WO2001044852A2 - Procede et dispositif de microscopie - Google Patents

Procede et dispositif de microscopie Download PDF

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Publication number
WO2001044852A2
WO2001044852A2 PCT/EP2000/012120 EP0012120W WO0144852A2 WO 2001044852 A2 WO2001044852 A2 WO 2001044852A2 EP 0012120 W EP0012120 W EP 0012120W WO 0144852 A2 WO0144852 A2 WO 0144852A2
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WO
WIPO (PCT)
Prior art keywords
sample
illumination
modulator
imaging system
focal plane
Prior art date
Application number
PCT/EP2000/012120
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German (de)
English (en)
Other versions
WO2001044852A3 (fr
Inventor
Achim Kirsch
Jürgen Müller
Original Assignee
Evotec Oai Ag
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 Evotec Oai Ag filed Critical Evotec Oai Ag
Priority to DE20023019U priority Critical patent/DE20023019U1/de
Publication of WO2001044852A2 publication Critical patent/WO2001044852A2/fr
Publication of WO2001044852A3 publication Critical patent/WO2001044852A3/fr

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Classifications

    • 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
    • G02B21/20—Binocular arrangements
    • G02B21/22—Stereoscopic arrangements

Definitions

  • the invention relates to a microscopy method for generating three-dimensionally spatially resolved optical images of a sample, in particular a microscopy method using a microscope with a lateral illumination modulator.
  • the invention further relates to optical imaging systems for obtaining three-dimensionally spatially resolved images of a sample.
  • the lighting of each point is ideally modulated with a different time sequence in such a way that there is no correlation with the time sequences of the lighting of all other points. For this it is necessary to choose independent random sequences for each point.
  • sets of finitely long sequences with vanishing correlation such as e.g. B. complementary Golay sequences or Hadamard sequences of the S-matrix type, which consist of a maximum of as many elements as the sequences are long, are selected. It is often sufficient because the crosstalk between two points decreases with distance, with short sequences only the correlation between points close to each other can be broken. Since these sequences also contain negative values that cannot be implemented purely optically in a transmission mask, a constant component must be added to the sequence. This causes the measured optical image to overlap a confocal with a conventional one
  • a disadvantage of this class of confocal microscopes is the contrast range of the detector required by the superimposition of the confocal and conventional signals. With a comparable signal component of the confocal and conventional image, the detector must be able to process twice the total signal compared to the desired confocal signal.
  • confocal microscopes Another disadvantage of confocal microscopes is that the confocal signal is only detected from a subset of all pixels. With confocal microscopes only Signals recorded simultaneously from approximately 2% (or even far below) to a maximum of 50% of the area of the focal plane. As a result, the measured intensity is up to a factor of 50 (or more) greater than the time-averaged intensity. In order to achieve a high data acquisition rate with these methods, it is generally necessary to work with a very high local excitation intensity. However, nonlinear processes such as the accumulation of the dye molecules in triplet states, which have a negative influence on the signal intensity, are gaining in importance.
  • the object of the invention is to provide an improved method for three-dimensional spatial microscopy and microscopes for implementing this method, with which both a high luminous efficacy and a high data acquisition rate can be achieved without the aforementioned disadvantages.
  • an optical imaging method in which a conventional first partial image of a sample is first recorded.
  • a conventional image is understood here to mean the light-microscopic image of the entire sample area under consideration.
  • the first partial image also contains interference signals which result from the fact that light which is emitted or scattered outside the focal plane, or light after multiple scattering inside and outside the focal plane Detector reached.
  • the focal plane is the image plane on which the microscope is focused for specimen imaging.
  • a second partial image is recorded on the sample, which contains only the undesired storages.
  • the sample is illuminated with laterally structured illumination and completely scanned.
  • the optical signal is recorded only from sample points that are not currently illuminated.
  • the desired three-dimensionally resolved image is finally obtained by compensating for the storages in the first field with the help of the second field.
  • the three-dimensional, spatially resolved image has a spatial resolution in the image plane and perpendicular to it.
  • Another object of the invention is to provide an optical imaging system with at least one light source for sample illumination, a device for recording a first conventional partial image of the sample, a lateral illumination modulator for structured illumination of the sample in the focal plane and a device for transmitting light from non- illuminated sample areas to a detector device.
  • the lighting modulator is formed by a DMD (Digital Mirror Device) or an LCD (Liquid Crystal Device), which are controlled according to a predetermined time pattern for the structured illumination of the sample in the focal plane.
  • the structured illumination takes place through a partially reflecting or transmitting mask, which is moved, for example, by rotation or translation.
  • a partially reflecting or transmitting mask which is moved, for example, by rotation or translation.
  • the illumination modulators are used in reflection or transmission depending on the sample, the imaging systems operate in reflection or transmission geometry and one or two light sources are provided, each with adapted illumination optics.
  • the invention has the following advantages.
  • the structure of the microscope is simplified.
  • lossy and application-specific dichroic beam splitters can be dispensed with, since the illumination and detection beam paths do not overlap.
  • a 50% beam splitter can be used to record the conventional first partial image, which reflects approximately 50% of the incident light and transmits approximately 50% almost independently of the wavelength.
  • the lighting and imaging efficiency is the same for both partial images.
  • a further advantage over the second class of confocal microscopes results from the fact that less requirements can be placed on the contrast range of the detector than with the conventional techniques, because, in contrast, the two partial images to be recorded either consist of a conventional image or of the in background contained in this image.
  • the scaling of the signal on the detector can Orientation of the conventional image and does not have to take into account further signal components added to it.
  • Another advantage results from the fact that signals from sample areas are recorded which are not irradiated with the maximum intensity. Nonlinear effects such as the accumulation in triplet states, which increasingly occur in dyes with a high excitation intensity and thus reduce the fluorescence signal, are avoided. This means that there are less stringent requirements for the photophysical properties of the dyes.
  • FIG. 1 a schematic representation of the light path in an optical imaging system according to a first embodiment of the invention with two light sources
  • FIG. 2 shows a schematic illustration of the light path in an optical imaging system according to a further embodiment of the invention with a light source
  • FIG. 3 a schematic representation of the light path in an optical imaging system according to a further embodiment of the invention with a rotating mask
  • FIG. 4 a schematic representation of the light path in an optical imaging system according to a further embodiment of the invention in a transmission geometry.
  • the imaging technology according to the invention can be implemented with lighting modulators which are used as transmitted light modulators (for example programmable aperture masks based on liquid crystals (LCD) or micromechanical switches), as reflection modulators (for example DMD) or mixed forms thereof (for example partially reflecting and transmitting panes) are executed.
  • transmitted light modulators for example programmable aperture masks based on liquid crystals (LCD) or micromechanical switches
  • reflection modulators for example DMD
  • mixed forms thereof for example partially reflecting and transmitting panes
  • FIG. 1 comprises a first light source 110, an illumination modulator 120 with a multiplicity of illumination elements 121, an imaging optics 130, a detector device 150 and a second light source 160.
  • the reference numeral 140 refers to a sample that is associated with the imaging system 100 is to be mapped.
  • light sources 110 and 160 z. B. use filtered white light lamps or laser light sources, both light sources preferably having the same type. zen, for example to excite a certain fluorescence in the sample 140.
  • a first illumination light path for uniform illumination of the sample 140 is formed by the first light source 110 via a field lens 111, the illumination empler 113 and the imaging optics 132.
  • the second light source 160 forms a second illumination light path for sample illumination with a laterally varying intensity m of the focal plane via a further field lens 161, the illumination modulator 120 and the imaging optics 130.
  • the lighting modulator 120 shown here is a DMD.
  • the DMD 120 contains a matrix arrangement of individual mirrors which can be tilted independently of one another between two stable positions, which form the modulator elements and of which only two mirrors 121 are shown in one of the pivot positions for reasons of clarity (121a and 121b).
  • the deflection from the DMD area depends on the specific design of the DMD used and can be, for example, ⁇ 10 ° with typical mirror dimensions of around 16 ⁇ m.
  • the structure and control technology of DMDs are known per se from the prior art, so that they are not further explained here.
  • the imaging optics 130 comprise a first field lens 131 and an objective 132.
  • the detector device 150 comprises an imaging optics 151, a filter 152 and a detector camera 153. With the detector camera 153, light from all sample areas or only from the unilluminated points of the focal plane is optionally recorded.
  • the imaging optics 151 can be used both with lenses and with mirrors such as, for example, an opener triplet according to the patent US Pat reflective elements. Due to the imaging properties of the Offner triplet, it is very well suited, for example, for use with a DMD.
  • the imaging system 130 is arranged in such a way that the illumination modulator 120 is real-sized in a microscope application, the sample 140 is imaged.
  • the two pivot positions 121a and 121b of the mirrors 121 can be differentiated according to their function for lighting or detection. In the position 121a of the mirrors 121 (see lower partial image in FIG. 1), these guide light from the lamp 110 with the imaging optics 130 onto the sample 140 (illumination). In contrast, the lighting modulators in position 121b are oriented such that light is reflected from the sample 140 to the detector device 150 (detection).
  • the light from the first light source 110 is directed onto the sample 140 via the field lenses 111, the illumination empler 113 and the imaging optics 132.
  • the lighting empler is here a prismatic beam splitter or a semitransparent mirror with a permeability of, for example, 50%.
  • a shutter 112 can be installed or the lighting empler 113 can be switched or pivoted.
  • the spatially three-dimensional image recording with an optical imaging system 100 according to Figure 1 is carried out by alternating sample imaging with the following settings:
  • a first imaging mode the lighting with the first light source 110 is released either with the shutter 112 or the switchable lighting empler 113, so that the entire sample is illuminated via the illumination empler 113.
  • all of the mirrors 121 of the DMD 120 are pivoted into the detection position 121b, so that no light reaches the sample 140 from the light source 160 and the entire sample is imaged on the detector device 150 (detection of the first, conventional partial image).
  • the light path from the first light source 110 to the sample 140 is blocked by the shutter 112 or the switchable mirror 113.
  • the sample 140 is now illuminated with the light source 160 via the mirrors 121 of the DMD 120 pivoted into the illumination position 121a. Which mirrors 121 of the DMD 120 are pivoted into the illumination position in chronological order and which points in the focal plane of the sample 140 are illuminated depends on the respective modulation pattern.
  • the modulation pattern determines the time-dependent illumination sequence or the time-dependent aperture pattern with which the focal plane of the sample 140 is illuminated.
  • the modulation pattern can be based, for example, on a so-called pseudo-random sequence with finite length, a Hadamard sequence of the S-matrix type or a random sequence. In particular, all sequences can be implemented, which are described in EP 0 911 667 AI and WO 97/31282.
  • the sample 140 is illuminated with time-dependent modulation patterns by imaging the micromirrors 121 into the focal plane. Simultaneously, the light emitted in the remaining sample 140 (scattered light and fluorescent light) is directed to the detector device 150 via the imaging optics 130 and the microscope mirrors 121 pivoted in the detection position 121b.
  • the detector camera 153 takes a two-dimensional one Image of the sample 140, with m being detected at any time in the focal plane only from those points that are not illuminated in each case. This second sub-picture thus essentially consists of the background contributions.
  • the partial images determined during the first or second sample acquisition are evaluated to determine an image of the focal plane.
  • the difference image is calculated from the first and second partial images.
  • the signals of the partial images are preferably initially weighted such that the signal components contained in the two partial images cancel each other outside the focal plane. This can be done as follows:
  • a test object is arranged in the form of a scattering or fluorescent flat disk or another arbitrary, as homogeneously scattering or fluorescent object as possible outside the focal plane.
  • the test object is alternately illuminated with the first and second light sources 110 and 160 and the respective signal distributions are recorded in accordance with the two imaging modes.
  • Corresponding normalization factors result from the quotient of the signal distributions, which are taken into account in the aforementioned difference formation.
  • a common normalization factor or specific normalization factors for individual pixels or groups of pixels are determined for the entire image.
  • FIG. 2 An alternative embodiment of an optical imaging system 100 according to the invention is shown in FIG. 2. at In this embodiment, only one light source 170 is provided, the light of which can be selected via the field lens 171, the switchable or pivotable illumination empler 172 and the imaging optics 131 or the deflecting mirror 173, the micromirror 121 of the DMD 120 pivoted in the illumination position and the imaging optics 130 is directed to the test.
  • the conventional illumination of the entire sample takes place via the illumination empler 172 designed as a prism beam splitter or partially transparent mirror, as has already been described for FIG. 1 (light source 110).
  • the illumination takes place via the micromirrors 121 of the DMD 120 pivoted into the illumination position 121a, as has already been described, while light from the non-illuminated areas of the focal plane is recorded by means of the detector device 150.
  • all elements of the DMD are pivoted 120 m to the detection position 121 b or the illumination empler 172 is brought out of the beam path. Even if it is not shown in FIG. 2, an optional construction with shutters is possible. Otherwise, the imaging system 100 according to FIG. 2 corresponds to the first embodiment described above, so that the individual components are identified by the same reference numerals as above.
  • Reference numeral 174 denotes a shutter. Electromechanical shutters (e.g. diaphragms or swiveling mirrors, operated e.g. with electric motors or lifting magnets) are preferably used as shutters.
  • the sample is also imaged, as described above, by recording two fields according to the two imaging modes and then into one spatially three-dimensionally resolved image of the sample.
  • the DMD 120 is in turn driven according to the modulation patterns already mentioned.
  • FIG. 3 A further embodiment of an imaging system 200 according to the invention with a partially transparent and partially reflecting rotating mask is shown in FIG. 3. It comprises a light source 210, a mask 220 as a lighting modulator, an imaging optics 230 and a detector device 250 consisting of an imaging optics 251, a filter 252 and a detector camera 253.
  • the reference numeral 240 refers to a sample that is to be imaged with the imaging system 200.
  • the light source 210 illuminates the mask 220 via the field lens 211, which is imaged into the sample 240 with the illumination optics 230. In the focal plane of the sample 240, there is thus an illumination pattern which corresponds to the reflection pattern imprinted on the mask 220.
  • the light emitted by the sample 240 is imaged on the mask 220 using the imaging optics 230.
  • the imaging optics 251 the mask 220 is imaged on the detector camera 253. Similar to the imaging optics 151, the imaging optics 251 can be constructed from both refractive and reflective elements.
  • the imaging modes described here differ in the optical properties of individual subareas (sectors) of the mask 220.
  • a preferred embodiment of the mask 220 is shown in the lower part of FIG. 3.
  • a first imaging mode results from the fact that the homogeneous partially reflecting sector a of the mask 220 is used for uniform, conventional illumination of the sample 240.
  • the light emitted by the sample 240 can partially pass through the homogeneous sector of the mask 220 and is imaged on the detector camera 253 (acquisition of the conventional partial image).
  • a sector b of the mask 220 is used, on which, for. B. by means of photolithographic techniques, the aforementioned modulation patterns were impressed. Fully mirrored and completely transmitting zones are arranged here according to the desired modulation pattern.
  • the modulation pattern can be selected as in the previously described embodiments, for example as a pseudo-random number sequence. Due to the rotation of the mask 220, the lateral arrangement of the aperture patterns on the disk 220 results in a time-dependent lighting sequence for the individual points of the focal plane.
  • this mask 220 acts in such a way that only light from points that are not currently illuminated pass through the mask and can be recorded with the detector device 250.
  • the second partial image recorded with the detector camera 253 thus essentially consists of the undesirable background contributions.
  • the further image processing for obtaining a three-dimensionally resolved image of the focal plane of the sample 240 from the two partial images is carried out according to the methods described above.
  • the positions of the illumination device 210, 211 and the detector device 250 can optionally be interchanged without any further changes.
  • FIG. 300 A further alternative embodiment of an optical imaging system 300 according to the invention for examinations in transmission geometry is shown in FIG. It comprises a light source 310 which illuminates an illumination modulator 320 via a field lens 311.
  • the focal plane m of the sample 340 is imaged via an imaging optics 330 m.
  • a second imaging optics 331 images this focal plane in the sample 340 onto a detection modulator 350, which is imaged onto a detector camera 363 via an imaging optics 361 and a filter 362.
  • the light source 310 is, for example, a filtered white light lamp or a filtered laser.
  • the modulators 320, 350 are designed here as LCDs. For fluorescence applications, the emission of sample 340 is filtered out with filter 362.
  • all pixels of the LCDs 320, 350 are set to transmission, so that the sample 340 is illuminated uniformly and a conventional first partial image is recorded with the detector camera 363.
  • the transmission of the elements of the lighting modulator 320 is varied, for example, according to the modulation patterns described above, and the points of the focal plane of the sample 340 are thereby illuminated with the corresponding time-dependent lighting sequences.
  • the transmission of the elements of the detection modulator 350 is controlled in such a way that only light from the points of the focal plane of the sample that are not directly illuminated at the respective time reaches the detector device 360.
  • This second partial image recorded with the detector camera 363 contains the background contributions.
  • a three-dimensionally resolved image of the focal plane of the sample 340 from the two partial images is carried out according to the methods described above.
  • the optical imaging system according to the invention can be implemented both as a stand-alone device and as an addition to an available microscope.
  • the DMD lighting modulator can be implemented, for example, with a “DLP XGA Electronics Subsystem Kit” (manufacturer: Texas Instruments, Dallas, TX, USA).

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

Abstract

Selon l"invention, pour représenter de manière optique en trois dimensions un échantillon (140), il est prévu d"éclairer uniformément l"ensemble de l"échantillon (140) et d"effectuer une première prise de vue partielle conventionnelle et d"éclairer latéralement de manière structurée ledit échantillon (14) dans un plan focal. L"éclairage des points individuels du plan focal varie selon des séquences prédéterminées. Une seconde prise de vue partielle intervient ensuite par détection de la lumière émise par des points d"objet non éclairés dans chaque cas dans le plan focal, puis une image de l"échantillon (140) résolue spatialement en trois dimensions est déterminée à partie de la première et de la seconde image partielle. L"invention concerne également des systèmes de représentation optiques utilisés pour mettre ledit procédé de représentation en oeuvre.
PCT/EP2000/012120 1999-12-15 2000-12-01 Procede et dispositif de microscopie WO2001044852A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DE20023019U DE20023019U1 (de) 1999-12-15 2000-12-01 Vorrichtung zur Mikroskopie

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DE1999160583 DE19960583A1 (de) 1999-12-15 1999-12-15 Verfahren und Vorrichtung zur Mikroskopie
DE19960583.1 1999-12-15

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Cited By (2)

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US6778724B2 (en) 2000-11-28 2004-08-17 The Regents Of The University Of California Optical switching and sorting of biological samples and microparticles transported in a micro-fluidic device, including integrated bio-chip devices
US7745221B2 (en) 2003-08-28 2010-06-29 Celula, Inc. Methods and apparatus for sorting cells using an optical switch in a microfluidic channel network

Families Citing this family (5)

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AU3609601A (en) 2000-03-06 2001-09-17 Olympus Optical Co., Ltd. Pattern forming member applied to sectioning image observing device and sectioning image observing device using it
DE102005040471B4 (de) * 2005-08-26 2007-06-21 Leica Microsystems (Schweiz) Ag Mikroskop
US7593156B2 (en) 2005-08-26 2009-09-22 Leica Microsystems (Schweiz) Ag Microscope with micro-mirrors for optional deflection and/or beam splitting
DE102006022590C5 (de) * 2006-05-15 2010-05-12 Leica Microsystems (Schweiz) Ag Beleuchtungseinheit für ein Mikroskop
DE102006022592B4 (de) * 2006-05-15 2008-02-07 Leica Microsystems (Schweiz) Ag Mikroskop mit Beleuchtungseinheit

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
US6778724B2 (en) 2000-11-28 2004-08-17 The Regents Of The University Of California Optical switching and sorting of biological samples and microparticles transported in a micro-fluidic device, including integrated bio-chip devices
US7745221B2 (en) 2003-08-28 2010-06-29 Celula, Inc. Methods and apparatus for sorting cells using an optical switch in a microfluidic channel network
US8426209B2 (en) 2003-08-28 2013-04-23 Celula, Inc. Methods and apparatus for sorting cells using an optical switch in a microfluidic channel network

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DE19960583A1 (de) 2001-07-05
DE20023019U1 (de) 2002-11-07

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