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WO2008152568A2 - Procédé et dispositif pour analyser un échantillon - Google Patents

Procédé et dispositif pour analyser un échantillon Download PDF

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Publication number
WO2008152568A2
WO2008152568A2 PCT/IB2008/052267 IB2008052267W WO2008152568A2 WO 2008152568 A2 WO2008152568 A2 WO 2008152568A2 IB 2008052267 W IB2008052267 W IB 2008052267W WO 2008152568 A2 WO2008152568 A2 WO 2008152568A2
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WO
WIPO (PCT)
Prior art keywords
sample
reference marker
light
optical device
light beam
Prior art date
Application number
PCT/IB2008/052267
Other languages
English (en)
Other versions
WO2008152568A3 (fr
Inventor
Toon Van Alem
Waltherus C. J. Bierhoff
Ivon F. Helwegen
Nenad Mihajlovic
Sjoerd Stallinga
Dirk L. J. Vossen
Original Assignee
Koninklijke Philips Electronics N.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 Koninklijke Philips Electronics N.V. filed Critical Koninklijke Philips Electronics N.V.
Publication of WO2008152568A2 publication Critical patent/WO2008152568A2/fr
Publication of WO2008152568A3 publication Critical patent/WO2008152568A3/fr

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders

Definitions

  • the invention relates to a method and device for analyzing a sample with an optical device.
  • a scanning microscope is used to image or analyze a sample by scanning a focused spot of light over the sample and collecting the light reflected and/or scattered by the sample on a detector.
  • the spot is focused on the sample by an objective lens.
  • the microscope comprises means for displacing the focused spot so as to scan the sample with the focused spot, those means for instance comprising means for rotating a mirror placed in the light path of the incident light beam, means for displacing the objective lens, etc.
  • the microscope may comprise as many displacing means as independent directions to be scanned.
  • the means for displacing the focused spot are driven by electric driving signals that result in a transverse or axial displacement of the focused spot in the sample.
  • Transverse and axial directions are defined relatively to the optical axis of the microscope.
  • a problem arises because the dependence of the actual position of the scanning spot on the driving signals is generally not known beforehand or during the scanning phase with sufficient precision. Indeed, this dependence can vary from apparatus to apparatus and in the course of using an apparatus. Phenomena which may change this dependence are, for instance, manufacturing tolerances, wear, thermal effects, acoustic vibrations, vibrations provoked by the operator, intensity drifts of the means generating the electric signals, influence of the inertial forces on the means for displacing the spot (in particular if the scanning is performed quickly), etc.
  • a scanning microscope is a precision apparatus, even a tiny phenomenon of the above type may change the dependence between the driving signals and the actual position of the spot in the sample in a range that influences the analysis. Indeed, given an electric signal, the position of the focused spot must be known with an accuracy that should be at least equal to the size of the scanning spot, which is typically l ⁇ m. If this accuracy is not met in the transverse directions, unwanted distortions of the image will arise, while if it is not met in the axial direction, an incorrect surface of the sample will be imaged.
  • US 2005/0078361 discloses a microscope comprising at least one transparent specimen support unit associated with a specimen, and a reference specimen including at least one planar area having a defined structure of known configuration, the reference specimen being detectable by light microscopy for at least one of calibration of the microscope.
  • the reference specimen it is thus possible on the one hand to calibrate the double confocal scanning microscope beam path prior to the actual specimen detection, and on the other hand to repeat repeat such a calibration operation during specimen detection for alignment.
  • the specimen detection is paused, and this paused time is used for repeating the calibration operation.
  • such a method is not completely satisfactory since real-time calibration is not rendered possible, while the device may be complicated, with the use of a laser light which has a wavelength different from that used for specimen detection.
  • a method for analyzing a sample with an optical device comprising: a) generating at least one light beam that irradiates, along a single light path, both the sample and a reference marker, b) simultaneously detecting light interacted with the sample, for analyzing the sample, and with the reference marker, for at least one of calibrating, aligning or adjusting the optical device.
  • real time calibration, alignment or adjustment may be performed, since the detection of light interacted with the reference marker and the sample is simultaneous, this simultaneousness being possible because of the simultaneous irradiation, along a single light path preferably having in a single direction, of the sample and the reference marker by the light beam.
  • it is not a particular light beam that is used for irradiating the reference marker but the same light beam as the one irradiating the sample, along the same light path. Since both elements are irradiated by the same light beam, the detection of the signals related to their interaction with light is simultaneous.
  • this direct feedback on the scanning spot notably permits to save time, since everything is performed simultaneously, and to improve the accuracy of the measurements, since even the changes that may occur in the adjusting of the optical device during the scanning phase will be detected in real- time.
  • the method of the invention may permit to measure the actual transverse (perpendicular) and/or axial positions of the light beam, thanks to the reference marker, simultaneously with the analysis of the sample.
  • the method of the invention comprises repeating steps a) and b) for scanning the sample with the light beam.
  • the reference marker is formed of a semi-transparent material.
  • the optical device comprising means for displacing the spot for scanning the sample with the light beam, the displacing means being controlled by a control signal
  • the method comprises determining a parameter of a function linking the control signal to the actual displacement of the spot.
  • the position of the reference marker in the optical device is known a priori with an accuracy at least as good as a required position accuracy for the scanning spot.
  • the method comprises separating the interacted light beam into two detected light beams incident onto two distinct detectors, a first detected light beam comprising light interacted with the sample and a second detected light beam comprising light interacted with the reference marker.
  • the light interacted with the sample and the light interacted with the reference marker are detected on a single detector.
  • the method comprises separating an optical signal corresponding to light interacted with the reference marker from an optical signal corresponding to light interacted with the sample by means of an interference pattern analysis.
  • the optical device being a multi-spot optical device
  • the method comprises generating simultaneously a plurality of light beams along a plurality of light paths, the reference marker being placed on a light path of at least one of the light beam scanning the sample.
  • the reference marker is placed on a light path of all the light beams scanning the sample.
  • the method comprises generating a single light beam for scanning the sample.
  • an optical device for analyzing a sample comprising:
  • a reference marker placed on a light path of the light beam so that the reference marker is irradiated by said light beam simultaneously with the sample, - means for simultaneously detecting light interacted with the sample, for analyzing the sample, and with the reference marker, for at least one of calibrating, aligning or adjusting the optical device.
  • the reference marker is formed of a semi- transparent material.
  • the reflection coefficient of the material forming the reference marker is comprised between 5 and 40%, preferably between 15 and 25%.
  • the reference marker comprises a plurality of parallel strips.
  • the optical device comprising an optical axis
  • the reference marker comprises a structure periodic in the direction of at least a direction transverse to the optical axis of the optical device, the structure for instance comprising a diffraction grating.
  • the optical device comprises at least three reference markers axially positioned so as to define a reference plane relative to which a required slice of the sample to be scanned can be defined.
  • the optical device comprises a focus detection system, for detecting light interacted with the reference markers, the focus detection system comprising an astigmatic focusing device.
  • the optical device comprises a sample support for supporting the sample, wherein the reference marker has the form of a layer deposited on the sample support, which is practical.
  • the reference marker is located between the sample support and the sample.
  • a spacer layer is provided between the reference marker and the sample.
  • the optical device comprises a confocal detector for detecting light interacted with the sample and/or light interacted with the reference marker.
  • the optical device is a scanning microscope.
  • a sample unit comprising a sample support, a sample and a reference marker layer deposited on the sample support between the sample and the sample support.
  • FIG. 1 is a schematic view of a scanning microscope
  • - Fig.2 is a schematic view of a first embodiment of means for displacing the focused spot in a transverse direction;
  • - Fig.3 is a schematic view of a second embodiment of means for displacing the focused spot in a transverse direction
  • - Fig.4 is a schematic view of an embodiment of means for displacing the focused spot in the axial direction
  • - Fig.5 is a schematic view of the objective, the sample and the reference marker in an embodiment of the microscope of the invention
  • - Fig.6 is a schematic view of an embodiment of the detection branch of the microscope of the invention
  • - Fig.7 is a schematic cross-section view of the elementary pattern of an embodiment of the reference marker of the microscope of the invention
  • Fig.8 is a schematic view of a split detector for detection of light interacted with the reference marker of Fig.7;
  • - Fig.9 is a diagram representing, on the upper part, the difference signal between the two half detectors of Fig.8, on the lower part, the function that links the actual position of the focused spot in the x direction to the value of the signal that controls the means for displacing the focused spot;
  • - Fig.10 is a schematic view of an embodiment of the detection branch of the microscope, comprising an astigmatic optical element;
  • - Fig.11 is a schematic view of the action of the astigmatic optical element of
  • - Fig.12 is a schematic view of the detection branch of Fig.10 showing the effect of defocusing the incident light beam
  • - Fig.13 is a schematic view of three spots on the detector of the detection branch of Fig.10, for three different focus positions and
  • - Fig.14 is a diagram representing a s-curve obtained with the signal detected on the detector of Fig.13.
  • the invention applies to an optical device for analyzing a sample and permits calibration, alignment or adjustment of the optical device. It applies particularly to an optical device comprising means for displacing the light beam irradiating the sample and permits calibrating, aligning or adjusting of those displacing means.
  • the invention will be described in relation with a scanning microscope, that is to say, an optical device where the means for displacing the light beam are adapted to scan the sample.
  • a scanning microscope is described with reference to Fig.l.
  • Light is emitted by a light source 1, for instance a laser diode.
  • the emitted light beam 2 is reflected at a beam splitter 3 and collimated into a parallel beam of light by a collimator lens 4 comprising an optical axis 4'.
  • Light that has interacted with the sample 8 is captured by the objective lens 6 and ends up, via the collimator lens 4 and the beam splitter 3, on a detector 9, preferably a silicon photo-diode.
  • the interaction may be, for instance, reflection or scattering. In another embodiment, the interaction may be transmission.
  • the light collected on the detector 9 permits to analyze the sample; analyzing the sample may consist, for instance, in imaging or detection. Imaging for instance consists in obtaining a 2D or 3D image of the sample 8.
  • Detection for instance consists in analyzing a particular feature of the sample 8 (for instance, detect the presence or not of a particular molecule), which may be obtained by the analysis of a particular optical interaction of the light with the sample; for instance, detection may comprise spectroscopy, etc.
  • the scanning microscope comprises means (not represented on Fig.l) for displacing the focused spot 7 in one of the directions x, y perpendicular (transverse) to the optical axis 5 and/or in the axial direction z, that is to say the direction z of the optical axis 5.
  • Those displacing means permit to scan the sample 8 with the focused spot 7, whether in one, two or three directions x, y, z and this is why such a microscope is called a scanning microscope.
  • the scanning microscope has been presented in relation with an objective comprising a single objective lens 6, but the microscope objective may comprise a plurality of lenses, any combination of which can be contemplated. This will in fact generally be the case, since scanning microscopes are precision apparatuses which may require complicated optics.
  • the microscope objective comprises a global optical axis 5.
  • the scanning microscope comprises means for displacing the spot, for instance means for rotating a mirror placed on the light path of the incident light beam, means for displacing the objective lens, etc. These means may be any of the mentioned examples, a combination or any other embodiment not mentioned.
  • the microscope may comprise as many displacing means as independent directions to be scanned.
  • displacing means for displacing the focused spot 7 in a transverse direction, here the x direction, which comprise a rotating mirror 13.
  • the mirror 13 is placed between the collimator lens 4 and the objective lens 6, which in that case are not aligned but positioned with their optical axes 4', 5 perpendicular to each other.
  • the mirror 13 is mounted on hinges such that it can rotate around an axis 13' perpendicular to the plane of the optical axes 4', 5 of the lenses 4, 6, as illustrated by arrow 13".
  • the mirror 13 In a first orientation the mirror 13 reflects the light beam towards the objective lens 6 such that the light beam makes a first angle with the optical axis 5 of the objective lens 6. The objective lens 6 focuses the light beam into a focused spot 7 at a first position A, following a light path designated P A and represented in full line on Fig.2. In the drawing this first angle is zero so that the spot position P A is on the optical axis 5.
  • the mirror 13 In a second orientation the mirror 13 reflects the light beam towards the objective lens 6 such that the light beam makes a second angle 14 with the optical axis 5 of the objective lens 6. The objective lens 6 focuses the light beam into a focused spot 7 at a second position B, following a light path designated P B and represented in dashed line on Fig.2.
  • the second position B is laterally displaced with respect to the first position A, in a direction transverse to the optical axis 5 of the microscope objective 6, here in the x direction.
  • the distance between the two positions A and B is proportional to the angle 14 between the light paths P A and P B , provided that this angle is small compared to 1, which is typically the case. Therefore, the rotation of the mirror 13 directly permits to influence the position of the focused spot 7; by driving the mirror 13, the spot 7 is thus scanned in the x direction.
  • the mirror 13 is driven by driving means, which are controlled by a control electric signal (or voltage).
  • the mirror 13 and its driving means form means for displacing the spot 7, controlled by the control signal, which permit to scan the sample with the light beam.
  • Fig.3 there is represented another embodiment of means for displacing the focused spot in a transverse direction, here in the y direction.
  • the collimator lens 4 and the objective lens 6 are facing each other, their optical axes 4', 5 being parallel to each other.
  • the objective lens 6 is mounted on a holder 15 that can be displaced (translated) in a direction perpendicular to its optical axis 5, here in the y direction.
  • the optical axis 5 of the objective lens 6 is at a first distance from the optical axis 4' of the collimator lens 4.
  • this distance is zero, that is to say, the optical axes 4', 5 are collinear.
  • the objective lens 6 focuses the light beam into a focused spot 7 at a first position C, following a light path designated Pc and represented in full line on Fig.3.
  • a second position (indicated in dashed lines) the optical axis 5 of the objective lens 6 is at a second distance from the optical axis 4' of the collimator lens 4.
  • the objective lens 6 focuses the light beam into a focused spot 7 at a second position D, following a light path designated P D and represented in dashed lines on Fig.3.
  • the sample 8 may be scanned with the focused spot 7 along the y direction.
  • the holder 15 is driven in translation by adapted driving means which are controlled by an electric signal (or voltage).
  • the holder 15 and its driving means form means for displacing the spot 7, controlled by the control signal, which permit to scan the sample with the light beam.
  • Fig.4 there is represented an embodiment of means for displacing the focused spot in the axial z direction (or in a direction parallel to the axial direction z).
  • the collimator lens 4 and the objective lens 6 are facing each other, their optical axes 4', 5 being collinear.
  • the objective lens 6 is mounted on a holder 15' that can be displaced (translated) in the axial direction z.
  • the axial position of the objective lens 6 is at a first distance from the sample 8 and the objective lens 6 focuses the light beam into a focused spot 7 at a first position E, following a light path designated P E and represented in full line on Fig.4.
  • the axial position of the objective lens 6 is at a second distance from the sample 8 and the objective lens 6 focuses the light beam into a focused spot 7 at a second position F, following a light path designated P F and represented in dashed lines on Fig.4.
  • the sample 8 may be scanned with the focused spot 7 along the z direction.
  • the holder 15' is driven in translation by adapted driving means which are controlled by a control electric signal (or voltage).
  • the holder 15' and its driving means form means for displacing the spot 7, controlled by the control signal, which permit to scan the sample with the light beam.
  • the above described means for displacing the focused spot can be combined between them and/or with any other suitable means.
  • the configuration of the microscope and its optics are adapted to the chosen displacing means.
  • the displacement of the scanning spot may either be stepwise, that is to say, the spot is imaged or analyzed at a position, then moved and imaged or analyzed at another position, etc., or continuous, the spot then being imaged or analyzed continuously as it scans the sample.
  • the scanning microscope is adapted to hold a sample support 11, which may be of any kind known in the art.
  • a sample support 11 may also be designated as a sample holder 11 or a substrate 11.
  • the sample support 11 comprises a glass plate 11, transparent to the light beam, and is placed on means 16 for holding and positioning the sample support 11.
  • the microscope comprises an optical reference marker 12 for calibration, alignment or adjustment of the optical device before, during or after the scanning of the sample 8 with the light beam.
  • An optical reference marker is a structure of known position, shape and characteristics that interacts with light that passes through, the interacted light being detected by the microscope and used for purposes of calibration, alignment or adjustment of the optical device.
  • the optical reference marker 12 is positioned on at least a light path P of the light beam scanning the sample 8.
  • the reference marker 12 is therefore positioned in the same direction as the sample 8, in the direction of said light path P.
  • the reference marker 12 and the sample 8 are aligned in the direction of said light path P. Therefore, when the light beam is irradiated on the sample 8 along said light path P, the reference marker 12 is irradiated simultaneously with the sample 8.
  • the reference marker 12 is positioned after the sample 8 on the light path P of the light beam. According to another embodiment, not represented on the drawings, the reference marker 12 is positioned before the sample 8 on the light path P of the light beam, that is to say, on Fig.5, above the sample 8.
  • the reference marker 12 is adapted to simultaneous irradiation, along a single light path, of the reference marker 12 and the sample 8 by a light beam and to simultaneous detection of the optical signals of the light that has interacted with them, with the further constraint that each optical signal is not changed too much by the existence of the other one.
  • the reference marker 12 may be formed of a semi-transparent material, that is to say, a material that reflects more than 0% but less than 100% of the light that passes through.
  • a semi-transparent material is characterized by its reflection coefficient, that is to say, the ratio between the amount of reflected light among the total amount of light incident on the material.
  • the reference marker 12 advantageously is semi-transparent, but for different reasons. In the first case (the reference marker 12 before the sample 8 on the light path P), a sufficient amount of light needs to pass through the reference marker 12 to illuminate the sample 8 for its analysis. In the second case (the reference marker 12 after the sample 8 on the light path P), the reflection must be sufficiently low to avoid illuminating the sample 8 on the return path of the light, in order not to influence too much the optical signal from the sample 8.
  • the reflection coefficient of the material is comprised between 5 and 40%, preferably between 15 and 25%; this permits to make a compromise between the desirable detection of the reference marker 12 and the sample 8; the value 5% is just above the interface glass-air while 40% is below half the power.
  • the reference marker 12 When the reference marker 12 is placed before the sample 8 on the light path P of the light beam, the reference marker 12 is semi-transparent so that the light which is incident on the reference marker 12 is transmitted through the reference marker 12 to also irradiate the sample 8. For instance, 20% of the light that impacts the reference marker 12 may be reflected by this latter and 80% transmitted to the sample 8 for the analysis (imaging or detection) of the sample 8.
  • the optical interaction phenomenon used to detect information resulting from the interaction of light with the reference marker 12 may be reflection but also diffraction, scattering or any other optical interaction.
  • sample unit may be defined as the entity positioned under the light path P of the light beam.
  • the sample unit here comprises the sample support 11, the sample 8 and the reference marker 12; the sample unit is positioned on and held by the means 16 for holding the sample support 11.
  • the sample unit comprises two sample supports, for instance formed by two glass plates sandwiching the sample 8 and the reference marker 12 therebetween.
  • a glass plate similar to the represented glass plate 11 of Fig.5 would be provided above the sample 8 and in contact with this latter.
  • a semi-transparent optical reference marker 12 placed in a light path P of the scanning light beam has the consequence that two relevant optical signals emanate from the sample unit, namely the optical signal from the sample 8 and the optical signal from the reference marker 12. These signals should be distinguished and analyzed.
  • the optical signals are separated and captured on two detectors by splitting the return beam into two parts, that is to say, two detected light beams, as will now be explained.
  • Fig.6 is represented the so-called detection branch of the microscope, that is to say, the elements of the microscope that permit to detect the light that has interacted with the sample 8 and the reference marker 12.
  • Light 17 that has interacted with the sample 8 and the reference marker 12 is, in the embodiment described, captured by the objective lens 6 and the collimator lens 4 and is incident on a beam splitter 18, adapted to separate the interacted light beam 17 into a first detected light beam 19 that comprises the contribution of the interaction of the incident light beam with the sample 8 and a second detected light beam 19' that comprises the contribution of the interaction of the incident light beam with the reference marker 12.
  • Polarization may be used for this separation.
  • the first detected light beam 19 is directed in a direction perpendicular to the optical axis 4' of the collimator lens 4 onto a first detector 9, while the second detected light beam 19' is directed in a direction collinear to the optical axis 4' of the collimator lens 4 onto a second detector 9'.
  • the first detector 9 detects the optical signal related to the interaction of the incident light beam with the sample 8, this signal containing information on the sample 8, while the second detector 9' detects the optical signal related to the interaction of the incident light beam with the reference marker 12.
  • the beam splitter 18 is herein a plate beam splitter, but it can also be a cube beam splitter or a diffractive beam splitter, for instance.
  • the microscope of course comprises a light source 1 for irradiating the sample 8 and the reference marker 12, in the embodiment described with a single light beam, means for displacing the focused spot 7 in the sample 8 and the reference marker 12 so as to scan the sample 8 with the light beam, those means being adapted to the configuration of the detection branch of the microscope.
  • the detection branch is provided on the same side as the light source 1 and the interacted light is collected within the same lenses 4, 6 which also serve to focus it on the sample 8 and the reference marker 12.
  • the light is collected on the side opposite to the side where the light is incident.
  • the imaging or detection is performed in transmission and not in reflection.
  • the reference marker 12 has to be arranged to present optical characteristics that are adapted to simultaneous irradiation, along a single light path, of the reference marker 12 and the sample 8 by a light beam and to simultaneous detection of the optical signals of the light that has interacted with them, with the further constraint that each optical signal is not changed too much by the existence of the other one.
  • the signal on the second detector 9' permits to calibrate, align or adjust the microscope. Notably, it permits to give the relation between the electric signal controlling the means for displacing the light beam and the actual position of the focused spot 7 on the sample support 11, the position of the reference marker 12 on the sample support 11 being known. It may also permit to give the relation between the patterns of light detected on the detectors 9, 9' and the size and shape of the objects generating those patterns, the size and shape of the reference marker 12 being known. Other applications of the reference marker 12 for the calibration, alignment or adjustment and their equivalents may be contemplated.
  • the reference marker 12 may be formed of a layer deposited onto the (glass plate) sample holder 11, on the side on which the sample 8 is supported such that the reference marker 12 is located between the sample holder 11 and the sample 8, as can be seen in Fig.5.
  • the reference marker 12 is formed of a layer deposited on the side of the sample holder 11 opposite to the side supporting the sample 8.
  • the reference marker 12 may be positioned before the sample 8 on the light path P of the light beam, for instance being formed by a layer deposited on a second glass substrate provided in that purpose.
  • the signal emanating from the sample 8, which is carried by the first detected light beam 19 and comprises the contribution of the interaction between the incident light beam and the sample 8, is detected with a confocal detector.
  • a confocal detector has an effective detection area smaller or comparable to the lateral size of the focused spot 7.
  • the effective detection area of the detector 9 is made small, for instance, by having a photo- detector 9 with a small sensitive area, or by placing a pinhole at the image plane corresponding to the sample; a pinhole is an opaque screen with a small, preferentially circular opening.
  • the advantage of a confocal detector is the increased axial resolution.
  • a confocal detector may also be provided to detect the signal from the reference marker 12.
  • the interacted light beam may be split into two detected light beams by a beam splitter, each part being collimated by a lens, pinholes being provided at the images planes corresponding to the sample plane and the reference marker plane.
  • the microscope may be an interference microscope.
  • two signals of comparable amplitude i.e. the signal coming from the reference marker 12 and the signal coming from the sample 8) are detected on the same detector, where they give rise to an interference pattern.
  • This interference pattern is recorded and processed to get the image or any useful information on the sample 8 as well as the image or any useful information on the reference marker 12.
  • the microscope is relatively sensitive to the phase information carried by the light (i.e. the optical path length difference between light from the reference marker 12 and light from the sample 8). This phase information can be related to (small) changes in the refractive index of the sample 8.
  • the optical signal corresponding to light interacted with the reference marker 12 is separated from the optical signal corresponding to light interacted with the sample 8 by an interference pattern analysis.
  • a spacer layer is placed in between the semi-transparent reference marker 12 and the sample 8.
  • This spacer layer may be made of a transparent and chemically inert substance. This prevents possible chemical reactions between the substance of which the reference marker 12 is made (e.g. a metal) and the sample 8 (e.g. biological tissue).
  • the reference marker 12 e.g. a metal
  • the sample 8 e.g. biological tissue.
  • a confocal detector it improves the axial resolution of the confocal detection for points of the sample 8 close to the reference marker 12, since in that case the reference marker 12 is out of focus by a distance of at least the thickness of the spacer layer.
  • the reference marker layer 12 preferably has a lateral extension exceeding that of the sample layer 8, but not necessarily.
  • the reference marker 12 comprises a layer deposited on the surface of the sample support 11, which layer is continuously deposited on the surface of the sample support 11.
  • the reference marker 12 comprises a plurality of isolated patches or strips of material at regular intervals.
  • the lateral structure of the reference marker layer 12 may be uniform, for measuring the axial (focus) position, or it may comprise a grating structure, or a set of pits, or something else that characterizes the structure of the reference marker 12 in the transverse directions, so as to generate an optical signal depending on the transverse position of the light beam; in that case, the reference marker 12 may comprise a periodic structure in one or both of the transverse directions.
  • the reference marker 12 comprises a layer that extends over the whole transverse surface of the sample 8 and that is structured as a two-dimensional grating (periodic in the x direction and periodic in the y direction).
  • a method such as the one that will be described below can then be used to count the number of periods in x and y starting from a certain begin point. In this way the x and y position of the scanning spot 7 can be measured.
  • the so-called astigmatic method, described below, can be used to measure the z-position of the scanning spot 7 with respect to the reference marker layer 12.
  • the reference marker 12 is an integral part of the microscope instead of being a part of the sample holder 11. In such a way, the actual position of the reference marker 12 does not depend on the position of the sample support 11 but only on the manufacturing tolerances of fixation of the reference marker 12 on the microscope.
  • the reference marker 12 extends, in at least one lateral direction x, y, along the whole length of the sample 8 in that direction.
  • the reference marker 12 extends, in one of the lateral directions x, y, along the whole length of the glass plate 11.
  • the axial depth of the reference marker 12 is inferior to the axial depth of the sample 8. It may for instance be a few nanometers deep. This permits to use a metal layer that is partly reflective.
  • the reference marker 12 comprises a plurality of discrete marks, their number depending on the accuracy required.
  • the reference marker 12 may comprise a plurality of parallel strips of a semi-transparent material.
  • the strips may be l ⁇ m wide, spaced apart by lO ⁇ m.
  • the parallel strips are of different structure in order to distinguish them.
  • the parallel strips are all identical; in such a case, in order to know the exact position of the focused spot on the sample holder 11, the scanning method may be started at an edge (or corner) of the sample holder 11 and the spot be scanned perpendicularly to the parallel strips, the strips crossed by the light beam being counted so as to deduce therefrom the exact position of the spot.
  • the parallel strips of the reference marker 12 fulfill a function similar to the one of the bars of a bar code.
  • the reference marker 12 for determining the relation between the electric signal controlling the means for displacing the focused spot 7 and the actual position of the spot 7, in the transverse directions as well as in the axial direction.
  • the reference marker 12 permits to determine the position of the focused spot 7 as a function of the electric signal driving the displacing means. Therefore, the reference marker 12 functions as a gauge for transverse or axial positioning of the focused spot.
  • the displacing means may be of any kind, for instance as the ones described with reference to Figs.2-4.
  • the reference marker 12 may comprise structures adapted to interact, that is to say reflect, diffract, scatter or otherwise modify the incident light, such that the position of these structures is known a priori with an accuracy at least as good as the required position accuracy of the scanning spot.
  • the optical reference marker 12 is scanned, light diffracted, scattered, or reflected by the structures that form the marker (at least partly) enters the optical system and is captured by the second photo-detector 9', thereby producing a signal that may be used to connect the control signal of the means for displacing the focused spot 7 to the a priori known position of the reference marker 12.
  • the reference marker 12 comprises a diffraction grating.
  • the grating 12 comprises a substrate 20 and a structure comprising an elementary pattern 21 reproduced periodically in one direction, here in the x direction.
  • the grating 12 is herein a binary grating, that is to say a grating that has a height profile consisting of two levels.
  • the elementary pattern 21 of the grating 12 here comprises a first portion 22 at a first height, a second portion 23 at a second height and a third portion 24 at the first height, the difference h between the two heights being called the step height h, the whole elementary pattern 21 extending on a distance p in the x direction.
  • the periodicity p is conventionally called the pitch p.
  • the pitch should satisfy p> ⁇ /2NA and is preferably close to ⁇ /NA for a better detection of the out of focus reference marker 12 by the microscope, where ⁇ is the wavelength of the light irradiated on the reference marker 12 and NA is the so-called numerical aperture of the objective lens 6.
  • the reflected cone is called the 0 th order
  • the cone at the angle + ⁇ is called the +l st order
  • the cone at the angle - ⁇ is called the -1 st order.
  • the 0 th order beam enters the microscope entirely, the ⁇ l st orders only insofar they overlap with the 0 th order.
  • the diffracted beams are incident on a split photo-detector 9', illustrated on Fig.8, that is to say, a detector 9' that is split into two segments 9'a, 9'b.
  • Fig.8 are represented the spot 25 corresponding to the 0 th order light beam, in full lines, and the spots 26, 27 corresponding to the +l st and -1 st order light beams, in dashed lines. It can be seen on Fig.8 that the ⁇ l st order spots 26, 27 overlap with the 0 th order spot, in overlapping regions 26', 27', respectively.
  • the intensity of the overlapping regions 26', 27' vary sinusoidally with the position of the focused scanning spot 7 with respect to the grating 12. There is also a phase delay between the sinusoidal variations in the two overlapping regions 26', 27' depending on the phase depth ⁇ of the grating 12.
  • the two segments 9'a, 9'b each give a signals S a , Sb.
  • the function x(V) can be determined, as can be seen on the lower part of Fig.9; x(V) is the function that links the actual position of the focused spot 7 in the x direction to the value of the electric signal V that controls the means for displacing the focused spot 7. Therefore, thanks to the reference marker 12, the x position of the focused spot 7 may be gauged.
  • a second optical reference marker 12 to gauge the y position of the scanning spot may also be provided, similar to the grating 12 that has just been described, but extending in the y direction.
  • three optical reference markers 12 may be provided, which comprise structures adapted to interact with, that is to say reflect, diffract, scatter or otherwise modify the incident light, such that the axial position of these markers is known a priori with an accuracy at least as good as the required position accuracy of the scanning spot.
  • the means for displacing the focused spot in the axial direction z can be used to adjust the axial position of the focused spot 7 to a prescribed value for a specific transverse position characterized by coordinates (x,y).
  • the microscope scans a two-dimensional slice of the sample 8 defined by a function z(x,y). Generally, this slice is a plane, i.e. the surface is not curved.
  • the positions of the at least three reference markers 12 define a reference plane relative to which the required slice z(x,y) can be defined.
  • the microscope simultaneously scans, with a single incident light beam, the optical reference markers 12 and the sample 8.
  • the scanning microscope comprises a focus detection system that measures the distance between the focused spot 7 and the (partially) reflecting surface of a reference marker 12, from the light reflected off this surface.
  • This focus detection system may comprise a so-called astigmatic focusing device for performing a so-called astigmatic method.
  • an astigmatic optical element is placed on the detection light path (i.e. the light path of the light, which has interacted with the sample 8 and reference marker 12, which returns in the microscope for being detected).
  • This astigmatic optical element may be placed before the detector 9', detecting the light that has interacted with the reference marker 12, as shown on Fig.10, where only the elements necessary to the comprehension of the described embodiment are represented.
  • This astigmatic optical element is, in this embodiment, a lens 28 with at least one surface of toroidal shape (i.e. a curved surface with a different curvature in the two directions perpendicular to its optical axis 28').
  • Such an astigmatic lens 28 may have one spherical surface and one cylindrical surface.
  • the astigmatic lens 28 may be replaced by a plan-parallel plate placed in the return beam at a substantial angle (e.g. 45°) with the optical axis or by a diffractive optical element.
  • Fig.11 shows the action of the astigmatic lens 28 on a light beam.
  • the light is focused in two mutually perpendicular focal lines 29, 30, a first focal line 29 in front of the detector 9' and a second focal line 30 behind the detector 9'.
  • the focal lines 29, 30 are perpendicular to each other and both are perpendicular to the axial direction z.
  • the first focal line 29 is parallel to the x axis and the second focal line 30 is parallel to the y axis.
  • Fig.l 1 where it can be seen that light rays 31, 31' in the plane comprising the y and z directions (y-z plane) are focused at the intersection of the first focal line 29 with the optical axis 28'.
  • Light rays 32, 32' in the x-z plane are focused at the intersection of the second focal line 30 with the optical axis 28'.
  • the cross-section of the bundle of rays is circular; this spot 33 is called the circle of least confusion.
  • Fig.12 shows the effect of defocusing the incident light beam with respect to the reference marker 12.
  • the focused spot 7 is no longer at the reference marker 12 but at a position (shown in dashed lines) in front of the reference marker 12.
  • the focal lines 29, 30 move to the positions 29', 30' in dashed lines, in the direction opposite to the incidence direction of the interacted beam on the detector 9'.
  • the spot on the detector 9' is no longer circular but elliptical, elongated in the direction of second focal line 30.
  • the focal lines move to positions in the direction of the incidence direction of the beam on the detector 9'; as a consequence the spot on the detector 9' is no longer circular but elliptical, elongated in the direction of the first focal line 29.
  • the long axis of the ellipse is in the direction of the focal line that is closest to the detector 9'.
  • the detector 9' is divided in four segments G, H, I, J and the focal lines 29, 30 are oriented along the diagonal directions of the detector segmentation.
  • An axial position signal can be obtained by the diagonal difference signal G+I-H-J (see Fig.13).
  • Fig.14 shows this axial position signal as a function of the driving signal V, which is the so-called s-curve.
  • the signal is zero (So) for the in focus configuration, is maximum (S M ) for a configuration in which the first focal line 29 is at the detector plane, and is minimum (S m ) for a configuration in which the second focal line 30 is at the detector plane.
  • the zero-crossing of the curve corresponds to the axial position of the reference marker 12.
  • the measured axial position driving signals V J then define the driving signal settings V(x,y) corresponding to this plane.
  • the offset ⁇ V(x,y) with respect to this setting thus gives the axial offset ⁇ z(x,y) between the sample slice that is scanned and the reference plane, in other words, a signal is obtain that measures the axial distance between the focused spot 7 and the semi-transparent marker 12.
  • the range for which the focus detection signal gives a substantially linear position signal the so-called s-curve length
  • the s-curve length must be larger than the thickness of the sample 8.
  • the s-curve length should be about 20 ⁇ m. This is significantly larger than the typical s-curve lengths in a focus detection system for optical storage applications, which is about 5 ⁇ m. This large s-curve length can be achieved by e.g. changing the curvature of the cylindrical surface of a typical astigmatic lens for optical storage applications.
  • the s-curve length should be at least as large as the sum of the sample layer thickness and the spacer layer thickness.
  • the optical microscope of the invention may be integrated with an electron microscope, be used in a bio-sensing application based on imaging or be used for the examination of pathology samples.
  • the invention has been presented in relation with a microscope generating a single light beam that is scanned in the sample.
  • the microscope is a multi-spot scanning microscope, that is to say, a microscope where a plurality of light beams are simultaneously irradiated on the sample along a plurality of light paths.
  • the plurality of light beams form, in such a case, an array of spots, the array for instance extending in the two transverse directions and the spots being spaced apart by regular spaces in the two directions.
  • a reference marker 12 may be placed on a light path of at least one light beam scanning the sample 8.
  • the reference marker 12 may be placed on a light path of all the light beams scanning the sample 8 for simultaneous irradiation of the reference marker 12 and the sample 8 by all the light beams.
  • a computer program may be stored/distributed on a suitable medium, such as an optical storage medium or a solid-state medium supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless telecommunication systems. Any reference signs in the claims should not be construed as limiting the scope.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention porte sur un dispositif optique pour analyser un échantillon (8), comprenant : des moyens pour irradier l'échantillon (8) par au moins un faisceau lumineux, un marqueur de référence (12) placé sur un trajet de lumière (P) du faisceau lumineux de telle sorte que le marqueur de référence (12) est irradié par ledit faisceau lumineux simultanément avec l'échantillon (8), des moyens pour détecter simultanément une lumière qui interagit avec l'échantillon (8), pour analyser l'échantillon (8), et avec le marqueur de référence (12), pour au moins l'un parmi l'étalonnage, l'alignement ou l'ajustement du dispositif optique.
PCT/IB2008/052267 2007-06-11 2008-06-09 Procédé et dispositif pour analyser un échantillon WO2008152568A2 (fr)

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JP3445100B2 (ja) * 1997-06-02 2003-09-08 キヤノン株式会社 位置検出方法及び位置検出装置
DE10100246A1 (de) * 2001-01-05 2002-07-11 Leica Microsystems Mikroskop und Verfahren zum Betreiben eines Mikroskops
JP4158514B2 (ja) * 2002-12-24 2008-10-01 ウシオ電機株式会社 両面投影露光装置
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