Method and apparatus for acquiring brightness information of a sample

Abstract

The invention relates in particular to a method for obtaining brightness information of a sample (48) using fluorescence correlation spectroscopy. In a first measurement block, the sample (48) is illuminated with a focused beam of excitation radiation using a lens (47), which is directed onto / into the sample (48) in an angular range of a first numerical aperture. A first range of the cross section of the beam is adjusted for this purpose in the entrance pupil (EP) of the lens (47). At the sample (48) in the excitation volume resulting from the illumination, detection radiation is generated and detected. The detected detection radiation is directed along a detection beam path (410) and mapped onto a spatially resolved detector (414) comprising a plurality of detector elements (1-32) that are equally spaced around an optical axis (oA) of the detection beam path (410) and that are readable independently of one another, and brightness information is obtained. Advantageously, the first numerical aperture is selected based on the sample (48) by obtaining values ​​of at least some optical properties of the sample (48), and the current first numerical aperture is determined and adjusted based on the obtained values. The invention further relates to a device designed to carry out the method according to the invention.

Description

Technical Field

[0001] The present invention relates to a method and an apparatus according to the preamble of the independent claims.

Background Art

[0002] Fluorescence correlation spectroscopy (FCS) is a method in the field of confocal microscopy, particularly confocal laser scanning microscopy, and its effect has been proven, for example, to examine the dynamics of the behavior of molecules within cells.

[0003] The method, which is also abbreviated as FCS hereinafter, has been continuously developed further, whereby various variants of FCS are available (for example, Non-Patent Document 1 and Non-Patent Document 2). One variant is known as "spot-variation FCS" (for example, Non-Patent Document 3). In this process, the excitation radiation is focused and directed towards the sample. Depending on the focusing and spreading range of the focus of the focused beam of the excitation radiation in the direction (x or y direction) transverse to the propagation direction (usually the optical axis) and the propagation direction (z direction), a so-called confocal volume (konfokales Volumen) is illuminated within the sample in conjunction with confocal detection. Luminance information (measurement values) is acquired for a plurality of different sizes of confocal volumes. For this purpose, the numerical aperture (NA) of the objective lens used for illumination purposes is changed. For example, a variable iris diaphragm, a plurality of different pinholes arranged on a revolver or a slider, or a telescope is provided in the excitation beam path and appropriately controlled. The luminance information is acquired by a photodetector (for example, a photodiode).

[0004] The drawback of this approach is that an individual long measurement needs to be performed for each volume size to be measured, and a typical duration of 10 to 100 seconds per individual measurement is required.

[0005] A publication by Scipioni et al. (Non-Patent Literature 4) discloses improvements to spot-variable FCS. For acquisition purposes, a planar detector with spatial resolution is used, and its multiple detection elements can be read and evaluated individually and independently of each other. In particular, a detector type called the "Airyscan detector" is used by Scipioni et al., which is positioned in the intermediate image plane of the detection beam path, and each of its detection elements acts as an individual pinhole (Non-Patent Literature 5). In this configuration, luminance information from a selection of multiple detection elements is evaluated, and virtual pinholes of different sizes are simulated by the selection of detection elements. In this way, it is possible to virtually generate multiple changing confocal measurement volumes even if the actually illuminated confocal excitation volume remains constant. The procedure in the publication by Scipioni et al. (2018) makes it possible to simultaneously acquire luminance information from up to four different measurement volumes in a single measurement. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lenne et al., 2006;EMBO J.25:3245-3256 [Non-Patent Document 2] Wenger et al., 2007;Biophys.J.92:913-919 [Non-Patent Document 3] Wawreznieck, L et al., 2005;Biophys.J.89:4029-4042 [Non-Patent Document 4] Scipioni et al., 2018;Nature Communications;DOI:10.1038 / s41467-018-07513-2 [Non-Patent Document 5] Huff, 2015, NatureMethods; Application Notes, December 2015 [Overview of the project] [Problems that the invention aims to solve]

[0007] In particular, since the lowest possible exposure to excitation radiation is desirable for biological samples, the present invention aims to propose options for further improving the acquisition speed, especially for fluorescence correlation spectroscopy. [Means for solving the problem]

[0008] This objective is achieved by a method for acquiring luminance information. Furthermore, an apparatus suitable for achieving this objective is proposed. This method and apparatus are the subject of the independent claim. Advantageous developments of the present invention are the subject of the dependent claim.

[0009] In particular, in possible methods for obtaining brightness information from a sample using fluorescence spectroscopy techniques, each method includes at least one measurement process, and each method performs at least one measurement block, each including several individual measurements. The first measurement block includes steps A to E.

[0010] In step A, the sample is illuminated by a focused beam of excitation radiation using an objective lens, and the excitation radiation is directed towards or into the sample within an angular range of a first numerical aperture. For this purpose, a first divergence range of the beam cross-section at the entrance pupil of the objective lens is set.

[0011] Step B involves the generation of detection radiation within the excitation volume of the sample by this illumination. This excitation volume is also referred to as the confocal volume or confocal excitation volume. Detection radiation is radiation that arises, in particular, from the influence of the excitation radiation. Thus, excitation radiation, for example, in the form of laser radiation, can cause detection radiation to be emitted by a phosphor used to mark the structure, organelles, molecules, and / or regions of the sample.

[0012] The detected radiation is captured in step C. Advantageously, the detected radiation is captured using the same objective lens also used for illumination, in order to keep the technical complexity of the setup of the optical device used within reasonable limits.

[0013] Step D includes guiding the captured detection radiation along the detection beam path and imaging the detection radiation onto a spatially resolved detector having multiple detection elements arranged in groups at equidistant from each other around the optical axis of the detection beam path and readable independently of each other. In this specification, arrangement in groups is understood to mean that at least two detection elements are positioned at the same distance from the optical axis, i.e., a group is determined by the same distance for multiple detection elements in a given group. Multiple detection elements do not need to be adjacent to each other. Furthermore, the distance from the optical axis to the individual detection elements in a group may vary within an acceptable range. One of the multiple detection elements may be located on the optical axis of the detection beam path and form its own group.

[0014] In step E, multiple luminance information is acquired as multiple measurements from multiple detection elements, and the multiple luminance information acquired from each element of at least one selected group of multiple detection elements positioned at the same distance from the optical axis is evaluated, and in particular, is combined with each other and / or from further groups by calculation. In this process, it is possible to simultaneously acquire additional multiple luminance information from further multiple detection elements, e.g., further multiple groups, which makes it possible to create multiple virtual pinholes, particularly those of different sizes and / or shapes. It is important that multiple luminance information from a group acquired at the same time is assigned to the relevant group. Furthermore, it is possible to acquire multiple luminance information from multiple detection elements of multiple groups and selectively store or temporarily store them, but it is also possible to use only the multiple luminance information from selected multiple detection elements within the analysis procedure.

[0015] This method includes, in particular, the option of selecting a first numerical aperture based on a sample by obtaining multiple values ​​of at least some optical properties of the sample and determining and setting the current first numerical aperture based on the multiple values ​​obtained.

[0016] For example, the optical properties of a sample are understood to mean the spatial dimensions of the sample, particularly its thickness, and its optical density. Furthermore, the optical properties are determined, for example, by the type of medium in which the sample is located and / or the constituent parts of the sample (e.g., cytosol). In addition, the optical properties of a sample may be considered, for example, the expected or determined number, type, and packing density of organelles or molecules, as well as the markers used (e.g., fluorophores).

[0017] For example, the values ​​of several optical properties of a sample can be measured or estimated in advance. Alternatively or additionally, all or some of the optical properties to be considered can be determined by appropriate simulations and provided for subsequent use. Obtaining multiple optical properties can also be understood as reading the corresponding multiple values ​​from a provided lookup table (LUT).

[0018] The number and types of optical properties provided for use in the method according to the present invention can be defined for each sample type (fixed selection) or for each sample (individually selected). However, it is also possible, for example, to consider a predetermined number and quality of optical properties and / or to adapt them during the execution of the method (dynamic selection).

[0019] In a preferred configuration of the method according to the present invention, steps A to E can be repeated in a second measurement block or a further number of measurement blocks, and step A includes adjusting the beam spread range at the entrance pupil of the objective lens to a different spread range than the beam spread range of the preceding measurement block, or changing the diameter of the beam of detected radiation captured with respect to the first measurement block or the preceding measurement block. Furthermore, the spread range of the beam of detected radiation to be captured is adapted to the size of the detection area of ​​the detector, so that the detection area is illuminated overall. This means ensures, for example, that all available detection elements can be used even in the case of a second spread range modified with respect to the first spread range, or that there is no undesirable undersampling of the beam of detected radiation.

[0020] If the illumination and imaging apparatus used to carry out the method according to the present invention provides only a plurality of discretely adjustable aperture diameters (see below) for setting the beam spread range, it is possible to best-adaptively select the combination of aperture diameter and Airyscan zoom setting manually or automatically.

[0021] The essence of the present invention lies in combining i) the use of a spatially resolved detector having multiple selectively and individually readable detection elements with ii) controlled and variable adjustment of the beam spread range of excitation radiation for performing fluorescence correlation spectroscopy (FCS). Depending on the specific configuration of the detector, for example, it is possible to simultaneously perform four measurement processes in a first measurement block, each having multiple individual measurements. Each individual measurement can acquire brightness information from one detection element. After its completion, for example, four further measurement processes can be simultaneously performed in a second measurement block in a short time. In this way, for example, eight measurement processes can be performed in a short time.

[0022] In various configurations of the method according to the invention, the spread range of the excitation radiation beam can be adjusted only in the excitation beam path or only in the common portion of the excitation radiation and detection radiation beam paths. In a further configuration, in order to reproduce the effect of adjusting the spread range of the excitation radiation beam, the spread range of the detection radiation beam can be changed in a controlled manner within the detection beam path.

[0023] Advantageously, as a spatially resolved detector, a so-called Airyscan detector can be used, in which case the arrangement of the plurality of detection elements is already adapted to the circular cross-section of the detection radiation beam. In a further embodiment of the invention, it is possible to use other detectors, which are arranged in a so-called pinhole plane (intermediate image) within the detection beam path, and the plurality of their detection elements can be individually selectively read. As an example, such a detector can be a SPAD (single photon avalanche diode) array. In a further embodiment of the invention, the plurality of detection elements can be arranged in a plurality of rows and a plurality of columns. It is possible to individually select and read a plurality of detection elements in a plurality of columns and / or a plurality of rows in order to assign a plurality of detection elements at approximately the same distance from the optical axis to respective groups. The plurality of measurement values read from the plurality of detection elements are assigned and stored to the detection elements and optionally to the groups, held available or provided for further processing.

[0024] In a further configuration, the method can be carried out using a further plurality of measurement blocks, i.e., a third, fourth measurement block, etc. In that case, the explanations described above and below in the specification are applied as appropriate.

[0025] If steps A through E are repeated in a second measurement block or multiple further measurement blocks, it is possible to determine and set the corrected current numerical aperture based on the multiple values ​​obtained. For example, this allows the user to systematically determine the configuration best suited to a particular sample and thus determine the best point spread function (PSF).

[0026] To keep the explanation concise, we will refer to at least two measurement blocks below. In the measurement block of the method configuration according to the present invention, a portion of the confocal excitation volume is captured in the form of each measurement volume (corresponding to the modified illumination spot) by a plurality of selected detection elements and a plurality of virtual pinholes thus obtained. The average diameter of each portion, for example, the average diameter of the beam waists (Strahltaille) of the associated plurality of measurement volumes, differs by at least 5 nm perpendicular to the optical axis. This ensures that additional measurements not yet obtained are obtained in each measurement process.

[0027] The same applies to the selection of multiple portions of the excitation volume to be captured when comparing the two measurement blocks. Therefore, the second beam divergence range at the entrance pupil is advantageously selected such that the average diameter of the multiple portions of the excitation volume captured in the first and second measurement blocks differs by at least 5 nm.

[0028] A possible configuration of the method according to the present invention includes, in step A of the second measurement block, selecting a second beam spread range at the entrance pupil of the objective lens to be smaller than the first beam spread range. As a result, the diameter of the excitation radiation beam, i.e., the beam spread range at the entrance pupil of the objective lens, is reduced. This also leads to a limitation of the numerical aperture (NA), within that angular range, in which the excitation radiation focused by the objective lens is directed towards the sample. The limitation of the NA simultaneously increases the point spread function (PSF) of the excitation.

[0029] An advantageous configuration of the method according to the present invention reduces the beam divergence of the excitation radiation relative to the divergence in at least one preceding measurement block. Simultaneously, the numerical aperture of the detected radiation is maintained independently of the multiple beam divergences of the excitation radiation. The design of such a method is supported by an apparatus (see below) in which the adjustment of each beam divergence of the excitation radiation is performed only in the excitation beam path.

[0030] In a further configuration of the method according to the present invention, the first numerical aperture may also be selected to be smaller than the second numerical aperture or the second numerical aperture of at least one subsequent measurement block.

[0031] The luminance information obtained by the method according to the present invention can be used, in particular, to perform fluorescence correlation spectroscopy. For this purpose, multiple luminance information obtained from multiple selected detection elements of different groups are related to each other with respect to space and / or time, which is incorporated below under the phrase "computational combination".

[0032] In a further configuration of the method according to the present invention, the detected radiation is generated and captured at at least two wavelengths. Measurements for different wavelengths are compared with one another in a position-related manner (e.g., by cross-correlation).

[0033] If steps A to E are repeated in a second measurement block or multiple further measurement blocks, it is possible to determine and set the current numerical aperture to be corrected for subsequent measurement blocks based on the multiple values ​​obtained. In this way, it is possible to individually address the characteristics and interactions of the sample, the marker used, and / or the illumination and imaging device (device that acquires luminance information).

[0034] In addition to the method, the present invention also relates to an apparatus for acquiring brightness information from a sample. This apparatus comprises an excitation beam path for guiding a beam of excitation radiation, the excitation beam path being provided with an objective lens for generating a focus in the sample space. Furthermore, a detection beam path for guiding a beam of detection radiation originating from the sample space and captured by the objective lens is a component of the apparatus. The detection beam path has a spatially resolved detector comprising multiple detection elements, from which multiple brightness information can be acquired and read independently of each other. Furthermore, the detection beam path includes means for controlled modification of the beam spread of the detection radiation. As a result of this effect, the beam spread of the captured detection radiation can be adapted to the size of the detection area of ​​the detector. In this way, the detection area can be illuminated overall.

[0035] The captured detection radiation enters the detection beam path as a result of the effect of the beam splitter (main color splitter). In this case, the main color splitter separates the captured detection radiation from the incident or reflected excitation radiation.

[0036] The apparatus according to the present invention is characterized by means for controlled adjustment of the beam cross-sectional divergence range at the entrance pupil of the objective lens. This means can be arranged in the excitation beam path, particularly advantageously. In alternative embodiments, this means can also be arranged in the common beam path or the detection beam path, with fewer associated disadvantages. An equally possible arrangement of this means in the detection beam path indirectly results in an effect equivalent to adjusting the beam cross-sectional divergence range at the entrance pupil of the objective lens.

[0037] Means for controlled adjustment of the beam cross-sectional divergence range at the entrance pupil of the objective lens may be, for example, an adjustable aperture such as an iris diaphragm, at least one aperture that can be introduced into the excitation beam path, a telescope, or an acousto-optic element. For example, an aperture that can be introduced into the excitation beam path may be mounted on a revolving nosepiece, a filter wheel, or a slider, or may be configured to be rotatable or foldable.

[0038] The shape of at least one aperture of the diaphragm can be a hole shape. In a further embodiment, the aperture may be a slot or an ellipse. The elongated cross-section of the excitation radiation beam provided by the slot-shaped or ellipse aperture allows for an elongated or nearly linear focus in the xy-plane.

[0039] In further embodiments, means for controlled adjustment of the beam cross-sectional divergence range may be an optical modulator (spatial light modulator (SLM)) or any other phase-modulating optical element. If these optical elements are controllable, it is also possible to generate multiple excitation volumes of free form (Freiformen) that can be selectively changed over time.

[0040] In this case, time-varying switching can be performed in different time regimes, thereby enabling different applications. For example, rapid switching of multiple excitation volumes can be achieved using laser multiplexing methods, that is, rapidly aligning them temporally (e.g., switching of multiple lasers at specific time windows "gates"). that This makes it possible. For example, this procedure can be used in TCSPC (time-correlated single-photon counting). Multiple (excitation) lasers can be switched on and off within a time window, for example, on the scale of molecular diffusion time. Thus, multiple excitation volumes can be switched individually or together, independently of each other, in any desired time sequence.

[0041] Means for adapting the beam spread of the captured detected radiation to the size of the detector's detection area can, advantageously, be a zoom optical unit, such as those already included in some confocal microscopes.

[0042] The apparatus may further comprise a control unit connected to means for adjusting the cross-section of the excitation radiation beam at the entrance pupil of the objective lens and / or means for adjusting the broadening range of the cross-section of the detection radiation beam. The control unit is configured such that the respective settings of the means are carried out based on a predetermined relationship between the broadening range of the excitation radiation cross-section and the broadening range of the detection radiation cross-section, by the control unit generating a plurality of corresponding control commands and transmitting them to a plurality of actuators of the associated adjustment means. The method according to the present invention can advantageously be performed efficiently and reproducibly by a control unit configured in this manner.

[0043] The beam spread range for each beam is generated by a control unit and transmitted to a means for adjusting the beam cross-sectional spread range, and adjusted by controlling the means for adjusting the beam cross-sectional spread range according to a set of appropriate control commands executed by the means for adjusting the beam cross-sectional spread range. Specific settings of the control commands can be modified by obtaining multiple values ​​of at least some optical properties of the sample and determining the current numerical aperture based on the obtained multiple values.

[0044] The method according to the present invention can be advantageously applied to perform fluorescence correlation spectroscopy (FCS). This process makes it possible to calculate the correlation between measurements from multiple selected detectors of different groups and / or between detectors of a common group. An example of such a procedure is known from the publication by Scipioni et al. (2018; see above).

[0045] A further application of the present invention involves adapting the confocal volume (i.e., PSF) to the current (biological) measurement task to be solved. As a result, the user can, for example, identify whether molecular clustering (Clusterbildung) is present. If the size of the confocal volume could not be varied, domain formation (Domainbildung) could not be recognized when the PSF size was unfavorable. For example, domains may be too small or too large relative to the system's PSF.

[0046] The approximate size of the domain can be determined for further applications. Users perform measurements on different PSF sizes (i.e., sample regions of different sizes) to determine the size of the corresponding domain or the heterogeneous distribution or clustering of molecules (e.g., also called "lipid rafts" depending on the application in biomembrane research). The observed effect is greatest when the PSF size is approximately half the size of the observed structure (Wawreznieck, L. et al., 2005; Biophys.J.89:4029-4042).

[0047] It is also possible to detect two or more dyes. For this purpose, for example, data acquisition can be performed using two detection channels (2-channel Airyscan). In this case, the objective is to correlate FCS measurements of various markers (dyes) at the same location with each other (cross-correlation). In this way, it is possible to detect correlations between the target marked biomolecules and to investigate whether the two molecules exist within a microdomain or "mesh," that is, whether they do not move independently of each other.

[0048] The advantages of this invention are to improve the speed of acquiring brightness information and to suppress exposure of the imaged sample to excitation radiation. Furthermore, the present invention discloses several application options. In certain possible applications, temperature and viscosity can be measured at the molecular level ("nanometer" and "nanometer," respectively). Thus, the diffusion coefficient can be determined based on diffusion measurements performed using the present invention. For example, several measured particles (e.g., dye molecules, fluorescent / luminescent particles, collectively referred to herein as "objects") can be used as a comparison standard. For the several particles used, the diffusion coefficient can be predetermined, for example, as a function of temperature, i.e., as a function of particle radius. Based on the known dependence of viscosity on temperature, the local temperature at the focal point can be immediately derived from the FCS measurement.

[0049] The present invention will be described in more detail below with reference to exemplary embodiments and drawings. [Brief explanation of the drawing]

[0050] [Figure 1] Figure 1 shows a schematic diagram of a first exemplary embodiment of the apparatus according to the present invention. [Figure 2] Figure 2 shows a schematic diagram of a second exemplary embodiment of the apparatus according to the present invention. [Figure 3] Figure 3 shows a schematic diagram of a third exemplary embodiment of the apparatus according to the present invention. [Figure 4] Figure 4 shows a schematic diagram of an Airyscan detector with exemplary numbering of multiple detection elements. [Figure 5] Figure 5 shows a schematic diagram of a spatially resolved 2D detector with exemplary numbering of multiple detection elements. [Figure 6] Figure 6 shows a schematic diagram of the first group of detection elements of the Airyscan detector. [Figure 7] Figure 7 shows a schematic diagram of the first group of detection elements of the 2D detector. [Figure 8] Figure 8 shows a schematic diagram of the two groups of detection elements in the Airyscan detector. [Figure 9] Figure 9 shows a schematic diagram of the two groups of detection elements in the 2D detector. [Figure 10] Figure 10 shows a schematic diagram of the three groups of detection elements in the Airyscan detector. [Figure 11] Figure 11 shows a schematic diagram of the three groups of detection elements in the 2D detector. [Figure 12] Figure 12 shows a schematic diagram of the four groups of detection elements in the Airyscan detector. [Figure 13] Figure 13 shows a schematic diagram of the four groups of detection elements in the 2D detector. [Figure 14] Figure 14 shows a schematic diagram of the process of combining multiple luminance information from two different groups of detection elements in the Airyscan detector. [Figure 15] Figure 15 shows a schematic diagram of the process of combining multiple luminance information from two different groups of detection elements in a 2D detector. [Modes for carrying out the invention]

[0051] The diagrams are simplified and only include the technical elements necessary for explanation. The beam paths are similarly simplified. A first exemplary embodiment of an apparatus for acquiring brightness information from a sample (Figure 1) comprises a light source 41, for example, a laser light source, from which a beam of excitation radiation is emitted and guided along an excitation beam path 42. Any multiple optical elements for shaping and / or collimation of the excitation radiation are not shown. Means 44 for controlled modification of the beam spread range are arranged in the excitation beam path 42, and in the exemplary embodiment, means 44 are in the form of a controlledly adjustable aperture. In further embodiments, a revolving nosepiece or slider may be provided, thereby introducing a plurality of different apertures into the excitation beam path 42. Alternatively, means 44 may be a controlledly adjustable telescope or acousto-optic element.

[0052] Means 44 are moved by the drive unit 416 outside the excitation beam path 42 (shown by the dashed line) and can result in multiple different numerical apertures, in each of their angular ranges the excitation radiation can be directed towards the sample 48 to be imaged. In further embodiments, means 44 may be adjustable with respect to the transmittance for excitation radiation, particularly with respect to the hole diameter (pinhole, iris diaphragm) or the length and width of the slit (adjustable slit aperture, acousto-optic element).

[0053] After passing through means 44, the excitation radiation is incident on the main color splitter 43, which is penetrating to the excitation radiation and allows it to pass through. Downstream of the main color splitter 43, the excitation radiation passes through a portion of the beam path of the device, which is called the common beam path 4210, along which the excitation radiation and the detection radiation (see below) are guided together or can be guided together.

[0054] The beam of excitation radiation, which has been deflected in front of it by the mirror 45, can then be deflected in a controlled manner by the subsequently positioned scanner 46 and directed towards the entrance pupil EP of the objective lens 47. The mirror 45 allows for a compact design and may be omitted in further forms of the apparatus if deflection of the excitation beam path 42 is not required or anticipated.

[0055] The lateral spread range is set by the effect of means 44, and the excitation radiation, deflected in a manner controlled by scanner 46, is focused by objective lens 47 into the sample space where the imaged sample 48 may be located on sample stage 49. The emitted and thus focused excitation radiation provides a confocal excitation volume.

[0056] The detection radiation generated within the sample 48 by the excitation radiation in the confocal excitation volume is captured by the objective lens 47 and guided along a detection beam path 410 that coincides with the excitation beam path 42 to the main color splitter 43 (shown by a dashed line).

[0057] In a further embodiment of the apparatus according to the present invention, the detection radiation can be captured by an additional objective lens (not shown). In such a case, the excitation beam path 42 and the detection beam path 410 can be completely separated from each other, or they can be reintegrated, for example, by an additional color splitter (not shown) to form a common beam path 4210.

[0058] In the illustrated exemplary embodiment, the detection radiation is converted into a stationary beam ("descannt") by passing through the scanner 46 and reaches the main color splitter 43. The main color splitter 43 is reflective to the wavelength of the detection radiation, which is different from the wavelength of the excitation radiation. The detection radiation reflected by the main color splitter 43 reaches a zoom optics unit 411 located in the detection beam path 410. The zoom optics unit 411 is adjustable by a zoom drive unit 412. In a further embodiment, the transmittance and reflectance of the main color splitter 43 can be reversed, resulting in the excitation radiation being reflected and the detection radiation passing through. Therefore, the beam paths 42 and 410 must be designed appropriately.

[0059] The means 44, scanner 46, zoom drive unit 412, drive units 415 and 416, and optionally the light source 41 are appropriately connected to a control unit 413 for exchanging data and control commands. For example, the control unit 413 is a computer or an appropriate control circuit.

[0060] The zoom optical unit 411 is a means for controlled modification of the beam spread of the detection radiation, which can be used to adapt the beam spread of the captured detection radiation to the size of the detection area of ​​the spatially resolved detector 414, which is similarly positioned in the detection beam path 410 in the intermediate image ("pinhole plane"). Its purpose is to illuminate the entire detection area as much as possible. Thus, the detection radiation is directed towards the detector 414 by the zoom optical unit 411 and adapted with respect to its beam spread by the zoom drive unit 412.

[0061] Optionally, the control unit 413 and the detector 414 may be interconnected to allow the control unit 413 to generate and / or verify a set of control commands, for example, based on brightness information acquired from the detector 414. For example, these control commands may control the light source 41, the means 44, the scanner 46, the zoom drive unit 412, and / or an optional drive unit 415 of the sample stage 49.

[0062] In a second exemplary embodiment of the apparatus according to the present invention, means 44 is positioned in a common beam path 4210 (Figure 2). In this case, both excitation radiation and detection radiation pass through means 44 when means 44 is introduced into the common beam path 4210 as shown.

[0063] According to a third exemplary embodiment, the means 44 may also be positioned within the detection beam path 410 (Figure 3). In this process, the current setting of the means 44 affects the change in the beam divergence range of the excitation radiation at the entrance pupil EP of the objective lens 47.

[0064] The steps and configuration of the method according to the present invention will be described below with reference to Figures 4 to 15. Figure 4 schematically shows a top view of the detection region of an Airyscan detector which can be used as a spatially resolved detector 414. Multiple luminance information from detection elements 1 to 32 can be read out individually and can be combined with each other ("binning") as needed. The beam spread range of the detected radiation can be selected to be incident on the detection region at 1.25 AU (Airy-Units). For example, each detection element 1 to 32 can detect an interval of 0.2 AU. The central detection element, indicated by reference numeral "1", is located on the optical axis (oA) of the detection beam path 410.

[0065] Figure 5 shows a further embodiment of a spatially resolved detector 414 (hereinafter also referred to as a 2D detector) with a matrix arrangement of detection elements 1 to 31, which can be realized, for example, in a SPAD array, a CMOS chip, or an sCMOS chip.

[0066] Figures 6 to 15 below provide technical details based on the detection elements 1 to 31 or 1 to 32 of the Airyscan detector type detector 414, and the substantially rectangular detector 414 with detection elements 1 to 31, i.e., the detector 414 having a row-by-row and column-by-column arrangement. For the numbers of the relevant specific detection elements below, please refer to Figures 4 and 5, respectively. In Figures 6 to 15 below, the central detection element 1 is shown filled with a checkerboard pattern for clarity.

[0067] To carry out the method according to the present invention, multiple luminance information obtained from detection elements 1 to 32 can be selectively combined by calculation. As shown in Figures 6 to 13 below, multiple virtual pinholes of different sizes can be generated by selectively adding multiple luminance information obtained simultaneously from detection elements 1 to 32. Advantageously, multiple luminance information is obtained from all detection elements 1 to 32 or 1 to 31, and the virtual pinholes are generated by the targeted selection of the obtained multiple luminance information. The obtained multiple luminance information can, of course, be used for the analysis of multiple different pinholes, which is why they are advantageously stored. For example, the luminance information from detection element 1 is used to evaluate all pinholes.

[0068] To simulate a pinhole with the smallest possible diameter, only luminance information from detection element 1 is used. Therefore, these multiple luminance pieces form the first group of detection elements of the Airyscan detector (Figure 6) or 2D detector (Figure 7) in each case. The luminance information from detection element 1 represents the first measured volume within the confocal excitation volume.

[0069] By combining multiple luminance information from detection elements 1 and (blacked out in Figure 8) 2-7, or detection elements 1 and (blacked out in Figure 9) 2-9, which constitute a group, a virtual pinhole with a larger diameter can be obtained through calculation. Detection elements 1-7 or 1-9 can also be referred to as a common group or a composite group. The common group of detection elements described above acquires multiple luminance information from the second measurement volume of the confocal excitation volume.

[0070] Multiple detection elements that function to represent virtual pinholes with larger diameters are shown in Figures 10 and 11. For example, three groups of detection elements, visualized using pattern filling or in black, form further common groups. In Figure 10 (Airyscan detector), these are detection element 1 of the first group, detection elements 2-7 of the second group, and detection elements 8-19 of the third group. In Figure 11 (2D detector), these are detection element 1 of the first group, detection elements 2-9 of the second group, and detection elements 10-12, 13-15, 20-23, and 28-30 of the third group. Each of the multiple virtual pinholes thus formed allows for the acquisition of multiple luminance information from a third measurement volume.

[0071] By selecting detection elements 1-32 (Airyscan detector, Figure 12) or detection elements 1-31 (2D detector, Figure 13) as four groups of detection elements in each case, a representation of a virtual pinhole with the maximum possible diameter can be obtained, thereby enabling the acquisition of multiple luminance information from a fourth measurement volume.

[0072] For example, the detection elements of four groups are visualized as patterned or in black. In Figure 12 (Airyscan detector), these are detection element 1 of group 1, detection elements 2-7 of group 2, detection elements 8-19 of group 3, and detection elements 20-32 of group 4. In Figure 13 (2D detector), these are detection element 1 of group 1, detection elements 2-9 of group 2, detection elements 10-12, 13-15, 20-23 and 28-30 of group 3, and detection elements 16-19, 23-27 and 31 of group 4.

[0073] When performing the method according to the present invention using the apparatus according to the present invention as shown in Figure 1, a first measurement block can be performed, as part of which four measurement volumes of the confocal excitation volume of the sample 48 are measured simultaneously. In this case, the zoom optical unit 411 is set, for example, so that the detection radiation is incident on the detection area of ​​the detector 414 at 1.25 AU. Each of the four virtual pinholes described above represents the diameter of the measurement volume, and the diameter of the measurement volume (spot diameter) depends on the current optical conditions of the microscope and the currently selected numerical aperture into which the excitation radiation enters the entrance pupil EP of the objective lens 47 (see below).

[0074] In step A, the sample 48 is illuminated with a focused beam of excitation radiation using the objective lens 47. In this process, the excitation radiation is directed to and / or into the sample 48 within an angular range of a first numerical aperture by setting a first divergence range of the beam's cross-section at the entrance pupil EP of the objective lens 47. For this purpose, for example, means 44 can be moved out of the excitation beam path 42 or positioned in the excitation beam path 42 with a first setting of its free aperture.

[0075] In step B, the detected radiation is generated within the confocal excitation volume of sample 48 by illumination. In step C, the detected radiation is captured by the objective lens 47 within an angular range of the first numerical aperture.

[0076] The captured detected radiation is guided along the detection beam path 410 in step D and imaged on the spatially resolved detector 414. Multiple luminance information is acquired from each element of the group of detection elements 1-31 and 1-32, particularly those arranged at approximately the same distance from the optical axis, and assigned to each detection element or group.

[0077] In the second measurement block, steps A to E are repeated, in step A, a second beam spread range (second NA) is set at the entrance pupil EP of the objective lens 47, which is different from and particularly smaller than the first beam spread range (first NA). This directs the excitation radiation to and / or into the sample 48 within an angular range of the second numerical aperture. For this purpose, for example, means 44 can be moved into the excitation beam path 42 or positioned in the excitation beam path 42 with its second setting of free aperture.

[0078] By limiting the lateral spread of the excitation radiation beam, a second numerical aperture (PMA) is obtained that is smaller than the first PMA, thereby increasing the size of the excitation PSF (point spread function).

[0079] According to the selected second numerical aperture, a number of control commands are generated by the control unit 413 and sent to the zoom drive unit 412 to adapt the zoom optical unit 411 so that significant illumination of the detection area of ​​the detector 414 is achieved again.

[0080] For example, the actual values ​​of the first and second numerical apertures and the resulting magnification of the zoom optical unit 411 are advantageously selected to yield multiple measurement volumes such that, as a whole, the optical parameters are sufficiently far apart from the measurement volumes of other measurement blocks and also sufficiently far apart from each other, in order to enable further significant measurement processes. In this way, four additional measurement volumes for evaluating the “spot variation FCS” technique described herein can be obtained in just one further measurement process.

[0081] In certain possible experiments, it is possible to use an objective lens 47 of "40 × / 1.2". In the first measurement block, for example, four measurement volumes with spot diameters of 170 nm, 190 nm, 225 nm, and 245 nm are captured at the first numerical aperture. In this case, the means 44 is moved out of the excitation beam path 42. In the second measurement block, the means 44 in the form of an aperture having 1 / 1.4 of the diameter of the pupil EP of the objective lens is swung into the excitation beam path 42 (second numerical aperture), and if the magnification of the zoom optical unit 411 is simultaneously adapted to 1.4x, it is possible to capture four further measurement volumes with spot diameters of 195 nm, 240 nm, 275 nm, and 285 nm using only one further measurement. The confocal excitation volume actually illuminated remains constant in the first and second measurement blocks.

[0082] This procedure can be modified in further configurations of the method according to the present invention. For example, the second numerical aperture can be selected using an aperture having 1 / 2.8 of the diameter of the pupil EP of the objective lens, and can be set to 1 AU for the zoom optical unit 411. In this way, four further measurement volumes having spot diameters in the range of 300 to 500 nm can be captured in the second measurement block.

[0083] Therefore, in iterations of the method using the first and second measurement blocks, the method according to the present invention advantageously allows for the acquisition of twice as many measurements as possible by comparable methods of the prior art. Nevertheless, this acquisition is approximately four times faster than when all eight measurement processes (see above) are performed individually using the known Spotscan FCS method.

[0084] The possible combinations obtained by calculating multiple luminance information from two different groups of detection elements in the Airyscan detector are schematically shown in Figure 14. For example, the detailed procedure is described in detail in Scipioni et al. (2018; Nature Communications; DOI: 10.1038 / s41467-018-07513-2).

[0085] In this case, it is possible to calculate multiple cross-correlations between the luminance information from the first group and the fourth group (see also above). In this case, the first group is formed from the luminance information from the detection element 1 arranged on the optical axis. The fourth group includes the detection elements 20 to 32. The multiple calculated cross-correlations between the luminance information from detection element 1 and the luminance information from detection elements 20 to 32 are represented by arrows.

[0086] In relation to this, Figure 15 shows the calculation of multiple cross-correlations between the luminance information from detection element 1 and the luminance information from detection elements 11, 14, 19, 21, 23, 27, 29 and 31 of the 2D detector. [Explanation of Symbols]

[0087] 1-32...Detection elements 41...Light source 42…Excitation beam path 4210... Common beam path 43... Main color splitter 44…Means (for controlled modification of the beam spread range of excitation radiation) 45...Mirror 46... Scanner 47…Objective lens 48… Sample 49…Sample stage 410...Detection beam path 411... Zoom Optical Unit 412... Zoom drive unit, actuator 413... Control Unit 414… Detector 415…(Drive unit of sample stage 49) 416... Actuator, drive unit EP…Entrance pupil (of objective lens 7) oA…(Optical axis of detection beam path 410)

Claims

1. A method for obtaining brightness information from a sample (48), the method comprising a first measurement block, The first measurement block is Step A involves illuminating the sample (48) with a focused beam of excitation radiation using an objective lens (47), wherein the excitation radiation is directed towards or into the sample (48) within an angular range of a first numerical aperture, by setting a first divergence range of the cross-section of the beam in the entrance pupil (EP) of the objective lens (47). Step B involves generating detection radiation within the excitation volume of the sample (48) by the illumination, Step C, which captures the detected radiation, Step D involves guiding the captured detected radiation along a detection beam path (410) and imaging the detected radiation onto a spatially resolved detector (414) positioned in the intermediate image plane, wherein the spatially resolved detector (414) has a plurality of detection elements (1 to 32) that are arranged at equidistant distances around the optical axis (oA) of the detection beam path (410) and can be read independently of each other. Step E involves acquiring brightness information from at least each element of a group of detection elements (1 to 32) arranged at the same distance from the optical axis (oA), Equipped with, The method is characterized in that the first numerical aperture is selected based on the sample (48) by obtaining a plurality of values ​​of at least some optical properties of the sample (48), and determining and setting the current first numerical aperture based on the obtained plurality of values.

2. Steps A to E are repeated in a second measurement block or a further number of measurement blocks. In step A, A second divergence range is set in the entrance pupil (EP) of the objective lens (47) that is different from the first divergence range of the beam, or the diameter of the beam of the captured detected radiation is changed with respect to the first measurement block or a preceding measurement block. The method according to claim 1, characterized in that the beam spread range of the captured detected radiation is adapted to the size of the detection area of ​​the detector (414), and as a result the detection area is illuminated overall.

3. The method according to claim 1, characterized in that a first group located on the optical axis (oA) is considered in order, and further radially adjacent groups are considered in order, and these groups are combined with the preceding groups to form a common group, thereby creating a plurality of virtual pinholes of increasing size in each measurement block, and as a result, within the confocal excitation volume determined by the focused beam of the excitation radiation, a plurality of brightness information for a specific measurement volume is acquired for each pinhole size by a plurality of detection elements (1 to 32) belonging to each common group.

4. The method according to 2, characterized in that multiple measurement volumes are captured in multiple measurement blocks, and the multiple average diameters of the multiple measurement volumes differ by at least 5 nm in a direction perpendicular to the optical axis (oA).

5. The method according to 2, characterized in that in step A of at least one further measurement block, the beam divergence range at the entrance pupil (EP) of the objective lens (47) is selected to be smaller than the beam divergence range in the preceding measurement block.

6. The method according to 2, characterized in that steps A to E are repeated in the second measurement block or a further plurality of measurement blocks, and the current numerical aperture to be corrected in the subsequent measurement block is determined and set based on the plurality of acquired values.

7. The method according to any one of claims 1 to 6, characterized in that multiple luminance information obtained from a selection of multiple detection elements (1 to 32) is related to each other with respect to at least one of space and time.

8. The method according to 7, characterized in that, in order to perform fluorescence correlation spectroscopy, at least one of the spatial and temporal relationships of multiple luminance information from a selection of multiple detection elements (1 to 32) is determined.

9. The method according to 8, characterized in that the detected radiation is generated and captured at at least two wavelengths, and measurements for different wavelengths are compared with each other in a position-related manner.

10. A device for acquiring brightness information from a sample (48), An excitation beam path (42) that guides the beam of excitation radiation, the excitation beam path (42) includes an objective lens (47) for generating a focus within the sample space, A detection beam path (410) that guides the beam of detection radiation generated from the sample space and captured by the objective lens (47), A spatially resolved detector (414) having a plurality of detection elements (1 to 32), wherein a plurality of luminance information can be acquired and read out independently from the plurality of detection elements, Means (411) for controlled modification of the beam spread of the detected radiation, wherein the means (411) for controlled modification of the beam spread of the detected radiation can be adapted to the size of the detection area of ​​the detector (414) in order to illuminate the detection area as a whole. Equipped with, The apparatus is characterized in that means (44) for controlled adjustment of the beam cross-sectional spread range in the entrance pupil (EP) of the objective lens (47) are arranged within the excitation beam path (42), within the detection beam path (410), or within the common portion of the excitation beam path (42) and the detection beam path (410).

11. The apparatus according to claim 10, characterized in that a beam splitter (43) is provided to separate the captured detection radiation from the excitation radiation and direct the beam of the detection radiation into the detection beam path (410).

12. The apparatus according to claim 10, characterized in that the means (411) for controlled modification of the beam spread range of the detected radiation is a zoom optical unit (411).

13. The apparatus according to 10, characterized in that the means (44) for controlled adjustment of the divergence range of the cross-section of the beam in the entrance pupil (EP) of the objective lens (47) is an adjustable aperture, at least one aperture that can be introduced into the excitation beam path, a telescope, or an acousto-optic element.

14. The apparatus according to 10, wherein a control unit (413) is provided, and the control unit is connected to the means (44) for adjusting the cross-section of the beam of the excitation radiation and the means (411) for adjusting the spread range of the cross-section of the beam of the detection radiation in the entrance pupil (EP) of the objective lens (47), and the control unit is configured such that the setting of each of the means (44, 411) is carried out based on a predetermined relationship between the spread range of the cross-section of the excitation radiation and the detection radiation.

15. Use of the apparatus according to any one of claims 10 to 14 for determining viscosity and the diffusion coefficient of at least one type of substance located in the sample.

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