumination optics include optical elements - in the simplest case a single lens up to a complex system of lenses, filters, apertures, etc. The function of incident illumination optics is to guide as much light from the fluorescence incident illumination source to the sample and there for a uniform illumination of the sample. By appropriate adjustment of this

Incident illumination optics, in particular their focal length and / or

Magnification, it can be ensured that passing through the object plane passing light that enters the transmitted light illumination optics, there of the annular aperture located there on a further propagation in the direction

Transmitted light source is prevented. If different objectives and / or different light rings are used with the microscope, the inner diameter of the phase ring will generally differ between the lenses. If the fluorescence incident optics is designed such that it can be changed in focal length and / or magnification, then the size of the light cone at the position of the phase ring can be chosen so that the preferably entire light cone in the inner region of the phase ring (and thus also in the interior Area of the light ring) is located. The invention further relates to a use of said ring stop in a microscope of the type mentioned for the purpose of avoiding the excitation of luminescence in the transmitted light source by light of the

Fluorescence Auflichtbeleuchtungsquelle. To avoid repetition, please refer to the above performances in connection with the

directed microscope according to the invention.

Finally, the invention relates to a method for microscope illumination using a microscope of the type mentioned above, wherein the

Transmitted light illumination optics and / or incident light illumination optics and / or the lens of the microscope and / or the position of the ring diaphragm on the transmitted light illumination axis is set such that the of

Fluorescence incident light illumination device generated fluorescence reflected light illumination beam path with its cross section after passing through the object plane of the microscope within the inner aperture region of the ring diaphragm of the phase contrast transmitted light illumination device. For further embodiments and advantages of the method according to the invention, turn to the above statements in connection with the

directed microscope according to the invention.

It is particularly advantageous if, in the fixed position, the ring diaphragm on the transmitted light illumination axis and for a given setting of the

Durchlichtbeleuchtungsoptik and the objective, the Auflichtbeleuchtungsoptik is set such that the fluorescence generated by the fluorescence Auflichtbeleuchtungseinrichtung fluorescence

Incident illumination beam path with its cross section after passing through the object plane of the microscope completely within the inner

Aperture area of the ring diaphragm of the phase contrast transmitted light illumination device is located. It is furthermore advantageous if the epi-illumination source is imaged essentially in the rear focal plane of the objective, in which the phase ring is also located. This rear focal plane is through the microscope objective and the transmitted light illumination optics or the condenser in the rear

Focal plane of the condenser shown, in which the annular aperture is located. By suitable adjustment of the reflected-light illumination optics, the image of the reflected-light illumination source can be selected such that its image is smaller than the diameter of the inner diaphragm region of the annular diaphragm. This in turn should be located in the rear focal plane of the lens image of the

Incident light source within the diameter of an inner portion of the phase ring lie. This inner region is the transparent region within the inner diameter of the phase ring.

Further advantages and embodiments of the invention will become apparent from the

Description and attached drawing.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the particular combination given, but also in other combinations or in isolation, without departing from the scope of the present invention.

The invention is illustrated schematically with reference to an embodiment in the drawing and will be described below with reference to the drawing.

Figure description shows schematically the construction of a microscope for examining a sample in phase-contrast transmitted light illumination and / or fluorescence incident illumination according to an embodiment of the invention, FIG. 2 schematically shows an annular diaphragm as can be used in a microscope according to FIG. 1, and FIG. 3 shows schematically the optical path of the fluorescence incident illumination in a microscope according to FIG. 1 according to an embodiment of the invention.

The microscope shown schematically in FIG. 1 has a phase-contrast transmitted light illumination device 11 and a fluorescence incident illumination device 12. The phase contrast transmitted light illumination device 11 has as essential elements a transmitted light illumination source 101, which in this embodiment represents a solid-state light source such as a white-light LED, as well as a

Transmitted light illumination optics 103, which in this embodiment a

Condenser represents, on. In the rear focal plane of the condenser is the annular aperture 102, also called light ring.

To examine a sample in phase-contrast transmitted light illumination, the microscope 10 has an objective 105 with a phase ring 106.

To examine a sample in fluorescence incident illumination, the microscope 10 has the said fluorescence incident illumination device 12, which contains as essential elements a reflected-light illumination source 121 and incident-light illumination optics 122. A beam splitter 110 arranged on the optical axis of the objective 105 is shown schematically and directs the fluorescence incident illumination beam path in the direction of the objective 105 and object plane 104. Radiated from a specimen in the object plane 104

Fluorescent light passes through the objective 105 and the beam splitter 110 into the tube 131 of the microscope 10. In a known manner, the eyepiece 131 (not shown) and / or a camera 132 can be arranged downstream of the tube 131. Of the Beam splitter 110 also avoids that light of fluorescent incident light source 121, which is reflected at components of the microscope, such as the lens 105, in the direction of tube 131 passes. In Figure 2, the annular aperture 102 of Figure 1 is shown schematically in plan. Clearly visible is the opaque inner diaphragm region 203, which is surrounded by an at least partially translucent substantially annular region. The annular region 202 in turn is followed by an annular opaque region 204. This geometry of the annulus 201 ensures that the sample is illuminated at certain aperture angles when the annulus is placed in the back focal plane of the condenser 103. In this way, as explained above, together with the phase ring 106, an object can be imaged and examined in phase contrast. The microscope 10 shown in FIG. 1 not only permits phase contrast but also the imaging or examination of an object in fluorescence incident illumination. As described above, a portion of the fluorescence incident illumination passes through an object located in the object plane 104 into the phase-contrast transmitted light illumination device 11. There, therefore, part of the fluorescence incident light illumination via the condenser onto the

Transmitted light source 101 passed. This is in principle also the case if in the rear focal plane of the condenser 103, an annular aperture 102 is arranged, since this annular aperture has light-transmissive areas. When incident light source 121 is incident on the transmitted light illumination source 101, the incident light illumination source 121 leads to luminescence when using solid-state light sources, as explained in detail at the outset, which in turn is disturbing

Backlight when taking pictures in fluorescence reflected light illumination makes noticeable. This can be prevented by selecting the microscope structure according to FIG. 1 such that the fluorescence generated by the fluorescence incident illumination device 12 is selected.

Incident illumination beam path with its cross section after passing through the object plane 104 lies within the inner diaphragm area 203 of the annular diaphragm 102. In this way, the fluorescence incident light illumination is blocked before reaching the transmitted light illumination source 101. It is expedient if the entire cross section comes to rest within the inner diaphragm area 203.

The following measures are suitable for this effect of shielding or blocking. In principle, there is an adjustment of all possible adjustable optical elements in the microscope, namely the transmitted light illumination optics 103, the microscope objective 105 and incident light illumination optics 122, which can each consist of a single lens to a complex system of lenses, filters, screens etc. Frequently, these lenses 103, 105 and 122 are adjustable in their focal length. Additionally or alternatively, individual lenses of these optics 103, 105, 122 may be displaced along the respective optical axes. It is most expedient to use incident-light illumination optics 122 for the purpose according to the invention, as explained below.

Figure 3 shows schematically a possible beam path of

Fluorescence illumination in a microscope according to FIG. 1. In this respect, reference can be made to the description of the figures for FIG. 1 with regard to all the details. The illustrated beam paths show the beam paths for a point in the middle and a point on the edge of incident light illumination source 121. The focal spot of reflected light illumination source 121 is imaged in the rear focal plane of objective 105, in which phase ring 106 is also located. This plane is again imaged by lens 105 and condenser 103 in the rear focal plane of the condenser 103, in which the annular aperture 102 is located. By appropriate adjustment of incident light illumination optics 122, the image is selected such that the image of the focal spot of the

Incident illumination source 121 is smaller than the diameter of the inner diaphragm portion 203 of the annular diaphragm 102, 201 (see Figure 2), the light cone of the incident illumination beam path at the position of the annular diaphragm 102 will also fall only on the inner diaphragm portion 203 of the annular diaphragm. In a Thus, according to a suitable embodiment, incident light illumination optics 122 are to be set such that the focal spot of reflected-light illumination source 121 in FIG

Substantially falls in the rear focal plane of the lens 105. Of course, as those skilled in the art will understand, this condition need not be accurate but only substantially met. However, the light cone of the

Incident illumination beam path at the position of the phase ring 106 may be preferably smaller than the diameter of the inner transparent

Area of this phase ring 106th

LIST OF REFERENCE NUMBERS

10 microscope

11 Phase contrast transmitted light illumination device 12 Fluorescence incident illumination device

100 transmitted light illumination axis, optical axis

101 transmitted light source

102 ring aperture

103 Transmitted light illumination optics, condenser

104 object level

105 lens

106 phase ring 110 beam splitter

116 inner region of the phase ring

121 incident light source

122 incident illumination optics

131 tube

132 camera

201 ring diaphragm

202 annular area

203 inner panel area

204 outer panel area

Claims

05/15/2018 / mb / mg

claims

Microscope (10) for examining a sample in Püasenkontrast- transmitted illumination and / or in fluorescence incident illumination with a phase contrast transmitted light illumination device (11) and a fluorescence Auflichtbeleuchtungseinrichtung (12), wherein

the phase contrast transmitted light illumination device (11) a

Durchlichtlichtuchtungsquelle (101) and a

Having transmitted-light illumination optics (103) with an annular diaphragm (102, 201), wherein the annular diaphragm (102, 201) has an opaque inner diaphragm region (203) surrounded by an at least partially transparent annular region (202), and wherein the fluorescent Incident light illumination device (12) a

Incident light source (121) and a Auflichtbeleuchtungsoptik (122), and wherein

the microscope (10) has a lens (105) with a phase ring (106), wherein

the fluorescence reflected-light illumination beam path generated by the fluorescent incident-light illumination device (12) with its cross-section after passing through the object plane (104) of the microscope (10) within the inner diaphragm region (203) of the annular diaphragm (102, 201) of the phase-contrast transmitted-light illumination device (11) lies.

The microscope (10) of claim 1, wherein the transillumination source (101) comprises or represents a solid state source of light.

3. A microscope (10) according to any one of the preceding claims, wherein the transmitted light illumination optical system (103) comprises a condenser, in the rear focal plane of the annular diaphragm (102, 201) is arranged.

4. A microscope (10) according to any one of the preceding claims, wherein the

Focal length and / or magnification of Auflichtbeleuchtungsoptik (122) is variable.

5. A microscope (10) according to any one of the preceding claims, wherein the

Focal length of the lens (105) is variable.

6. Use of the annular diaphragm (102, 201) of a microscope (10) according to one of claims 1 to 5 for preventing the excitation of

Transmitted light source (101) by light of

Incident illumination source (121) in that the opaque inner diaphragm region (203) of the annular diaphragm (102, 201) the

Transmitted light source (101) from the light of

The incident light source (121) shields.

7. Use according to claim 6, wherein the transmitted light illumination optics (103) and / or the Auflichtbeleuchtungsoptik (122) and / or the lens (105) is adjusted such that the fluorescence Auflichtbeleuchtungsstrahlengang generated by the fluorescence Auflichtbeleuchtungseinrichtung (12) with its cross section after passage through the object plane (104) of the microscope (10) within the inner diaphragm region (203) of the annular diaphragm (102, 201) of the phase-contrast transmitted light illumination device (11).

8. A method for microscope illumination using a microscope (10) according to one of claims 1 to 5, wherein the

Transmitted light illumination optics (103) and / or the

Incident light illumination optics (122) and / or the objective (105) is set in such a way that the fluorescence intensity of the fluorescence Incident light illumination beam path with its cross section after passing through the object plane (104) of the microscope (10) within the inner diaphragm region (203) of the annular diaphragm (102, 201) of the phase-contrast transmitted light illumination device (11).

9. The method according to claim 8, wherein in the fixed position of

Ring aperture (102, 201) on the transmitted light illumination axis (100) and with a predetermined setting of the transmitted light illumination optics (103) and the objective (105) the incident illumination optics (122) is set such that the fluorescence Auflichtbeleuchtungsstrahlengang generated by the fluorescence Auflichtbeleuchtungseinrichtung (12) with his

Cross section after passing through the object plane (104) of the microscope (10) completely within the inner diaphragm area (203) of the

Ring diaphragm (102, 201) of the phase contrast transmitted light illumination device (11) is located.

10. The method of claim 9, wherein the incident illumination optics (122) is adjusted such that the incident illumination source (121) is arranged in the rear focal plane of the objective (105), in which the phase ring (106) is arranged within an inner region (116). this phase ring (106) is imaged.