EP3615977A1 - Microscope et procédé d'éclairage de microscope - Google Patents

Microscope et procédé d'éclairage de microscope

Info

Publication number
EP3615977A1
EP3615977A1 EP18728319.7A EP18728319A EP3615977A1 EP 3615977 A1 EP3615977 A1 EP 3615977A1 EP 18728319 A EP18728319 A EP 18728319A EP 3615977 A1 EP3615977 A1 EP 3615977A1
Authority
EP
European Patent Office
Prior art keywords
microscope
light illumination
fluorescence
phase
incident
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP18728319.7A
Other languages
German (de)
English (en)
Inventor
Benjamin DEISSLER
Arnold Müller-Rentz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leica Microsystems CMS GmbH
Original Assignee
Leica Microsystems CMS GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Leica Microsystems CMS GmbH filed Critical Leica Microsystems CMS GmbH
Publication of EP3615977A1 publication Critical patent/EP3615977A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/02—Objectives
    • G02B21/025—Objectives with variable magnification
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/14—Condensers affording illumination for phase-contrast observation
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/18—Arrangements with more than one light path, e.g. for comparing two specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G02B21/241—Devices for focusing
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B2207/00—Coding scheme for general features or characteristics of optical elements and systems of subclass G02B, but not including elements and systems which would be classified in G02B6/00 and subgroups
    • G02B2207/113—Fluorescence

Definitions

  • 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.
  • stained specimens are usually examined with a microscope in transmitted-light brightfield illumination.
  • 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.
  • DIC Differential Interference Contrast
  • 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.
  • 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 °.
  • 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.
  • the diffraction in the sample also causes a phase shift depending on the refractive index.
  • Backlight causes interference in the image plane.
  • 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.
  • 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.
  • LEDs Light-emitting diodes
  • advantages include higher light output with lower power consumption and longer life.
  • 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.
  • the solid state light source used for transmitted light illumination can be used by the
  • 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
  • Shutter which is switchable on the transmitted light illumination axis.
  • 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
  • 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
  • a microscope the use of a ring diaphragm in such a microscope and a method for microscope illumination with the
  • 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 can be used before incident radiation of fluorescence Auflichtbeleuchtungs recommended.
  • 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 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 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 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
  • 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
  • the microscope setup is selected such that the fluorescence Auflichtbeleuchtungsstrahlengang generated by the fluorescence Auflichtbeleuchtungschreibs adopted 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 füranderbeleuchtungs leads lies.
  • the transmitted light illumination optical system comprises a
  • the annular aperture is arranged in the rear focal plane.
  • 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.
  • 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
  • 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.
  • Incident illumination optics in particular their focal length and / or
  • 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
  • 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.
  • the Aufanderbeleuchtungsoptik is set such that the fluorescence generated by the fluorescence embark
  • 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
  • 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.
  • FIG. 2 schematically shows an annular diaphragm as can be used in a microscope according to FIG. 1
  • 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.
  • the microscope 10 has an objective 105 with a phase ring 106.
  • 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.
  • the eyepiece 131 (not shown) and / or a camera 132 can be arranged downstream of the tube 131.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • these lenses 103, 105 and 122 are adjustable in their focal length.
  • 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
  • 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.
  • incident light illumination optics 122 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.
  • incident light illumination optics 122 are to be set such that the focal spot of reflected-light illumination source 121 in FIG.
  • Incident illumination beam path at the position of the phase ring 106 may be preferably smaller than the diameter of the inner transparent

Landscapes

  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un microscope (10) pour l'étude d'un échantillon par éclairage en lumière transmise à contraste de phase et/ou éclairage en lumière incidente à fluorescence, comprenant un dispositif d'éclairage en lumière transmise à contraste de phase (11) et un dispositif d'éclairage en lumière incidente à fluorescence (12), le dispositif d'éclairage en lumière transmise à contraste de phase (11) comprenant une source d'éclairage en lumière transmise (101) ainsi qu'une optique d'éclairage en lumière transmise (103) avec un diaphragme annulaire (102, 201), le diaphragme annulaire (102, 201) comprenant une zone d'ouverture (203) interne opaque qui est entourée par une zone annulaire (202) au moins partiellement opaque, et un dispositif d'éclairage en lumière incidente à fluorescence (12) comprenant une source d'éclairage en lumière incidente (121) ainsi qu'une optique d'éclairage en lumière incidente (122), et le microscope (10) comprenant un objectif (105) comprenant un anneau de phase (106), le trajet optique d'éclairage en lumière incidente créé par le dispositif d'éclairage en lumière incidente à fluorescence (12) se trouvant avec sa section transversale dans la zone d'ouverture (203) interne du diaphragme annulaire (102, 201) du dispositif d'éclairage en lumière transmise à contraste de phase (11) après le passage à travers le plan d'objet (104) du microscope (10), et un procédé pour l'éclairage de microscope correspondant.
EP18728319.7A 2017-05-16 2018-05-16 Microscope et procédé d'éclairage de microscope Withdrawn EP3615977A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102017110638.3A DE102017110638B3 (de) 2017-05-16 2017-05-16 Mikroskop und Mikroskopbeleuchtungsverfahren
PCT/EP2018/062663 WO2018210906A1 (fr) 2017-05-16 2018-05-16 Microscope et procédé d'éclairage de microscope

Publications (1)

Publication Number Publication Date
EP3615977A1 true EP3615977A1 (fr) 2020-03-04

Family

ID=62486548

Family Applications (1)

Application Number Title Priority Date Filing Date
EP18728319.7A Withdrawn EP3615977A1 (fr) 2017-05-16 2018-05-16 Microscope et procédé d'éclairage de microscope

Country Status (5)

Country Link
US (1) US20200201014A1 (fr)
EP (1) EP3615977A1 (fr)
CN (2) CN110622055B (fr)
DE (1) DE102017110638B3 (fr)
WO (1) WO2018210906A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3798713A1 (fr) 2019-09-27 2021-03-31 Leica Microsystems CMS GmbH Microscope d'examen d'un échantillon et procédé correspondant

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3699761B2 (ja) * 1995-12-26 2005-09-28 オリンパス株式会社 落射蛍光顕微鏡
JP4608043B2 (ja) * 1999-09-24 2011-01-05 オリンパス株式会社 顕微鏡用焦点検出装置
JP5132480B2 (ja) * 2008-08-26 2013-01-30 オリンパス株式会社 顕微鏡
DE102011079941A1 (de) * 2011-07-27 2013-01-31 Leica Microsystems Cms Gmbh Mikroskopbeleuchtungsverfahren und Mikroskop
DE102011079942B4 (de) * 2011-07-27 2016-12-15 Leica Microsystems Cms Gmbh Mikroskopbeleuchtungsverfahren und Mikroskop
DE102013002640A1 (de) * 2013-02-15 2014-08-21 Carl Zeiss Microscopy Gmbh Verfahren zum betreiben eines lichtmikroskops und optikanordnung
DE102013110497B4 (de) * 2013-04-03 2023-04-27 Jörg Piper Verfahren und Vorrichtung zur Erzeugung einer variablen und simultanen Phasenkontrastabbildung in Kombination mit einer der Abbildungen Dunkelfeldabbildung oder Hellfeldabbildung oder Polarisationsabbildung
JP6131204B2 (ja) * 2014-02-28 2017-05-17 富士フイルム株式会社 観察装置

Also Published As

Publication number Publication date
WO2018210906A1 (fr) 2018-11-22
US20200201014A1 (en) 2020-06-25
CN110622055B (zh) 2023-01-06
CN110622055A (zh) 2019-12-27
DE102017110638B3 (de) 2018-09-27
CN115793223A (zh) 2023-03-14

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