WO2005012974A1 - Systeme d'imagerie de type microscope a epifluorescence comprenant une source lumineuse a halogenure metallique - Google Patents

Systeme d'imagerie de type microscope a epifluorescence comprenant une source lumineuse a halogenure metallique Download PDF

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Publication number
WO2005012974A1
WO2005012974A1 PCT/EP2004/051559 EP2004051559W WO2005012974A1 WO 2005012974 A1 WO2005012974 A1 WO 2005012974A1 EP 2004051559 W EP2004051559 W EP 2004051559W WO 2005012974 A1 WO2005012974 A1 WO 2005012974A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
imaging
metal halide
light
halide lamp
Prior art date
Application number
PCT/EP2004/051559
Other languages
English (en)
Inventor
Hendrik Sibolt Van Damme
Original Assignee
Pamgene B.V.
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 Pamgene B.V. filed Critical Pamgene B.V.
Publication of WO2005012974A1 publication Critical patent/WO2005012974A1/fr

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/24—Base structure
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes

Definitions

  • Epifluorescent microscope imaging system system for conducting automated sample analysis and illumination device.
  • the invention relates generally to epifluorescent imaging systems, to a system for conducting automated sample analysis and to an illumination device for an epifluorescent imaging apparatus, as well as the use of a metal halide lamp.
  • Examples of such systems and such a device are known.
  • the known system is an integrated microarray system, in which incubator and image-acquisition optics are integrated. It is capable of kinetic signal detection while varying the temperature and other parameters of an array containing the sample to be analysed.
  • probe DNA molecules including fluorescent tags are bound onto a substrate .
  • Light from the illumination system is projected onto a filter cube, which comprises an excitation and a detection filter, as well as a dichroic mirror.
  • the excitation filter lets through light within a frequency range suitable for exciting the fluorescent tags.
  • the detection filter lets through light in a frequency range emitted by the fluorescent tags.
  • the emitted light is captured by a CCD (charge coupled device) camera for automated signal processing.
  • CCD charge coupled device
  • a metal halide light bulb serves as a source of light.
  • the metal halide light bulb lies substantially in a horizontal plane with the filter cube and is an arc lamp working at high pressure.
  • a problem of the known system is that the light is emitted only from the small arc between the electrodes .
  • an epifluorescent imaging system arranged to have an imaging axis along which, in use, light travels between an optical element comprising a dichroic mirror and a sample position of a sample to be analysed, which imaging system comprises an illumination system, comprising a fitting for a metal halide lamp, characterised in that the epifluorescent imaging system comprises a metal halide lamp comprising a gas discharge tube, fitted in the fitting.
  • a metal halide lamp consisting of a, preferably ceramic, discharge tube does not have a bright arc, but a tube, which gives light as a whole. Therefore no sophisticated optics are needed to create a homogeneous light bundle.
  • the invention provides an epifluorescent imaging system, arranged to have an imaging axis along which, in use, light travels between an optical ele- ment comprising a dichroic mirror and a sample position of a sample to be analysed, which imaging system comprises an illumination system, comprising a fitting for a metal halide lamp and at least one optical element determining an optical path between a location of light emission of a fitted metal halide lamp and the filter element, wherein the optical path comprises a first section closest to the location of light emission characterised in that the fitting is suitable for a metal halide lamp provided with a gas discharge tube at the location of light emission and in that, in an operating position of the system, the first section of the optical path is oriented predominantly opposite the direction of gravitation.
  • This epifluorescent imaging system enables the attainment of a homogeneous light bundle by use of metal halide lamps with a gas discharge tube.
  • metal halide particles precipitate onto the bottom of the discharge tube. These particles absorb light and lead to inhomogonei ies in the emitted bundle of light that is passed through the filter cube and projected onto the sample site.
  • the system according to the invention is particularly useful for conducting bioassays, as the metal halide lamp with a gas discharge tube has the property of emitting light of a high intensity, which is homogeneously distributed across the spectrum, making it suitable for a wide range of fluorophores.
  • such lamps have a long lifetime, especially if they are of the low pressure type.
  • the optical path determined by the at least one optical element comprises a further section closer to the filter element, the further section being at an angle to the imaging axis.
  • the optical elements comprise a cold mirror. This cold mirror transmits most of the infra-red radiation. This has the advantage of preventing heating of the other optical elements in the imaging system, notably the fil- ter element. The properties of these other elements will thus remain more constant, and their lifetime is extended.
  • the system comprises a system of lenses positioned along the imaging axis for projecting an image of a sample at the sample position through an eyepiece, wherein the system of lenses has an optical magnification substantially equal to one, and in the preferred variant, the system of lenses is formed by an objective lens, positioned between the sample position and the filter element, and an imaging lens, positioned between the filter element and the eyepiece, wherein the objective lens and imaging lens have substantially identical properties.
  • the eyepiece may be an eyepiece for a human observer, but is preferably an eyepiece for attaching a camera, e.g. a CCD camera.
  • a camera e.g. a CCD camera.
  • the system for conducting automated sample analysis according to the invention comprises an epifluorescent imaging system according to the invention.
  • the system for conducting automated sam- pie analysis is adapted for processing at least one sample tray comprising at least one well containing a sample, wherein the illumination system comprises a diaphragm for limiting the diameter of a light beam transmitted between the filter element and the sample to a smallest diameter of the well.
  • the illumination system comprises a diaphragm for limiting the diameter of a light beam transmitted between the filter element and the sample to a smallest diameter of the well.
  • the illumination device for an epifluorescent imaging apparatus is characterised in that the fitting is suitable for a metal halide lamp provided with a gas discharge tube at the location of light emission and in that, in the operating position, the first section of the optical path is oriented predominantly opposite the direction of gravitation.
  • the illumination device is preferably adapted to be connected to an imaging apparatus of an existing device. The assembly of imaging apparatus and illumination device then forms an epifluorescent imaging system according to the invention.
  • a metal halide lamp comprising a gas discharge tube in an epifluorescent imaging system, arranged to have an imaging axis along which, in use, light travels between an optical element comprising a dichroic mirror and a sample position of a sample to be analysed, which imaging system comprises an illumination system, comprising a fitting for the metal halide lamp.
  • a preferred use for the system is as part of an integrated device for automated conducting of bioassays.
  • the preferred device receives a substrate plate 1.
  • the substrate plate 1 preferably comprises a number of wells 2, at the bottom of which is positioned a substrate 3.
  • the substrate 3 is pref- erably of a porous flow-through type.
  • DNA probes have been bound to the substrate 3 in certain spots on the top surf ce of the substrate 3.
  • a sample fluid comprising a number of sample molecules which have been tagged with fluorescent markers, is deposited into the well 2.
  • the integrated device preferably comprises means for incubating the sample in the wells 2, by heating the substrate plate.1 to a desired temperature.
  • the sample fluid is pumped back and forth across the substrate 3 a number of times, during which the sample molecules selectively bind to the DNA probes in the substrate 3. Subsequently, the integrated device washes the substrate 3, so that only the sample molecules that have reacted with specific DNA probes in a hybridisation reaction are left behind.
  • the imaging system according to the invention is used for quantitative and qualitative analysis of the hybridisation reactions that have taken place, to determine how many sample molecules have bound to which of the DNA probes in a substrate 3. It is noted that a different sample fluid may be placed in each of the wells 2, so that each well 2 is analysed in turn.
  • the substrate plate 1 may comprise any number of wells 2, but preferably comprises four wells, or it may be an industry-standard substrate plate comprising 96 wells.
  • the substrate plate 1 is placed in a first sample po- sition in which one of the wells 2 is substantially centred on an upright axis 4.
  • the integrated device for conducting bioassays has a base, and is positioned on its base in such a manner that the upright axis 4 is substantially vertical, i.e. aligned with the direction of gravitation.
  • the substrate plate 1 is at the bottom of the assembly, but it may be at the top, However, the latter mentioned variant is less advisable, as the substrate plate 1, and thus the contents of the wells 2 will be upside down. It is noted that light is projected into the well 2 from above, because the substrate 3 is generally not very transparent to light.
  • the epifluorescent imaging system in the integrated device for conducting automated bioassays comprises at least one filter cube 5. There may be a number of filter cubes 5 in a carousel, enabling one to be selected from a collection of filter cubes with different properties.
  • the imaging system comprises means for accurately positioning the filter cube 5 in a plane substantially perpendicular to the upright axis 4, such that it is centred on the upright axis 4.
  • the filter cube 5 comprises a dichroic mirror 6, which reflects light in the first frequency range (i.e. light used to excite the fluorophores) and is transparent in the second frequency range (i.e. the frequency range in which the light emitted by the fluorophores lies) .
  • the filter cube 5 further comprises an excitation filter 7, which filters out light with a frequency outside the first frequency range. It also comprises a detection filter 8, which only lets through light in the second frequency range.
  • An absorber 9 prevents multiple reflections inside the filter cube 5.
  • the illumination system comprised in the imaging system includes a fitting 12, accommodating, in use, a metal halide lamp 13. Any type of metal halide lamp may be used, for example a mercury- argon lamp.
  • a metal halide lamp may be used, for example a mercury- argon lamp.
  • the discharge tube is disposed in an outer envelope of the lamp 13.
  • the outer envelope is made of glass, and the discharge tube is a ceramic discharge tube, rather than quartz.
  • the discharge tube forms an arc chamber.
  • the effects of the invention can be achieved through the use of lamps with discharge tubes of both cylindrical and sphe- roid shape.
  • the type of lamp used has the advantage that the operating temperature can be higher and that more light- generating metallic ions are provided, leading to a smoother spectrum, and greater luminous efficacy.
  • metal deposits accrue at the bottom of the discharge tube 14, which deposits ab- sorb light emitted by the lamp 13. However, this does not affect the intensity or homogeneity of the light emitted, as only the upwardly emitted light is captured.
  • the further section of the light path is at an angle to the upright axis 4 and defines a reference plane in which the further section lies and which is perpendicular to a plane through the upright axis 4 and the further section.
  • the first section 10 is substantially parallel to the upright axis 4 and located on the same side of the reference plane as a base of the imaging sys- tern, in the shown example on the same side as the sample position.
  • the light emitted by the lamp 13 travels along the first section 10 of the optical path to the filter cube 5 through a condensor 15 and a diaphragm 16.
  • the diaphragm 16 ad- justs the beam diameter, to ensure that the diameter of the beam that eventually falls onto the bottom of a well 2, is equal to, or slightly smaller than, the diameter of the bottom of the well 2.
  • the light changes direction upon reflection in a cold mirror 17.
  • a cold mirror as is well-known in the art, has the property of transmitting infra-red radiation.
  • the cold mirror 17 performs two functions. It firstly changes the direction of the light from vertical to horizontal, and does so in a way that allows the illumination system to be compact.
  • the use of a cold mirror 17 as opposed to a filter is advanta- geous, as the back of the cold mirror 17 can easily be cooled, for example by forced convection or a heat sink attached to the non-reflecting side of the cold mirror 17.
  • Infra-red radiation may, optionally, be removed further by an infra-red filter 18 positioned after the cold mir- ror 17 in the second section 11 of the light path.
  • the cold mirror 17 prevents the infra-red filter 18 from overheating and cracking.
  • a field lens 19 focuses the light onto the filter cube 5, where it first passes through the excitation filter 7, before being reflected in the dichroic mirror 6.
  • the objective lens 20 forms part of a lens system along the upright axis 4, of which the other compo- nent is a tube lens 21.
  • a CCD camera 22 Positioned directly above the tube lens 21 is a CCD camera 22, which captures the image projected onto it by the objective lens 20 and tube lens 21 through an eyepiece (not shown) .
  • the objective lens 20 and tube lens 21 form a lens system having a magnification substantially equal to one, which implies that they are identical.
  • the numerical aperture of the objective lens 20 be higher than the effective numerical aperture as determined by the component of the filter cube 5 li it- ing the diameter of the light beam between the filter cube 5 and the objective lens 20. This prevents vignetting, i.e. darkening of the image around the image, which would affect the measurement of the number of sample molecules bound to DNA probes at the outer edges of the substrate 3.
  • the illumination system is comprises in a separate illumination device, which is detachable from the other parts of the epifluorescent imaging apparatus.
  • the imaging apparatus comprises an inlet port (not shown) , through which light from the illumination device is let in.
  • the illumination device comprises an outlet port through which light is emitted.
  • the inlet and outlet port are aligned when the illumination device is connected to the imaging apparatus by means of appropriately shaped fittings, which also determine the orientation of the illumination device.
  • the illumi- nation device comprises at least the fitting 12 and the cold mirror 17 of the illumination system shown in the drawing.
  • the fittings determine the orientation of the illumination device such that the first section 10 of the optical path from the metal halide lamp 13 is vertical and the second section 11 is horizontal, when the illumination device is connected to the imaging apparatus .
  • the invention is not limited to the above-described embodiment which can be varied within the scope of the attached claims. For instance, various means can be used to determine the position of the filter cube 5, such as a Maltese cross or a servo motor.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un système d'imagerie à épifluorescence conçu pour présenter un axe d'imagerie (4) le long duquel, en cours d'utilisation, la lumière se déplace entre un élément optique (5) comprenant un miroir dichroïque (6) et une position pour un échantillon à analyser. Ce système d'imagerie comprend un système d'éclairage qui comporte une attache (12) pour une lampe à halogénure métallique (13). Ledit système d'imagerie à épifluorescence comprend une lampe à halogénure métallique qui comporte un tube à décharge gazeuse (14), fixée dans l'attache (12).
PCT/EP2004/051559 2003-07-22 2004-07-20 Systeme d'imagerie de type microscope a epifluorescence comprenant une source lumineuse a halogenure metallique WO2005012974A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP03102258.5 2003-07-22
EP03102258 2003-07-22
US49384403P 2003-08-08 2003-08-08
US60/493,844 2003-08-08

Publications (1)

Publication Number Publication Date
WO2005012974A1 true WO2005012974A1 (fr) 2005-02-10

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Family Applications (1)

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PCT/EP2004/051559 WO2005012974A1 (fr) 2003-07-22 2004-07-20 Systeme d'imagerie de type microscope a epifluorescence comprenant une source lumineuse a halogenure metallique

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WO (1) WO2005012974A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2028517A3 (fr) * 2007-08-22 2009-06-03 Olympus Medical Systems Corporation Dispositif de source lumineuse

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5885531A (en) * 1995-03-16 1999-03-23 Bio-Rad Laboratories, Inc. Fluorescence imaging instrument utilizing fish
US6388807B1 (en) * 1999-10-12 2002-05-14 Leica Microsystems Heidelberg Gmbh Confocal laser scanning microscope
US6496309B1 (en) * 1999-06-18 2002-12-17 Genomic Solutions, Inc. Automated, CCD-based DNA micro-array imaging system
US20030058530A1 (en) * 2001-09-25 2003-03-27 Yoshihiro Kawano Microscope switchable between observation modes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5885531A (en) * 1995-03-16 1999-03-23 Bio-Rad Laboratories, Inc. Fluorescence imaging instrument utilizing fish
US6496309B1 (en) * 1999-06-18 2002-12-17 Genomic Solutions, Inc. Automated, CCD-based DNA micro-array imaging system
US6388807B1 (en) * 1999-10-12 2002-05-14 Leica Microsystems Heidelberg Gmbh Confocal laser scanning microscope
US20030058530A1 (en) * 2001-09-25 2003-03-27 Yoshihiro Kawano Microscope switchable between observation modes

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2028517A3 (fr) * 2007-08-22 2009-06-03 Olympus Medical Systems Corporation Dispositif de source lumineuse
US7789532B2 (en) 2007-08-22 2010-09-07 Olympus Medical Systems Corp. Light source device

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