WO2022152714A1 - Dispositif de microscope - Google Patents

Dispositif de microscope Download PDF

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Publication number
WO2022152714A1
WO2022152714A1 PCT/EP2022/050479 EP2022050479W WO2022152714A1 WO 2022152714 A1 WO2022152714 A1 WO 2022152714A1 EP 2022050479 W EP2022050479 W EP 2022050479W WO 2022152714 A1 WO2022152714 A1 WO 2022152714A1
Authority
WO
WIPO (PCT)
Prior art keywords
light
microscope device
sample
image
reflection element
Prior art date
Application number
PCT/EP2022/050479
Other languages
English (en)
Inventor
Rainer PROF. UHL
Original Assignee
Miltenyi Biotec B.V. & Co Kg
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 Miltenyi Biotec B.V. & Co Kg filed Critical Miltenyi Biotec B.V. & Co Kg
Publication of WO2022152714A1 publication Critical patent/WO2022152714A1/fr

Links

Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/1006—Beam splitting or combining systems for splitting or combining different wavelengths
    • G02B27/1013—Beam splitting or combining systems for splitting or combining different wavelengths for colour or multispectral image sensors, e.g. splitting an image into monochromatic image components on respective sensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/021—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using plane or convex mirrors, parallel phase plates, or particular reflectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/30—Measuring the intensity of spectral lines directly on the spectrum itself
    • G01J3/36—Investigating two or more bands of a spectrum by separate detectors
    • 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
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/361—Optical details, e.g. image relay to the camera or image sensor
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/10—Beam splitting or combining systems
    • G02B27/14—Beam splitting or combining systems operating by reflection only
    • G02B27/141—Beam splitting or combining systems operating by reflection only using dichroic mirrors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4406—Fluorescence spectrometry
    • 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 invention relates to a camera-based microscope which employs four-color distinction capabilities to provide a maximally contrast-rich fluorescence respectively transmitted light image.
  • a multicolor microscope is disclosed in WO 2020/038752, which relates to a microscope device having dual emission detection capabilities. It employs two cameras and places a dichroic beam splitter into the finite optical space between the microscope and the two cameras, which record the two desired spectral regions. In order not to distort the transmitted spectral image, the dichroic is kept as thin and the reflection angle as small as possible. However, since a thin substrate tends to compromise flatness and hence the quality of the reflected image, an optimal thickness always reflects a compromise between the quality of the transmitted and the reflected image.
  • a microscope device comprising: a microscope objective (1); one or more light sources; at least 3 dichroic beam splitters (5, 10, 16) and at least 2 cameras (4, 17) characterized in that the light generated by the light source interacts with the sample (3) thereby producing a sample beam wherein - a first image having a spectral range of light (A) is separated from the sample beam with a first dichroic beam splitter (5) and guided via reflection element (8) to the light detector (4) of the first camera (9), thereby resulting in residual beam X - a second image having a spectral range of light (B) is separated from residual beam X with a second dichroic beam splitter (10) and guided via reflection element (13) to light detector (4) of the first camera (9) thereby resulting in residual beam Y
  • Such microscope devices are especially useful for detecting multiple spectral ranges emitted by a sample which is often the case in sequencing DNA/RNA molecules. To avoid damaging of the sample, the interaction between the light and the sample should be kept as short as possible. Since the device of the invention can detect at least 4 different spectral ranges simultaneously, the use of the microscope device as disclosed herein in a sequenc- ing-by-synthesis process it is a further object of the invention.
  • Fig. 1 shows the general path of light of the microscope of the invention
  • the microscope is equipped to detect four spectral ranges, for example 405, 488, 561 and 638 nm or 375 nm, 473 nm, 532 nm, und 660 nm.).
  • the cut-on wavelengths are chosen to 488, 561 and 638 nm so as to separate the emission into the four spectral regions between the excitation wavelengths.
  • the respective spectral ranges of light of the images are between 473 nm and 532 nm; 532 nm and 594 nm, 594 nm and 660 nm; 633 nm and 660 nm.
  • At least one of the third dichroic beam splitters (5, 10, 16) is arranged (tiled) in the path of light as to minimize or avoid the chromatic error of the light detected by the camaras. Since the chromatic error depends on the spectral range / the wavelength of the light, the angular position of the dichroic beam splitters (5, 10, 16) may be same or different.
  • first, second and third dichroic beam splitter (5, 10, 16) with the respective residual images may be independently between + 45° and - 45°, preferable independently between + 30° and - 30° or independently between + 25° and - 25°. or independently between + 15° and - 15°.
  • the tilt angles of the first and second dichroic beam splitters (5, 10) and/or second and third dichroic beam splitters (10, 16) with the respective residual beams may be in opposing directions.
  • the light sources preferable provide light having a spectral range of wavelengths of 300 to 800 nm like white light, laser light, or LED light.
  • the sample may be subjected to the light “as is” or may be provided with fluorescence or phosphorenscence agent to mark regions or interest.
  • preferable light sources producing light with longer wavelengths are used, such as 525 and 635 nm.
  • the sample beam may be or comprise fluorescence or phosphorenscence radiation originating from the sample (3) or radiation transmitted or reflected by the sample (3).
  • the microscope device may be provided with at least one focusing element (2) into the beam-path of the sample beam upstream of the first beam splitter.
  • At least one focusing element is placed into the beam-path of the sample beam.
  • the focusing elements may consist or comprise at one lens or at least one objective or a combination thereof. In Fig. 1 this is shown with focusing element (2) providing image (6).
  • the microscope device according to the invention may be provided with one or more optical element (21, 22, 23, 24) into the beam-path of first, second, third and/or forth images A, B, C, D.
  • optical elements are capable of focal plane or image plane shift and can optionally be inserted and removed from the beam-paths with an appropriate device.
  • Suitable optical elements have a higher refractive index than the surrounding medium and may consist of coated or uncoated glass or polymer.
  • the optical elements can be provided with a filter for chromatic correction of any optical distortion which caused by the dichromatic beam splitters
  • Fig 1 shows a microscope in its most basic form, comprising an objective (1) and a tube-lens (2), which generates an image of an object (3) in the focal plane (4) of the tube-lens (2).
  • the chip (9) is aligned such that its position lies flat in the image plane (4) of the optical system.
  • B is directed to the other (in Fig. 1 the right) half of the camera detector-chip (9).
  • the tilt-angle of the two dichroic elements (5) and (10) is kept to at about 25° and at opposing angles so as to compensate for chromatic aberrations.
  • the thickness of the dichroic beam splitters is kept as small as possible (usually 1 to 3 mm) so as to minimize thickness-related aberrations in transmission, but thick enough to maintain flatness of the reflecting surfaces, which is of paramount importance for image quality of the reflected fraction of the beam.
  • the transmitted beam (D) is reflected onto the second camera-chip (17) with the help of a single reflecting element (18), whereas the reflected beam (C) requires 2 more reflections by means of the two reflecting elements (19) and (20).
  • the cure for this is to bring appropriate bandpass-filters or optical elements (21, 22 and 23) into the beam-path, and/or equip the reflecting elements with the same filter- layer as dichroic the beam splitter.
  • reflection element (8) is provided with a filter layer having the same optical properties as first dichroic beam splitter (5) and /or reflection element (13) is provided with a filter layer having the same optical properties as second dichroic beam splitter (10).
  • a short-pass filter can replace the bandpass, if the dichroic beam splitter is a short-pass, one needs long- pass filter.
  • the microscope device allows to differentiate the image of a color-labelled object with respect to up to four spectral regions, both in fluorescence-emission and in transmitted light absorption.
  • the optical path-lengths are identical for all spectral ranges (color-channels) and all images lie in the plane of the respective detector chip (camera).
  • the optical layout may be used to correct for longitudinal color-imperfections by adjusting the optical path-lengths accordingly.
  • a volitional detuning of the path-lengths may be used to look at two or more focal depths simultaneously and to reconstruct contrast-enriched images from images taken at different focal positions.
  • a dichroic ensemble designed for separating the emission excited by a 405 and a 488 nm laser, divides the light of a white light emitting diode into two spectral regions below and above 488 nm.
  • the thickness of the filter-element (22) determines the path-length difference of beams A and B.
  • optical element (21) and/or (23) can be provided with optical element (21) and/or (23).
  • the microscope devices of the invention are especially useful in methods for detecting multiple spectral ranges which are emitted during sequencing of DNA/RNA molecules, in particular in sequencing -by- synthesis processes to obtain DNA or RNA sequence information of a biological sample.
  • the sequencing-by-synthesis process is performed by hybridization of nucleotides provided with different dyes to the DNA or RNA of the biological sample and wherein the dyes emit light upon excitation by the one or more light sources in the spectral ranges A, B, C and D.
  • Such sequencing-by-synthesis process and the required dyes are known to the person skilled in the art

Landscapes

  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

La présente invention concerne un dispositif de microscope comprenant un objectif de microscope (1) ; une ou plusieurs sources de lumière ; au moins trois séparateurs de faisceau dichroïque (5, 10, 16) et au moins deux caméras (4, 9) caractérisées en ce que la lumière générée par la source de lumière interagit avec l'échantillon (3) ce qui permet de produire un faisceau d'échantillon, une première image ayant une plage spectrale de lumière (A) étant séparée du faisceau d'échantillon par un premier séparateur de faisceau dichroïque (5) et guidée par l'intermédiaire d'un élément de réflexion (8) au détecteur de lumière (4) de la première caméra (9), ce qui permet d'obtenir une image résiduelle X, une deuxième image ayant une plage spectrale de lumière (B) étant séparée de l'image résiduelle X par un deuxième séparateur de faisceau dichroïque (10) et guidée par l'intermédiaire d'un élément de réflexion (13) à un détecteur de lumière (4) de la première caméra (9) ce qui permet d'obtenir une image résiduelle Y, une troisième image ayant une plage spectrale de lumière (C) étant séparée du faisceau résiduel Y par un troisième séparateur de faisceau dichroïque (16) et guidée par l'intermédiaire d'un élément de réflexion (19, 20) au photodétecteur de lumière (4) de la deuxième caméra (17), ce qui permet d'obtenir une image résiduelle de guidage D d'image résiduelle D en tant que plage spectrale de lumière par l'intermédiaire d'un élément de réflexion (18) vers le détecteur de lumière (4) de la deuxième caméra (17).
PCT/EP2022/050479 2021-01-12 2022-01-12 Dispositif de microscope WO2022152714A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102021100349 2021-01-12
DEDE102021100349.0 2021-01-12

Publications (1)

Publication Number Publication Date
WO2022152714A1 true WO2022152714A1 (fr) 2022-07-21

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Family Applications (1)

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PCT/EP2022/050479 WO2022152714A1 (fr) 2021-01-12 2022-01-12 Dispositif de microscope

Country Status (1)

Country Link
WO (1) WO2022152714A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090309049A1 (en) * 2006-07-20 2009-12-17 Koninklijke Philips Electronics N.V. Multi-color biosensor
US20180067327A1 (en) 2015-03-23 2018-03-08 East Carolina University Multi-wavelength beam splitting systems for simultaneous imaging of a distant object in two or more spectral channels using a single camera
WO2020038752A1 (fr) 2018-08-20 2020-02-27 Till Gmbh Dispositif de microscope

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090309049A1 (en) * 2006-07-20 2009-12-17 Koninklijke Philips Electronics N.V. Multi-color biosensor
US20180067327A1 (en) 2015-03-23 2018-03-08 East Carolina University Multi-wavelength beam splitting systems for simultaneous imaging of a distant object in two or more spectral channels using a single camera
WO2020038752A1 (fr) 2018-08-20 2020-02-27 Till Gmbh Dispositif de microscope

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Cytometry", 1 April 2011, JOHN WILEY & SONS, INC., Hoboken, NJ, USA, ISBN: 978-0-471-14295-9, article TURAN ERDOGAN: "Optical Filters for Wavelength Selection in Fluorescence Instrumentation", pages: 2.4.1 - 2.4.25, XP055322815, DOI: 10.1002/0471142956.cy0204s56 *
ANONYMOUS: "ORCA-D2 Dual CCD Camera - Product Brochure", 5 March 2012 (2012-03-05), XP055554401, Retrieved from the Internet <URL:https://www.biovis.com/images/cameras/e_orcad2.pdf> [retrieved on 20190211] *
YUSUKE NIINO ET AL: "Simultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light", PLOS ONE, vol. 4, no. 6, 1 January 2009 (2009-01-01), US, pages e6036 - e6036, XP055268424, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0006036 *

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