Method and microscope for recording trajectories of individual particles in a sample

Abstract

The invention relates to a method for recording the movement path of a single particle in a sample and an optical microscope for carrying out the method. Starting from an at least approximately known starting position, the particle is scanned with an intensity distribution of a scanning light having a local intensity minimum. When illuminated with the scanning light, the particle to be tracked generates a detectable optical signal, from the intensity of which the updated coordinates of the particle are calculated. According to the invention, the scanning is terminated when a second, simultaneously detected measurement variable meets an abort criterion.

Description

[0001] Technical Field of the Invention

[0002] This invention relates to a method for temporal tracking of individual particles in a sample based on the MINFLUX principle. The invention also relates to an optical microscope for performing this method.

[0003] Existing technology

[0004] Observation and tracking of individual particles in a sample (English: Single Particle Tracking Single-particle tracking (SPT) is an important technique for studying molecular dynamics in biological systems. For this purpose, light-scattering particles, such as metallic nanoparticles, can be used; however, single molecules of fluorescent particles or fluorescent dyes are typically used. With fluorescent dyes, particles that would otherwise be undetectable can also be labeled, making them suitable for particle tracking. Biological applications include the study of enzyme-catalyzed reactions, DNA transcription, ion channel activity, and vesicle transport.

[0005] The applicability of the methods used here depends heavily on the achievable observation period and the temporal resolution when observing individual particles. For this purpose, two methods are known in the art: microscopy-based imaging and point detector-based methods. The former allows for the observation of individual particles over a larger image area, thus enabling the tracking of freely diffusing particles over relatively long periods. However, the achievable temporal resolution is limited by the camera frame rate, which is why it is impossible to study fast processes using this method.

[0006] Conversely, point detector-based methods, particularly when using time-resolved single-photon counting, can achieve high time resolution down to the picosecond range. However, in many applications, the observation period is limited to the time a single particle remains in the focal point of the illumination light. If the particle can diffuse freely, the observation period is extremely short. To overcome this limitation, the particles to be tracked, especially single molecules, are often immobilized, i.e., bound to a fixed carrier or held in one place by means of optical tweezers. However, for optical tweezers to be used, a single biomolecule must be coupled to a (larger) carrier particle, which severely limits its application in natural living systems.

[0007] Alternatively, the observation period for a single particle using a single-point detector can be extended by employing an adjustable sample holder and closed-loop regulation to actively keep the molecule in focus. NP Wells et al. first achieved tracking of a single fluorescent molecule in "Time Resolved 3D Molecular Tracking in Live Cells" published in Nano Lett. 10, 4732 (2010), where Cy5-dUTP molecules in 92% glycerol (a very viscous medium) could be tracked in real time for approximately 100 ms. Because dye molecules diffuse much faster in less viscous media, a single dye molecule can no longer be tracked quickly enough and quickly leaves the capture range of the regulation used for sample tracking. Therefore, the piezoelectric-based regulator used in that article cannot be applied to less viscous (and therefore more practically relevant) media. The maximum track duration is also short, approximately 100 ms, particularly due to the limited photon budget, i.e., the limited number of fluorescent photons emitted by the dye molecule before it is finally bleached.

[0008] To track individual particles, especially single dye molecules, more quickly, fast beam deflection devices, particularly electro-optic or acousto-optic deflectors, are now available, with positioning times in the (sub)microsecond range, but positioning ranges limited to a few micrometers. To enable scanning of individual particles over a larger area of ​​the sample, these fast, non-mechanical scanning devices can be combined with galvanometers, which allow for (slower) pre-positioning of the focused excitation light over a larger area of ​​the sample.

[0009] Reference DE 10 2011 055 367 A1 describes a variation of single-particle tracking known by the abbreviation MINFLUX, in which a particle is illuminated with a scanning light distribution having a minimum intensity, and the photons emitted by the particle are recorded. By shifting the intensity distribution relative to the sample, the rate of photons emitted by the particle is kept to a minimum, thus tracking the minimum of the particle moving within the sample. As the positioning confidence increases, the location of the minimum intensity can be moved closer and closer to the tracked particle, and by increasing the power of the scanning light, further precise positioning is achieved.

[0010] The MINFLUX method is uniquely advantageous due to its exceptional photon efficiency, allowing for precise localization of individual particles down to the single-digit nanometer range, even with relatively few photons. Because the scanning is performed at the minimum intensity of the light distribution (ideally zero intensity), and this minimum is located only in the vicinity of the particle, only low light intensity is applied to the particle. Quantitative analysis [see “MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells” by K. Gwosch et al., Nat. Methods 17, 217 (2020)] shows that the standard deviation of localization accuracy in the MINFLUX method... follow The relationship, among which It is the number of photons detected. This is the number of iteration steps. Therefore, after four iterations, That is, the uncertainty in positioning decreases with the square of the number of photons. Conversely, when determining the position of a single particle based on a wide-field image of the particle (e.g., in STORM / PALM microscopy or by scanning with Gaussian focused light), the standard deviation increases only with the number of photons detected. The square root is reduced Due to this special photon efficiency, even single fluorescent dye molecules (which emit only a very limited number of fluorescent photons before irreversible bleaching) can be located more positions using the MINFLUX method than using other methods.

[0011] Especially when using a rapid scanning device, the position of a single particle can be repeatedly determined at very short intervals, currently available techniques achieve approximately once every 100 µs. Due to its high photon efficiency and fast positioning speed, the MINFLUX method is particularly suitable for real-time tracking of individual fluorescent dye molecules in a sample. Here, individual dye molecules can also be tracked in low-viscosity media. It is known from existing techniques that the trajectory of a single dye molecule recorded using the MINFLUX method has achieved over 25,000 single-position determinations.

[0012] A variation of the MINFLUX method is described in DE 10 2017 104 736 B3, in which the scanning of isolated fluorescent dye molecules is not performed by irradiation with an intensity distribution of excitation light having a local minimum, but by irradiation with two substantially complementary intensity distributions of excitation light and fluorescence suppression light. Here, the intensity distribution of the excitation light has a local maximum, while the intensity distribution of the fluorescence suppression light has a local minimum at the same location. Specifically, the fluorescence suppression light can be STED light, which prevents the excited fluorescent dye molecule from emitting fluorescent photons in the edge regions of the intensity distribution of the excitation light by triggering stimulated emission. Similarly, in this embodiment of the MINFLUX method, the fluorescence intensity emitted by the dye molecule depends on the distance from the local minimum of the fluorescence suppression light, but here the fluorescence decreases instead of increasing with increasing distance to the minimum. M. Weber et al. presented an experimental implementation of this concept in “MINSTED fluorescence localization and nanoscopy” published on bioRxiv, doi: 10.1101 / 2020.10.31.363424 (2020).

[0013] While long trajectories of numerous individually located particles are generally desirable, the question remains whether the trajectory contains information meaningful for a given problem along its entire length. For example, if the distance between the particle and its binding site is too large, the study of possible interactions between the tracked particle and the binding site in the sample will not provide relevant information. Furthermore, if the cell enters an unrepresentative state during trajectory recording (e.g., due to cell division or apoptosis), the information derived from the trajectory may be irrelevant or even misleading. Additionally, excessive scanning can lead to unnecessarily long measurement durations, requiring unduly intense scanning light on the sample.

[0014] Invention Task

[0015] Therefore, the object of the present invention is to demonstrate a method and an optical microscope for tracking the motion of individual particles in a sample, wherein tracking of the particle is interrupted or aborted when tracking no longer provides information relevant to a given problem. Thus, the object of the method and optical microscope according to the invention is to demonstrate a method that defines appropriate abort criteria for tracking particles.

[0016] Solution

[0017] The objective of this invention is achieved by the method and optical microscope described below.

[0018] Invention Description

[0019] This invention is based on the concept that when tracking the motion of particles in a sample, it is not always possible to determine which parts of the recorded trajectory contain relevant information to answer a given question simply from the data points of the trajectory. Instead, it is necessary to further understand the background information in the sample. This background can be spatial information about the (direct) surrounding environment of the particle being tracked, or functional information characterizing, for example, the functional state of organelles (e.g., the open state of ion channels) or the functional state of the entire cell.

[0020] To this end, the present invention includes a method for recording trajectories (i.e., time-resolved position curves of individual particles in a sample), wherein a second measurement variable is detected in the sample, and the recording of the trajectory is interrupted or terminated when the second measurement variable or a control value calculated based on the second measurement variable meets a termination criterion. This timely termination of trajectory recording saves both time and photons.

[0021] Here, light-scattering particles (e.g., metal nanoparticles, silica or latex nanoparticles, or, in many applications, preferably fluorescent particles) can be used as the particles to be tracked. The term "fluorescent particle" should be understood broadly here; for example, fluorescent particles include single molecules of reagents, ligands, active substances, or biomolecules (such as proteins, DNA, lipids) labeled with fluorescent dyes, as well as fluorescently labeled supramolecular structures, aggregates, or molecular complexes such as micelles, vesicles, or lipid rafts. In particular, fluorescent particles also include molecular fluorophores, i.e., single molecules of fluorescent dyes, and fluorescent quantum dots (Qdots). A key factor is that the particles appear in discrete and substantially identical units.

[0022] To implement the method according to the invention, it is necessary to ensure that the particles are isolated, i.e., the spacing between adjacent particles is greater than the optical diffraction limit, so that adjacent particles can be identified as separate objects in optical imaging. Thus, for example, the spacing requirement can be met by using such a low concentration of particles that these particles are statistically sufficiently spaced from each other. If the fluorescent dye can be photoinduced to switch between a fluorescent and non-fluorescent state in at least one direction, spatially isolated fluorescent molecules of the fluorescent dye can also be generated by photoactivation and / or photodeactivation. By photoactivating a very small number of fluorescent dye molecules or photodeactivating most of the fluorescent dye molecules, a small number of dye molecules in a fluorescent state with the required spacing can be prepared. Here, this spacing requirement also applies to molecules of different fluorescent dyes that can be excited by excitation light of the same wavelength to produce fluorescence within the same fluorescence wavelength range. For appropriate methods for separating fluorescent dye molecules in a sample, those skilled in the art can refer to a comprehensive existing technique of localization microscopy (PALM, STORM, and related methods).

[0023] In order to record the trajectory of a single particle according to the method of the present invention, it is necessary to know at least approximately the initial position of the single particle at the start of the measurement. For example, the initial coordinates can be obtained by scanning the sample with focused light in a conventional laser scan or from a previously recorded wide-field image; specific methods in this regard can also be found in the prior art of PALM microscopy, STORM microscopy and MINFLUX microscopy.

[0024] Starting from initially determined particle coordinates, the particles are scanned with scanning light at one or more scanning positions, where the intensity distribution of the scanning light in the sample has local minimums, ideally zero. If a scanning position is mentioned below, it refers to the location of this local minimum. When illuminated with scanning light, the particles to be tracked produce detectable light signals, such as scattered light signals in the case of light-scattering particles or fluorescent signals in the case of fluorescent particles.

[0025] When scanning particles with scanning light, the number or intensity of light signals detected at each scanning position is used as the first measurement variable. The detected light signal decreases or increases depending on whether the particle is located at the center of the scanning position and thus near or at the minimum intensity of the scanning light, or whether it is far from the minimum intensity and thus exposed to a higher intensity of scanning light. In this respect, the number of detected photons or the light intensity represents a measure of the distance between the particle and the minimum intensity and is used to determine subsequent scanning positions and the updated coordinates of the scanned particle. Here, the determination of the updated coordinates of the particle can be performed after a fixed or variable (i.e., adjusted during the scanning process) number of scanning positions, after each individual scanning position, or even after each detected photon. By repeatedly positioning the particle and resetting subsequent scanning positions, the scan follows the movement of the particle in the sample, thereby reproducing the particle's trajectory.

[0026] The above-described method steps are essentially the same as those known in the art for single-molecule tracking based on the MINFLUX method, such as the one described by F. Balzarotti et al. in Science 355,606 (2017) entitled “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photonfluxes”.

[0027] The method according to the present invention differs from the prior art in that a second measurement variable in the sample is detected during the recording of the particle's trajectory, and the recording of the trajectory is interrupted or terminated when the second measurement variable or a control value calculated based on the second measurement variable meets a termination criterion. In the simplest case, the termination criterion is that the second measurement variable or control value is below a minimum value or above a maximum value. Alternatively, if the second measurement variable or control value again exceeds the minimum value or again falls below the maximum value, the scan can continue, where lag may occur in practice. More complex criteria can also be set as termination criteria, particularly taking into account the history of the second measurement variable.

[0028] According to the invention, the second measurement variable is detected at a location in the sample during trajectory recording, wherein the detection can be continuous, i.e., asynchronous with particle scanning, or incorporated into the particle scanning process. While not mandatory, the second measurement variable is preferably detected at least once each time the particle coordinates are updated. The detection of the second measurement variable can be performed in a point form at the particle's current coordinates or at the current scan position, but it can also be detected in an extended region of the sample and averaged over that region, for example, in the environment surrounding the previously determined particle coordinates or in a previously defined fixed region in the sample. For example, this region can be defined by the outline of a cell or organelle (e.g., the nucleus).

[0029] The purpose of the abort criterion is, for example, to terminate or at least temporarily interrupt the recording of trajectories if further localization of observed particles no longer provides information relevant to the given problem, or if changes in the sample render the information derived from further localization incomparable to that derived from previous localization—meaning it is no longer meaningful for the experiment or may even lead to erroneous conclusions. By probing a second measurement variable in parallel and applying the abort criterion, redundant scans can be avoided, thus preventing unnecessary exposure of samples to scanning light. Simultaneously, the measurement duration can be reduced, or more particle trajectories can be recorded within a given time. For many problems requiring statistical analysis of a large number of trajectories, this time advantage allows for the recording and analysis of larger datasets without extending the overall measurement duration.

[0030] In a preferred embodiment of the method according to the invention, for each location, the particle is scanned at multiple scanning positions within a typically ≤250 nm neighborhood surrounding the previously determined particle coordinates, wherein the polygons (in two-dimensional cases) or polyhedra (in three-dimensional cases) formed by the scanning positions include the previously determined particle coordinates. This ensures that precise particle localization is possible. The number of photons emitted by the particle at the scanning position or the light intensity allows for the calculation of the updated particle coordinates; for this, those skilled in the art can refer to methods and algorithms known in the prior art (particularly concerning the MINFLUX technique). The steps are repeated: further scanning positions are set within the neighborhood surrounding the respective current coordinates; the particle is scanned with scanning light at the newly set scanning positions; the light emitted by the particle at each scanning position is recorded; and the updated coordinates are determined. Here, after each localization step, the setting of the respective subsequent scanning positions is based on the previously determined particle coordinates. Optionally, the light power of the scanning light can also be increased. By repeatedly localizing the particles and resetting the scanning positions, the scanning follows the movement of dye molecules in the sample, thereby mapping the particle trajectory.

[0031] In an alternative implementation, after each scan of the particle at a scan position, the updated coordinates of the particle and the subsequent scan position are determined. It is impossible to completely locate the particle from only the photons detected at a single scan position; however, based on the scan history, the relative position of the minimum intensity to the assumed current particle position, and the number or intensity of photons detected at the current scan position, it is possible to infer which direction the scan position must move.

[0032] The setting of the scanning positions and the intensity of the scanning light depend heavily on the diffusion or transport velocity of the particles in the sample and the frequency at which the particles can be localized. While it is desirable to arrange the scanning positions as densely as possible around the current positions of the individual particles and to use high light power for precise localization, this also increases the risk that particles may leave the capture area between consecutive localizations. Although in the localization of static, immobile dye molecules, the scanning positions gradually approach the positions of the dye molecules, and the light power of the scanning light can be increased to improve localization accuracy, this is not always feasible when tracking moving particles. Instead, the scanning positions and the light power of the scanning light must be adjusted so that the particles remain within the capture radius between consecutive localizations. Here, the higher the localization frequency and the lower the particle velocity, the denser the scanning positions can be arranged around the particles, and the higher the light power can be adjusted. These two parameters can also be adjusted between consecutive localizations, i.e., during trajectory recording.

[0033] In a preferred embodiment of the method according to the invention, the second measurement variable is an optical measurement variable, particularly a fluorescence signal detected in the second detection channel. This fluorescence signal can originate from (another) fluorescent dye in the sample and is particularly used to label structures in the sample. However, it can also be an autofluorescence signal, i.e., the intrinsic fluorescence signal of the cell. For example, if the cell nucleus, cell membrane, organelles, structural proteins, ion channels, lipid rafts, or other structures in the sample are stained with (another) fluorescent dye, their fluorescence provides background information when tracking individual particles in the sample. The absence of such staining or a fluorescence signal below the minimum level of such staining at the location of the scanned particle can, for example, indicate that the scanned particle is located outside (or has left) a region of interest within a cell, and can therefore be used as a termination criterion for tracking the particle. For example, to study ligand-receptor interactions, ligands and receptors can be stained with different fluorescent dyes, and individual molecules of fluorescently labeled ligands can be tracked in the sample using the method according to the invention. Here, tracking of a single fluorescently labeled ligand molecule can be limited to the period during which the ligand is adjacent to the receptor and can interact with the receptor, as indicated by the fluorescence (control) signal of the receptor. If the control value indicates that the ligand molecule has left the corresponding receptor, then tracking can be stopped.

[0034] The termination criterion does not necessarily have to be directly defined on the second measured variable, but can also be related to a control value calculated based on the second measured variable, the calculation of which may include other measured variables. In particular, the control value can be calculated from the fluorescence ratio in two detection channels, where fluorescence is detected in different detection wavelength ranges. Here, an advantageous implementation is obtained when the fluorescence of a proportional indicator dye is used in the calculation of the control value. For example, the ratio of the fluorescence signals of such an indicator dye in two wavelength ranges can provide information, for example, about ion concentrations (especially calcium, magnesium, zinc, and sodium ions), pH, or membrane potential. The termination criterion associated with this control value allows the tracking of individual particles to be limited not only to spatially confined areas in the sample, but also to certain functional states of cells or organelles, such as the open state of ion channels or the integrity of the cell membrane. Cellular status in terms of cell cycle progression and division, or cell viability or vitality, can also be assessed using suitable indicator dyes or fluorescence-based reagents (such as the fluorescent ubiquitin cell cycle indicator FUCCI) (e.g., LIVE / DEAD assay, ThermoFisher Scientific). Here, double fluorescence staining can be used to simultaneously confirm apoptosis and necrosis of cells, allowing cells to be classified as dead, necrotic, or apoptotic based on fluorescence signals in one or two fluorescence channels. If control values ​​are derived from the fluorescence of one or more of these fluorescent cell state, viability, or vitality markers, then the tracking of individual fluorescent particles can be limited to intact cells or observation periods where the observed cells can be classified as healthy or at a certain stage of the cell cycle, according to the method of the invention. In this way, misunderstandings of measurement data due to the recording of unrepresentative data can be avoided.

[0035] Besides fluorescence intensity, other parameters of fluorescence emission can also be used to define termination criteria, particularly fluorescence lifetime or fluorescence anisotropy. These parameters are typically sensitive to changes in the binding state or molecular environment of the dye molecule or the molecules or particles labeled with fluorescent dyes, due to alterations in possible reaction pathways leading to reduced mobility or fluorescence quenching.

[0036] When using fluorescent particles, alternative embodiments of the method according to the invention can also be selected, wherein the scanning light is formed by the superposition of distributions of excitation light and fluorescence suppression light with local minimum intensities known from STED microscopy, thus preventing fluorescence emission from the focal edge range. Fluorescence suppression light can be understood as any type of light that blocks, reduces, or completely suppresses fluorescence emission from fluorescent particles. In particular, fluorescence suppression light can be stimulating light that induces stimulated emission of electronically excited dye molecules, causing the dye molecules to switch (return) to the electronic ground state, thereby preventing spontaneous fluorescence emission. Also in this configuration, scanning of a single fluorescent particle provides a fluorescence signal that depends on the distance between the particle and the minimum intensity of the fluorescence suppression light. However, the relationship between signal intensity and distance is inverse; that is, the fluorescence signal decreases as the distance from the minimum intensity of the fluorescence suppression light increases.

[0037] Besides fluorescence, the second measurement variable used to derive the stop criterion can also be another optical measurement variable, particularly second harmonic generation (SHG), third harmonic generation (THG), Rayleigh or Raman scattering, coherent anti-Stokes Raman scattering (CARS), reflected light, differential interference contrast (DIC), or polarization contrast. The advantage of using one of these optical measurement variables to set the stop criterion is that it eliminates the need for (further) staining of the sample with fluorescent labels, and these signals are unaffected by photobleaching. While these contrast modes do not achieve the same specificity as directional fluorescent labels, some contrast modes still exhibit a degree of selectivity. For example, SHG signals do not appear on structures with molecular symmetry centers, while certain non-centrosymmetric structures (such as collagen, myosin, and tubulin) provide particularly high signals in SHG contrast.

[0038] To generate a second optical measurement variable, the sample is typically illuminated with additional light besides the scanning light. The characteristics of this additional light are matched to the corresponding contrast mode and / or the scanning light. Here, the additional light should selectively excite the signal of the second optical measurement variable, rather than being emitted photons by the tracked particles. Therefore, under normal circumstances, the wavelength of the additional light differs from that of the scanning light. When using SHG or THG signals as the second measurement variable, the wavelengths that excite these signals are typically in the red or infrared spectral range, thus easily decoupling them from the scanning light that generates fluorescence or is scattered by particles. The light used to excite the second measurement variable can be directed into the sample along with the scanning light through a single objective; however, the sample can also be illuminated with additional excitation light through a separate optics. This separate optics can be a second objective facing the objective used to illuminate the sample with the scanning light, or arranged at an angle to the objective such that the scanning light and the additional excitation light used to generate the second measurement variable intersect in the sample. Here, the angle of intersection of the beams is between 15° and 165°, preferably between 45° and 135°, and particularly preferably between 80° and 100°. In this illumination of the sample (e.g., implemented in a light sheet microscope), the exposure of the sample to additional excitation light can be reduced, the spatial resolution of the second measurement variable can be improved, and the signal background generated by the additional excitation light when scanning the sample can be reduced.

[0039] The second measurement variable can also be a non-optical variable. For example, this variable can be an electrical measurement variable, particularly voltage, current, resistance, capacitance, inductance, or the frequency, phase, or amplitude of a voltage, current, or alternating electromagnetic field. The current in the sample can be measured, for example, in a patch-clamp arrangement, where a micropipette electrically isolates a single ion channel in the cell membrane of the cell under study from the surrounding medium, and electrodes measure the current passing through the ion channel. After the detected current is amplified and processed into a control value using a sensitive measurement amplifier, a stop criterion for executing the method according to the invention can be set based on this control value.

[0040] The present invention also relates to an optical microscope configured to perform the method according to the invention. For this purpose, the optical microscope includes an objective lens and a light source for scanning light, which can be used to excite particles in a sample to emit photons. According to the invention, the scanning light in the sample has a local intensity minimum, which is generated by means of a beam-shaping device arranged in the beam path of the scanning light. Such beam-shaping devices are known to those skilled in the art from the prior art; examples here are phase filters or programmable phase modulators (SLMs), which are also used, for example, in STED microscopy. The optical microscope also includes a scanning device that can be used to position the scanning light in the sample and scan particles in the sample at different scanning positions.

[0041] The optical microscope has a detection channel that detects a first optical measurement variable from the sample. This optical measurement variable is the light signal emitted by the particle to be tracked due to illumination with scanning light, and is therefore typically a scattered light signal or a fluorescence signal. For this purpose, an avalanche photodiode operating in photon counting mode is particularly suitable as a detector, as it can have exceptionally high sensitivity. However, an analog photomultiplier tube can also be used as a detector, provided it has sufficient sensitivity for single-molecule detection. Furthermore, according to the invention, the optical microscope has a detection channel for detecting a second measurement variable in the sample; this second measurement variable is used to set a stop criterion when implementing the method according to the invention.

[0042] If an optical microscope is configured to record the trajectory of fluorescent particles, then the optical microscope is a fluorescence microscope, and the scanning light includes at least a fluorescence excitation light that can excite the particles to emit fluorescence. A particular implementation is obtained if the scanning light is formed by superimposing a fluorescence excitation light and a fluorescence suppression light from another light source, wherein (only) the fluorescence suppression light has a local minimum intensity in the sample. As previously mentioned, fluorescence suppression light can be understood as any type of light suitable for blocking, reducing, or completely suppressing the fluorescence emission of fluorescent dyes. In particular, fluorescence suppression light can be a stimulus light that induces the stimulated emission of electronically excited dye molecules. Similarly, in the previous implementation, scanning of a single fluorescent particle provides a fluorescence signal that depends on the distance between the particle and the minimum intensity of the fluorescence suppression light; however, the relationship between signal intensity and distance is inverse, i.e., the fluorescence signal decreases as the distance from the minimum intensity of the fluorescence suppression light increases.

[0043] If an optical microscope is set up to record the trajectory of light-scattering particles, a scanning light is used to generate a scattered light signal on the particles. This scattered light signal can have the same wavelength as the scanning light (Rayleigh scattering) or a wavelength shifted relative to the scanning light (Rayleigh scattering, coherent anti-Stokes Raman scattering). Optical microscopes can also have multiple contrast modes, which can be used in parallel or alternately.

[0044] To realize the potential of this method in terms of achievable positioning accuracy, the scanning device is typically required to have a positioning accuracy of 1 nm or less, and corresponding reproducibility. On the other hand, the positioning time is preferably in the microsecond range to enable rapid scanning and positioning sequences of particles. These accuracy and speed requirements for the scanning device of the optical microscope according to the invention cannot be met, or are only insufficiently met, by mechanical beam deflection units such as galvanometers alone. Therefore, beam deflection units that do not require moving parts (e.g., electro-optic deflectors (EOD) or electro-acoustic deflectors) are suitable for the scanning device. With these beam deflection units, the required positioning time can be easily achieved, but the maximum deflection angle is very limited. For this reason, a preferred embodiment of the optical microscope according to the invention has both a galvanometer scanner for positioning the beam over a larger image field and an electro-optic deflector for (rapid) scanning of individual particles in the beam path. Alternatively, there can be a device that integrates the functions of the scanner and the beam deflection device; for example, such a device can be constructed from deformable mirrors. In a further alternative implementation, the scanner's functionality can be taken over, for example, by a movable sample stage.

[0045] In a preferred embodiment of the optical microscope, the second measurement variable is also an optical measurement variable, such as the fluorescence of another fluorescent marker in the sample. Preferably, the detection of this other fluorescence is performed in a wavelength range different from the emission generated by the particles due to irradiation with scanning light, so that the two variables can be separated from each other. If the emission of the particles and the excitation of the other fluorescent marker are temporally or spectrally separable, then the two detection channels can also be designed to be identical or not separated. In addition to fluorescence, other optical signals can be detected as the second measurement variable, particularly second harmonic generation (SHG), third harmonic generation (THG), Rayleigh or Raman scattering signals, coherent anti-Stokes Raman scattering (CARS), reflected light signals, differential interference contrast (DIC) signals, or polarization contrast signals.

[0046] Most implementations of a second measurement variable that is also an optical measurement variable require one or more additional light sources to generate a detection signal in the sample as the second measurement variable. These additional light sources can be, for example, lasers for exciting fluorescence or ultrashort pulse lasers for generating SHG or THG signals. The light from these additional light sources can be directed into the sample along with the scanning light through a single objective lens; however, the sample can also be illuminated by the light from these additional light sources through a separate optics. This separate optics can be a second objective lens facing the objective lens used to illuminate the sample with scanning light, or it can be arranged at an angle such that the scanning light and the light used to generate the second measurement variable intersect in the sample at an angle between 15° and 165°, preferably between 45° and 135°, and particularly preferably between 80° and 100°. Such a configuration is implemented, for example, in a light sheet microscope.

[0047] In an alternative embodiment, the second measurement variable is a non-optical measurement variable. In particular, the non-optical measurement variable is preferably an electrical measurement variable, i.e., voltage, current, capacitance, inductance, or the frequency, amplitude, or phase of a voltage, current, or alternating electromagnetic field. For example, current can be measured in a patch-clamp arrangement, where a single ion channel in the cell membrane of the cell under study is electrically isolated from the surrounding medium using a micropipette, and the current through the ion channel is measured by electrodes and converted into a control value for performing the method according to the invention after processing with a sensitive measurement amplifier.

[0048] Advantageous extensions of the invention are derived from the claims, description, drawings, and related descriptions of the drawings. The advantages described in the features and / or combinations of features of the invention are merely exemplary and may have alternative or cumulative effects.

[0049] For the disclosure of the original application documents and patent applications (but not the scope of protection), the following applies: Further features can be found in the accompanying drawings, particularly the relative arrangements and operational connections shown. Features of different embodiments of the invention or combinations of features from different patent application claims may also deviate from the chosen reference relationships in the patent application claims, and are hereby set forth. This also applies to features shown in separate drawings or mentioned in their descriptions. These features may also be combined with features from different patent application claims. Similarly, for further embodiments of the invention, features listed in the patent application claims may be omitted, but this does not apply to the independent patent claims of granted patents.

[0050] Reference marks included in the claims of a patent application do not constitute a limitation on the scope of protection of the patent application claims. They are only used to make the patent application claims easier to understand. Brief description of the attached diagram

[0052] Figure 1 The process of the method according to the present invention is illustrated schematically.

[0053] Figure 2 The various possibilities for recording a second measurement variable are shown.

[0054] Figure 3 An optical microscope according to the present invention is shown.

[0055] Attached Figure Description

[0056] Figure 1 A portion of cell 1 is shown, in which particle 2 is located, and the movement of particle 2 is to be tracked. The starting position 3 of the particle is at least approximately known (e.g., from a previously recorded wide-field image (not shown here), so scanning of particle 2 can begin at the starting position 3. For this purpose, the particle is illuminated with scanning light 5 at multiple scanning positions 4, with only a few particles shown in the figure as examples. According to the invention, scanning light 5 comprises an intensity distribution 7 having local intensity minima 8. Specifically, particle 2 here is a fluorescent particle that is excited to emit fluorescence 9 when illuminated with scanning light 5 (which is therefore excitation light 6). Alternatively, scanning light 5 may also be a superposition of excitation light 6 and intensity distribution 7 having local intensity minima 8 of fluorescence suppression light (especially stimulation light). Alternatively, the particle may also be a light-scattering particle. Then, when particle 2 is illuminated with scanning light 5, the scattered light signal will be detected instead of fluorescence 9.

[0057] At each scan position 4, fluorescence 9 emitted by fluorescent particles 2 is detected. Based on the previous scan position and the number of fluorescent photons or the detected fluorescence intensity, the updated coordinates of fluorescent particles 2 are determined, and one or more further scan positions 4 are set. Here, the coordinates of particles 2 can be updated after scanning at multiple scan positions 4, or the coordinates of fluorescent particles 2 can be updated in response to each detected fluorescent photon by setting subsequent scan positions. Therefore, by connecting the successively determined coordinates, the trajectory 10 of fluorescent particles 2 is generated.

[0058] According to the present invention, during the scanning of fluorescent particles 2, a second measurement variable 11 is detected as a control value 12, and the fluorescence 13 of another fluorescent dye 14 used for staining cells 1 15 is also detected. Here, another fluorescent dye 14 is selected such that its fluorescence 13 appears in a wavelength range different from the fluorescence 9 of particle 2, and does not affect the fluorescence 9 of particle 2 detected at scan position 4. If the fluorescence 13 of the other fluorescent dye 14 detected at its respective current scan position 4 exceeds a minimum value 16, it indicates that the tracked particle 2 is moving within cell 1, and the trajectory 10 is recorded by scanning particle 2. However, once the fluorescence 13 of the fluorescent dye 14 drops below the minimum value 16, it indicates that particle 2 is outside cell 1, and the scan is stopped 17. Further trajectories 18 of particles 2 outside cell 1 are no longer tracked.

[0059] Figure 2 Various choices of second measurement variables 11 are schematically illustrated to set the termination criteria for tracking individual particles 2 in sample 19. Although control values ​​can also be derived from multiple measurement variables, the measurement variables shown in the figure are primarily considered as alternatives.

[0060] In the illustrated embodiment, scanning light 5 illuminates sample 19 from below through objective lens 20 (in reverse configuration). Cell 1 is located in the sample, with cell nuclei 21 and some filamentous structures 22 exemplarily shown as organelles of cell 1. Fluorescent particles 2 are also shown, their trajectories recorded by scanning at different scanning locations within cell 1 using scanning light 5. For this purpose, the focused scanning light 5 in sample 19 is moved to the scanning position by tilting the beam 23 of scanning light 5 using a beam deflection device (not shown). Fluorescence 9 emitted by fluorescent particles 2 is collected by objective lens 20 and detected by a detector (not shown).

[0061] During the scanning and tracking of fluorescent particles 2 in sample 19, a second measurement variable 11 is recorded to set the stop criterion for the scan. This second measurement variable 11 may also be the fluorescence 13 of a second fluorescent dye, used to label individual components (e.g., organelles) or the entire cell 1. The excitation light 6 required to excite the second fluorescent dye may be incident on sample 19 along with the scanning light 5 in a common beam 23; the fluorescence 13 of the second dye may also be collected using objective lens 20. The fluorescence 13 is then used as a control value 12, on which the stop criterion for scanning and tracking fluorescent particles 2 is set. This stop criterion may, for example, be a minimum value below control value 12, thereby indicating that fluorescent particles 2 have been removed from the cell 1 or organelle labeled with the second fluorescent dye.

[0062] The second measurement variable 11 can also be transmitted light 26 collected by the second objective lens 25 and detected by another detector (not shown) in the transmission direction 24, instead of fluorescence 13. The intensity of the transmitted light 26 is used as the control value 12 for setting the stop criterion. Here, as the transmitted light 26, the scanning light 5 transmitted through the sample can be detected, but another illumination light 27 can also be incident on the sample 19 together with the scanning light 5. Irradiation with the further illumination light 27 can be performed in the form of dark field illumination in addition to bright field illumination; in this way, better control value contrast can be achieved if necessary. By arranging polarizers in the illumination beam path and the transmitted beam path, the transmission light contrast can also be designed as polarization contrast. Finally, the illumination light 27 can also be selected in such a way that a nonlinear optical signal is generated in the sample, in particular an SHG signal 28 (Second Harmonic Generation) or a THG signal (Third Harmonic Generation).

[0063] The second measurement variable 11 is not necessarily an optical measurement variable. For example, an electrical measurement variable, particularly current 32 or voltage, can also be used as the control value 12. To this end, the derivation of the current through ion channel 29 in a patch-clamp arrangement is shown in the figure as an example. For this purpose, a single ion channel 29 is isolated from the surrounding medium using a micropipette 30, and the ion current through ion channel 29 is derived using electrode 31 and detected as current 32. The signal processed by measurement amplifier 33 can be used as the control value 12 to set the termination criteria for scanning and tracking fluorescent particles 2 in sample 19. Therefore, in the example shown, the tracking of fluorescent particles 2 can be limited to the active range of ion channel 29.

[0064] Figure 3The structure of an optical microscope (designed herein as a fluorescence microscope 34) for implementing the method according to the invention is schematically shown. A laser source 35 provides scanning light 5, which is excitation light 6 for fluorescence excitation. The beam 23 of the scanning light 5 passes through a beam deflection device, here implemented in the form of two series-connected electro-optic deflectors (EODs) 36, for deflecting the beam 23 in either the horizontal or vertical direction. After passing through the EODs 36, the wavefront is shaped by a separate phase modulation element 37 (here in the form of a liquid crystal modulator 38 (Spatial Light Modulator, SLM)) such that when subsequently focused by the objective lens 20, an intensity distribution with local minimum intensity values ​​is generated in the sample 19 for the scanning light 5. The beam reflected or diffracted by the liquid crystal modulator 38 is coupled to the main beam path 40 of the fluorescence microscope 34 via a beam coupler 39. Advantageously, the beam coupler 39 is designed as a narrow-band reflective dielectric notch filter whose reflection range overlaps as little as possible with the wavelength range of the fluorescence 9, such that only a small portion of the fluorescence 9 extending in the beam path 40 in the opposite direction to the scanning light 5 is reflected out of the main beam path 40. The scanning light 5 is guided into the rear aperture of the objective lens 20 using the scanning lens 41, the scanner 42, and the tubular lens 43.

[0065] In the configuration shown, the galvanometer-based scanner 42 is used to relatively slowly, but coarsely, locate the focused scanning light 5 onto fluorescent particles in the sample within a large image field, while the EOD 36 is used to quickly locate the minimum intensity in the vicinity of the fluorescent particles. Here, the EOD 36 allows for high-speed localization, but the localization range is limited to a few micrometers. The fluorescence 9 from the sample, received by the objective lens 20, propagates along the main beam path 40 in the opposite direction to the scanning light 5. The fluorescence 9 is focused by lens 45 through a confocal perforated plate 44, collimated by another lens 46, separated from the scattered light by a filter 47, and detected by a detector 48.

[0066] In the fluorescence microscope 34, a second fluorescence channel is provided for detecting the fluorescence 13 of a second fluorescent dye, where fluorescence 13 serves as a stop criterion for scanning individual fluorescent particles in the sample 19. For this purpose, the fluorescence microscope 34 has another light source 49, whose light is coupled into the main beam path 40 via a second beam coupler 50. The fluorescence 13 of the second fluorescent dye excited by the light from the light source 49 is separated from the fluorescence 9 of the fluorescent particles by a beam splitter 51 and detected by a detector 52 after being filtered by a filter 47. The fluorescence microscope 34 also has a control unit 53, which generates control signals for driving the scanner 42 and EOD 36, and receives detector signals 55 from detectors 48 and 52. The control unit 53 is configured to position the scanning light 5 onto each fluorescent particle using the scanner 42, and to track individual particles in the sample by repeatedly scanning the sample with the scanning light 5 in the vicinity, and performing position determination based on the scanning position and the intensity or number of photons of the fluorescence 9 detected at the scanning position. During the tracking of a single fluorescent particle, the control unit 53 receives fluorescence 13 from sample 19 as a second measurement variable and stops tracking the particle when the stop criterion is reached (here, the minimum intensity of the second measurement variable, i.e., fluorescence 13).

[0067] Reference tag list

[0068]

Claims

1. A method for recording the trajectory (10) of a single particle (2) in a sample (19), the method starting from at least a approximately known starting position (3) of the particle (2), comprising the following repeatedly performed method steps: The particle (2) is scanned at one or more scanning positions (4) by an intensity distribution (7) of scanning light (5) having a local intensity minimum (8); The number or intensity of photons emitted by the particle (2) due to irradiation with the scanning light (5) at each of the scanning positions (4) is used as the first measurement variable; The updated coordinates of the particle are determined based on the number of photons detected or the intensity of the detected light at the scanning position (4), and the updated coordinates are added to the trajectory (10). Its features are, During the recording of the trajectory (10), a second measurement variable (11) is detected in the sample (19), and the recording of the trajectory (10) is interrupted or terminated when the second measurement variable (11) or the control value (12) calculated based on the second measurement variable (11) meets the termination criterion.

2. The method according to claim 1, characterized in that, During the process of repeatedly determining the updated coordinates of the particle (2), the optical power of the scanning light (5) is adjusted.

3. The method according to claim 1, characterized in that, The particle (2) is a fluorescent particle or a fluorescent dye molecule.

4. The method according to claim 2, characterized in that, The particle (2) is a fluorescent particle or a fluorescent dye molecule.

5. The method according to claim 3, characterized in that, The scanning light (5) is the fluorescent excitation light that excites the particles to emit fluorescence.

6. The method according to claim 4, characterized in that, The scanning light (5) is the fluorescent excitation light that excites the particles to emit fluorescence.

7. The method according to claim 3, characterized in that, The scanning light (5) is a superposition of fluorescence excitation light and fluorescence suppression light, wherein the fluorescence suppression light includes an intensity distribution (7) with a local intensity minimum (8).

8. The method according to claim 4, characterized in that, The scanning light (5) is a superposition of fluorescence excitation light and fluorescence suppression light, wherein the fluorescence suppression light includes an intensity distribution (7) with a local intensity minimum (8).

9. The method according to any one of claims 1-8, characterized in that, For each update of the particle (2), the particle is scanned at multiple scan positions (4) around the last determined coordinates.

10. The method according to any one of claims 1-8, characterized in that, After each scan of the particle (2) at the scan position (4), the coordinates of the particle (2) are updated.

11. The method according to any one of claims 1-8, characterized in that, If the second measured variable (11) or the control value (12) is lower than the minimum value (16) or exceeds the maximum value, the termination criterion is met.

12. The method according to claim 11, characterized in that, If the second measured variable (11) or the control value (12) exceeds the minimum value (16) again or falls below the maximum value again, the scan continues.

13. The method according to any one of claims 1-8 and 12, characterized in that, The second measurement variable (11) is detected at the location of the scanning light (5) in the sample (19).

14. The method according to any one of claims 1-8 and 12, characterized in that, The second measurement variable (11) is detected in the environment around the current coordinates of the particle (2) in the sample (19).

15. The method according to any one of claims 1-8 and 12, characterized in that, The second measurement variable (11) is detected at a fixed location or within a fixed range of the sample (19).

16. The method according to any one of claims 1-8 and 12, characterized in that, The second measurement variable (11) is an optical measurement variable.

17. The method according to claim 16, characterized in that, The second measurement variable (11) is the fluorescence signal.

18. The method according to claim 17, characterized in that, The control value (12) is calculated based on the fluorescence signal and another fluorescence signal.

19. The method according to claim 17, characterized in that, The control value (12) is a variable calculated based on fluorescence intensity or fluorescence lifetime of one or more fluorescence signals.

20. The method according to claim 18, characterized in that, The control value (12) is a variable calculated based on fluorescence intensity or fluorescence lifetime of one or more fluorescence signals.

21. The method according to claim 16, characterized in that, The second measurement variable (11) is a second harmonic generation signal (SHG), a third harmonic generation signal (THG), a Rayleigh or Raman scattered light signal, a coherent anti-Stokes Raman scattering (CARS), a reflected light signal, a differential interference contrast signal (DIC), or a polarization contrast signal.

22. The method according to any one of claims 17-21, characterized in that, A detection signal is generated by illuminating the sample (19) with light as a second measurement variable (11), the light being incident on the sample (19) at an angle between 15° and 165° with the incident direction of the scanning light (5).

23. The method according to any one of claims 1-8 and 12, characterized in that, The second measurement variable (11) is an electrical measurement variable.

24. The method according to any one of claims 1-8, 12 and 17-21, characterized in that, The second measurement variable (11) or the control value (12) calculated based on the second measurement variable (11) is a survival index, viability index or cell cycle index of the cells (1) located in the sample (19).

25. The method according to claim 2, characterized in that, During the process of repeatedly determining the updated coordinates of the particle (2), the optical power of the scanning light (5) is increased.

26. The method according to claim 18, characterized in that, The control value (12) is calculated based on the ratio of the fluorescence signal to another fluorescence signal.

27. The method according to claim 19 or 20, characterized in that, The control value (12) is pH value, ion concentration, membrane potential or fluorescence anisotropy.

28. The method according to claim 22, characterized in that, The light is incident on the sample (19) at an angle between 45° and 135° with the incident direction of the scanning light (5).

29. The method according to claim 28, characterized in that, The light is incident on the sample (19) at an angle between 80° and 100° with the incident direction of the scanning light (5).

30. The method according to claim 23, characterized in that, The electrical measurement variables are voltage, current (32), resistance, capacitance, inductance, or frequency, phase, or amplitude of AC voltage, AC current, or AC electromagnetic field.

31. An optical microscope (34), comprising: Objective lens (20) The light source (35) of the scanning light (5) A beam shaping device for forming an intensity distribution (7) of the scanning light (5) in the sample (19) having a local intensity minimum (8). A scanning device for positioning the scanning light (5) in the sample (19). The detection channel is used to detect the first optical measurement variable in the sample (19). The probe channel is used to detect the second measurement variable (11) in the sample. Its features are, The optical microscope (34) is configured to perform the method according to any one of claims 1 to 30.

32. The optical microscope (34) according to claim 31, characterized in that, The scanning light (5) is designed as a fluorescent excitation light.

33. The optical microscope (34) according to claim 32, characterized in that, The scanning light (5) is designed as a superposition of fluorescence excitation light and fluorescence suppression light, wherein the fluorescence suppression light includes an intensity distribution with a local intensity minimum (8).

34. The optical microscope (34) according to any one of claims 31-33, characterized in that, The optical microscope (34) has an additional light source designed to generate a detection signal in the sample as a second measurement variable (11).

35. The optical microscope (34) according to claim 34, characterized in that, The light from the additional light source is directed into the sample (19) at an angle between 15° and 165° to the incident direction of the scanning light (5).

36. The optical microscope (34) according to any one of claims 31-33, characterized in that, The second measurement variable (11) is a non-optical measurement variable.

37. The optical microscope (34) according to claim 35, characterized in that, The light from the additional light source is directed into the sample (19) at an angle between 45° and 135° to the incident direction of the scanning light (5).

38. The optical microscope (34) according to claim 37, characterized in that, The light from the additional light source is directed into the sample (19) at an angle between 80° and 100° to the incident direction of the scanning light (5).

39. The optical microscope (34) according to claim 36, characterized in that, The second measurement variable (11) is an electrical measurement variable.

40. The optical microscope (34) according to claim 39, characterized in that, The electrical measurement variables are voltage, current (32), resistance, capacitance, inductance, or frequency, phase, or amplitude of AC voltage, AC current, or AC electromagnetic field.

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