Localization of singularized fluorophores by continuous movement of a focused light beam around the molecule.

Abstract

To determine the molecular position of the singularized fluorophore, a light beam comprising the fluorescence excitation light and the fluorescence influence light is shaped and focused to form a light intensity distribution with a central intensity minimum of the fluorescence influence light. The central intensity minimum moves continuously along a track (31) which extends repeatedly around an estimated position (34). The individual photons of fluorescence emitted by the singularized fluorophore upon excitation with the fluorescence excitation light are aligned and the position of the intensity minimum of the central intensity minimum is recorded for the excitation of each aligned individual photon. Correspondingly, the estimated position (34) around which the track extends is updated based on the recorded position of the intensity minimum, the extension of the track (31) around the estimated position (34) is decreased and the effectiveness of the fluorescence influence light is increased. [Representative diagram] Figure 9

Description

[Technical Field]

[0001] The present invention relates to a method for determining the molecular location of singularized fluorescent dye molecules in an object. More particularly, the invention relates to a method comprising the features of the preamble of independent claim 1. Furthermore, the invention relates to a laser scanning microscope for determining the molecular location of singularized fluorescent molecules in an object. More particularly, the invention relates to a laser scanning microscope comprising the features of the preamble of independent claim 15. [Background technology]

[0002] U.S. Patent No. 10,900,901 discloses a high-spatial-resolution method for determining the location in n spatial dimensions of singularized molecules in a sample that can be excited by excitation light to emit luminescent light. A preliminary local region containing the singularized molecules is determined. The excitation light is irradiated onto the sample with an intensity distribution having a zero point and regions of increased intensity adjacent to the zero point on both sides of each of n spatial directions. First, the zero point is positioned at a preliminary location on a known side of the preliminary local region. Next, the current position of the zero point is sequentially shifted within the preliminary local region in each of the n spatial dimensions in response to separate but quasi-simultaneously aligned photons of the luminescent light, repeatedly moving the zero point between the current positions. The position of each zero point is shifted as soon as m photons of the luminescent light are aligned with the current position of the zero point. m is very small, sometimes as small as 1. Continuously shifting the zero point position can increase the maximum intensity of the excitation light. Moving the zero point position reduces the distance to the actual position of the molecule in the sample. As the maximum intensity of the excitation light is increased, the reduced distance is redistributed over a higher bandwidth of different intensities of the luminescence light from the singularized molecule. When one begins to determine the location of the singularized molecule, a larger area of ​​the sample containing the singularized molecule can be scanned in each spatial dimension along a circular or spiral track using a Gaussian intensity distribution of the excitation light, i.e., a simple focused beam of excitation light. The location of the singularized molecule is then inferred from the progression of the intensity of the luminescence light along the track.

[0003] WO 2020 / 064108 discloses a method and apparatus for shaping and shifting a light intensity distribution at a focal region of an objective lens. Multiple discrete portions of coherent input light are directed toward multiple non-identical pupil regions of the objective lens pupil. The light intensity distribution at the focal region of the objective lens is shaped to exhibit local intensity minima surrounded by intensity maxima. At least two of the multiple discrete portions of coherent input light are individually modulated in at least one of phase and amplitude by separate electro-optic modulators that shift the local intensity minima along a circle centered on the optical axis of the objective lens. Photons emitted from a single fluorophore present in the focal region are detected. Each detected photon is aligned to the position of the local intensity minimum within the focal region. The average position of the aligned positions is calculated and obtained as the position of the single fluorophore within the focal region.

[0004] Weber et al.'s paper, "MINSTED fluorescence localization and nanoscopy" (Nat. Phot., 2021, https: / / doi.org / 10.1038 / s41566-021-00774-2), describes a concept for stimulated emission depletion (STED)-based fluorescence localization and super-resolution microscopy that achieves spatial precision and resolution down to the molecular scale. The minimum intensity of the STED donut, or the STED minimum, serves as a movable reference coordinate for lateral fluorophore localization. The co-located excitation and STED beams circle around the estimated fluorophore position with a given radius at a rotation frequency of 125 kHz. The estimated position and radius are updated each time a photon is detected. The radius begins at half the diffraction-limited diameter of the effective point spread function (E-PSF) of the STED microscope used. The estimated position is shifted, and a fraction of the radius is reduced after each photon is detected. Furthermore, the STED intensity increases, and the E-PSF diameter decreases. Therefore, even though the maximum intensity gradually increases and the slope of the E-PSF becomes steeper, the fluorophore always maintains a moderate STED intensity. Magnifying the fluorophore improves the accuracy of determining the fluorophore's position. While MINSTED can achieve high accuracy, repeated irradiation of a very small localized area with high-intensity STED light can degrade the actual accuracy due to thermal effects. According to literature, the wavelength of the pulsed excitation light is 635 nm, and the STED wavelength of the pulsed STED light is 775 nm. For the Atto647N fluorophore, the maximum peak intensity of the fluorescence emission peak is approximately 660 nm, and the STED wavelength is at the far-red end of the emission peak. The remaining peak intensity corresponds to approximately 10% of the maximum peak intensity. Objective of the Invention

[0005] It is an object of the present invention to provide a method and a laser scanning microscope for determining the molecular location of a singularized fluorophore in an object with greater precision and with a minimum amount of photons emitted by the fluorophore, thereby minimizing photochemical stress on the fluorophore and / or photothermal stress on the object containing the molecule. Summary of the Invention [Means for solving the problem]

[0006] The object of the invention is achieved by a method comprising the features of independent claims 1, 5 and 10 as well as by a laser scanning microscope comprising the features of claim 24.

[0007] Preferred embodiments of these methods are as defined in the dependent claims.

[0008] The present invention relates to a method for determining the molecular location of singularized fluorophores in an object.

[0009] A singularized fluorophore is spatially separated from other fluorophores that can be excited by the same excitation light so that they emit indistinguishable fluorescence. The fluorophore whose molecular location is to be determined may be naturally singularized, for example, due to a low concentration of fluorophores in the object. Alternatively, the fluorophore may be actively singularized by bleaching or temporarily switching off neighboring fluorophores, or by switching on only a single fluorophore while leaving neighboring fluorophores in the dark. Methods well known to those skilled in the art include methods for singularizing fluorophores and determining whether only one singularized molecule is actually present in the measurement volume.

[0010] For embodiments of the invention that include only excitation light, a suitable minimum distance from a singularized fluorophore to a neighboring fluorophore is on the order of the wavelength of the excitation light. In embodiments of the invention that also include fluorescence quenching light, shorter distances may be appropriate. In general, the appropriate distance from a singularized fluorophore to a neighboring fluorophore must ensure that the neighboring fluorophore is not excited to emit fluorescence that cannot be registered separately from the fluorescence emitted by the singularized fluorophore whose position is to be determined.

[0011] The method according to the present invention includes providing a light beam including fluorescence excitation light and fluorescence influence light. The fluorescence influence light may be the same as the fluorescence excitation light, or the fluorescence influence light may be fluorescence quenching light provided in addition to the fluorescence excitation light. The additional fluorescence quenching light may be, for example, STED (stimulated emission depletion) light that affects the fluorescence of fluorophores by causing fluorophores excited by the excitation light to return from an excited state to a ground state before they can spontaneously emit fluorescence.

[0012] The method according to the present invention further includes a step of shaping and focusing the light beam to form a light intensity distribution having a central intensity minimum of the fluorescence quenching light. Furthermore, the method according to the present invention further includes a step of continuously shifting the light intensity distribution relative to the object so that the central intensity minimum moves continuously along a track that repeatedly extends about the estimated position of the singularized fluorophore. The track repeatedly extends about the estimated position. In this case, the track may surround or orbit the estimated position. However, the track may be centered on the estimated position and not orbit at a fixed distance. Specifically, the track may temporarily approach or even pass through the estimated position. However, the entire area spanned by or encompassed by the track will extend about the estimated position in all spatial dimensions in which the molecular position of the singularized fluorophore is determined.

[0013] The method according to the invention further comprises the step of registering a plurality of individual photons of fluorescence emitted by the singularized fluorophore upon excitation with the fluorescence excitation light, and recording the intensity minimum position of the central intensity minimum for the excitation of each of the registered individual photons, so that as much information as possible about the location of the molecule being determined can be extracted from each registered individual photon.

[0014] In response to each of the aligned individual photons, the estimated position around which the track runs is updated based on the recorded intensity minimum position, the extent of the track around the estimated position is decreased, and / or the fluorescence influence effectiveness of the fluorescence influence light is increased.

[0015] If the fluorescence influence light is the same as the fluorescence excitation light, the estimated position moves away from the recorded intensity minimum position. If the fluorescence influence light is fluorescence quenching light, the estimated position moves towards the recorded intensity minimum position. The new estimated position will, with a reasonable probability, be closer to the actual molecule position than the previous estimated position. Therefore, the extension of the track, i.e., the extension of the area centered on the estimated position and surrounded by the track, may be reduced. At the same time, or instead, the fluorescence influence effectiveness of the fluorescence influence light may be increased. Since the light intensity distribution formed by the method according to the present invention has a central intensity minimum of the fluorescence influence light, the intensity of the fluorescence influence light increases as the distance to the central intensity minimum increases. An increase in the fluorescence influence effect means that the effect of increasing the intensity of the fluorescence influence light is strengthened.

[0016] In general, there are various ways to increase the fluorescence influence effectiveness of the fluorescent influence light. For example, the intensity of the fluorescent influence light may be increased. Thus, the effectiveness of the fluorescent influence light does not necessarily have to relate to the relative effectiveness of the fluorescent influence light, i.e., a measure of the effect divided by the intensity of the fluorescent influence light, but may also relate to the absolute effectiveness or effect of the fluorescent influence light. Alternatively, or in addition, the fluorescence influence effectiveness of the optical influence light may be increased, in which case the effective fluorescence influence cross-section of the fluorescent influence light is increased. This can be achieved, for example, by changing the wavelength or wavelength composition of the fluorescent influence light.

[0017] When the wavelength or wavelength composition of the fluorescent light varies, attention must be paid to chromatic aberration in the optical settings. In general, the optical settings should be adjusted to have the wavelength or wavelength composition of the fluorescent light that maximizes the effective fluorescent influence cross section of the fluorescent light. Therefore, the optical settings should usually be fine-tuned to match the smallest wavelength of the fluorescent influence light.

[0018] Alternatively, or in addition, the time sequence of the fluorescence influence light pulses and the fluorescence excitation light pulses may vary. Such variations can significantly affect the fluorescence influence efficiency even when the intensities of the fluorescence influence light and the fluorescence excitation light are maintained constant. This applies both when the fluorescence influence light is fluorescence excitation light and when the fluorescence influence light is fluorescence quenching light. By varying only the time sequence of the fluorescence influence light pulses and the fluorescence excitation light pulses while maintaining the intensities of the fluorescence influence light and the fluorescence excitation light constant, the light power irradiated and absorbed by the object can be kept constant. Therefore, for example, an increase in the fluorescence influence efficiency does not result in an increase in thermal effects. The concept of increasing the fluorescence influence efficiency of the fluorescence influence light is particularly effective when the fluorescence influence light is fluorescence quenching light. Furthermore, the fluorescence quenching light pulse should not be simply longer or shorter than the fluorescence lifetime of the fluorophore. Rather, it should be much longer, i.e., at least three, five, or even ten times longer than the excitation light pulse. Detection of fluorescence photons can be gated to select only photons emitted after each fluorescence quenching light pulse, blocking signals from fluorophores that have not yet experienced a full fluorescence impact light pulse. Maintaining a detection time gate after the fluorescence quenching light pulse and shifting the excitation maximizes fluorescence detection.

[0019] The above concept of keeping thermal effects constant is particularly useful when implementing embodiments of the present invention in which tracks extend in all three spatial dimensions around the estimated position of the singularized fluorophore to determine the molecular position of the singularized fluorophore in all three spatial dimensions.

[0020] In summary, the method of the present invention uses every photon to update the estimated position of the fluorophore, and every update of the estimated position is used to strengthen the track around the estimated position and / or increase the effect of the fluorescent influence light around the central intensity minimum, which in combination allows the position of the molecule to be determined quickly with high accuracy of a few nanometers to about one nanometer.

[0021] STED fluorescence quenching is typically performed at the STED wavelength at the far-red end of the fluorophore's emission spectrum to avoid direct excitation of the fluorophore by the STED light and to maximize the detection spectrum bounded by the excitation and STED wavelengths. Due to the small stimulated emission cross-section, STED at the far-red end of the emission spectrum is less efficient and requires high STEM power, which can lead to serious thermal problems such as those mentioned above.

[0022] In methods according to the invention using STED light, the STED wavelength is set to or successively reduced to a wavelength at which the fluorescence emission peak of the singularized fluorophore still has at least 25%, preferably at least 30%, more preferably at least 35% of its maximum peak intensity. When the STED wavelength is successively reduced to increase the fluorescence influence effectiveness of the STED light, one may start with a wavelength that meets at least one of these criteria.

[0023] The method of the present invention using STED light does not expose singularized fluorophores to high STED intensities. Furthermore, no other fluorophores are present in the region of STED light intensity maxima surrounding the central intensity minimum of the STED light. Therefore, an STED wavelength can be selected that is very close to the peak emission of the fluorophore. In other words, the method of the present invention can increase the stimulated emission cross-section and therefore reduce the STED power required for a given effective PSF FWHM. In the method of the present invention, reducing the STED power by about one order of magnitude is sufficient to avoid the serious thermal problems mentioned above and realize the full potential accuracy of the method of the present invention.

[0024] Edge filtering, or more preferably, notch filtering, of the fluorescence light before aligning multiple individual photons can suppress the STED wavelength light, ensuring that the STED wavelength within the emission peak of the fluorophore does not generate significant background during the alignment step. Notch filtering of the fluorescence light is particularly effective at suppressing the STED wavelength light when the STED light is provided with a narrow STED wavelength bandwidth. When the STED light is pulsed, gating of the alignment of multiple individual photons can be performed to select photons emitted after each pulse of STED light, further suppressing background from the STED light during the alignment step. However, in general, the STED light can be applied as a continuous wave (cw). Specifically, embodiments of the present invention in which the STED wavelength is continuously reduced can be implemented using cw STED light, due to the commercially available frequency-tunable cw lasers.

[0025] In the method according to the present invention, the estimated position around which the track extends may be updated based on the recorded intensity minimum position, and the estimated position is shifted in a certain direction by a predetermined distance fraction of the distance between the estimated position and the recorded intensity minimum, depending on whether the fluorescence-influencing light is a fluorescence excitation light or an additional fluorescence quenching light. This distance fraction ranges from 3% to 33%. Preferably, the distance fraction ranges from 10% to 20%, i.e., approximately 15%. If the distance fraction is small, the estimated position will shift more slowly towards the molecular position, and therefore more photon alignments will be required to determine the molecular position with a certain accuracy. If the distance fraction is large, the estimated position will shift more quickly, but there is a risk that the estimated position will repeatedly pass the molecular position from different or opposite directions.

[0026] In the method according to the invention, the extension of the track around the estimated position may be reduced by an extension lying in the range of 1% to 10%, preferably in the range of 2% to 5%, and the step by which the extension of the track around the estimated position is reduced is therefore at least somewhat smaller than the step by which the estimated position is shifted for each aligned photon.

[0027] The fluorescence influence effectiveness of the fluorescence influence light may be particularly increased, and the extension of the effective excitation point spread function of the light intensity distribution and / or the effective point spread function of the aligned fluorescence may be reduced accordingly, i.e., preferably by the extension portion. Thus, the extension of the track centered on the estimated position and the effective excitation point spread function of the light intensity distribution are scaled down synchronously for each photon. If the fluorescence influence light is fluorescence excitation light, the effective excitation point spread function of the light intensity distribution is the point spread function of the central intensity minimum of the fluorescence excitation light. If the fluorescence influence light is additional fluorescence silencing light, the effective excitation point spread function of the light intensity distribution is the point spread function of the central intensity maximum of the fluorescence excitation light modified by the point spread function of the fluorescence silencing light. That is, it is confined to a narrow spot or peak close to the central intensity minimum of the fluorescence silencing light.

[0028] In the method according to the invention, the extent of the track around the estimated position is decreased until a predetermined minimum extent is reached, and / or the fluorescence influence effectiveness of the fluorescence influence light is increased until a predetermined maximum fluorescence influence effectiveness is reached. The extent of the track around the estimated position is correlated with the accuracy with which the molecular position is determined. Thus, reaching a predetermined minimum extent means reaching a predetermined accuracy in determining the molecular position. Due to photochemical stress on the fluorophores and / or photothermal stress on the object or sample containing the molecules, and / or due to limited light power of the available fluorescence influence light, it may not be possible, or even if possible, there may be no need, to exceed the predetermined maximum fluorescence influence effectiveness.

[0029] In the method according to the invention, the movement of the central intensity minimum along the track can continue across the aligned individual photons, regardless of the update of the estimated position and the reduction of the track extension and / or the increase in the intensity of the fluorescence-influenced light. This does not mean that the estimated position is updated in increments, nor that the track extension is reduced in increments. In particular, a rapid movement of the central intensity minimum along the track would be of no benefit, since a gradual change of the estimated position, i.e., the center of the area surrounded by the track and the extension of the track or the area surrounded by the track, would be completely impossible or at least very complicated. The same applies to the increase in the effectiveness of the fluorescence-influenced light.

[0030] In the method according to the present invention, the repetition rate at which the track extends or the central intensity minimum moves along the track around the estimated position of the singularized fluorophore is at least 50%, preferably at least 100%, of the photon rate at which individual photons are aligned. This means that, on average, no more than two, preferably no more than one, photon is aligned for each loop of the track around the estimated position. Thus, the positions of individual photons and the intensity at the minimum position of the central intensity minimum are clearly separated along the track. For the same purpose, signals representing aligned individual photons may be sampled at a sample rate at least 10 times, preferably at least 100 times, the repetition rate at which the central intensity minimum moves or the track extends around the estimated position of the singularized fluorophore. A sample rate that is 100 times the repetition rate at which the track extends around the estimated position corresponds approximately to the angular resolution of the recorded intensity minimum positions of the central intensity minimum, i.e., 360° / 100=3.6°.

[0031] In a method according to the invention, the track may extend in all three spatial dimensions around the estimated position of the singularized fluorophore. Preferably, the extension of the track around the estimated position in all three spatial dimensions reflects the expansion of the effective excitation point spread function of the light intensity distribution in the three spatial dimensions. Even more preferably, the extension of the track around the estimated position reflects the surface area of ​​the effective excitation point spread function where the ratio of signal change to signal intensity is maximized.

[0032] In practice, between responses to two consecutive individual photons of the plurality of individual photons, the track may run on the surface of an ellipsoid having x and y semi-axes in the focal plane along which the light beam focuses and a z semi-axis in the z direction along which the light beam focuses at the focal plane. The x and y semi-axes may be the same length, and the z semi-axis may be longer than the x and y semi-axes. The track may rotate about the z direction at an x-y rotational frequency that is no higher than the z rotational frequency at which the track rotates about an axis rotating in the focal plane at the z rotational frequency. It is desirable to reduce the non-integer ratio between the z rotational frequency and the x-y rotational frequency so that the region around the estimated position is not omitted for a long time and the track repeats approximately every approximately several axial cycles about the z direction and several lateral cycles about the axis rotating in the focal plane.

[0033] Alternatively, the track may run along a Lissajous curve, preferably passing through the estimated position, between responses to two consecutive photons among the multiple individual photons. A moderate and reasonable ratio between the rotational or pulse frequencies in the x, y, and z directions shortens the repetition period of the Lissajous curve while maintaining a sufficient balance with the sampling density centered on the estimated position. If the Lissajous curve passes through the estimated position, or at least passes through an internal point on the extension line of the entire track centered on the estimated position, the uncertainty of the molecular position determined based on a small number of aligned photons is reduced. This is particularly true when the fluorescence influence light is the same as the fluorescence excitation light.

[0034] In either case, if there is no response to the aligned photons, the track may become a closed loop around the estimated position.

[0035] Before shaping and focusing the light beam to form a light intensity distribution with a central intensity minimum for the fluorescence-affected light, the beam of fluorescence excitation light may be focused to form an excitation-limited light intensity distribution with a central intensity maximum. The excitation-limited light intensity distribution may be continuously shifted relative to the object. A preliminary plurality of individual photons of fluorescence emitted from singularized fluorophores upon excitation with the fluorescence excitation light may be separately aligned using a photodetector array. The photodetector array provides spatial resolution relative to an Airy disk onto which the fluorescence emitted at the central intensity maximum is imaged. The alignment position of the detector array and the intensity maximum position of the central intensity maximum are recorded for the excitation of each individual photon in the aligned preliminary plurality of individual photons. An estimated position to start is then determined from the alignment position of the detector array and the recorded intensity maximum positions for the aligned preliminary plurality of individual photons. The spatial resolution of the alignment of the individual photons within the Airy disk is used to obtain additional information regarding the estimated position to start when performing the core steps of the method according to the present invention.

[0036] The method according to the invention can be used to determine the positions of many fluorophores one by one. Once the molecular position of a singularized fluorophore has been determined, the molecular positions of other singularized fluorophores in the object may be further determined by singularizing and / or advancing another fluorophore.

[0037] A laser scanning microscope for determining the molecular location of a singularized fluorophore in an object according to the present invention includes a light source configured to provide a light beam including fluorescence excitation light and fluorescence influence light. The fluorescence influence light may be the same as the fluorescence excitation light or may be provided in addition to the fluorescence excitation light. The beam shaper and objective lens of the laser scanning microscope are configured to shape and focus the light beam to form a light intensity distribution having a central intensity minimum of the fluorescence quenching light. The scanner of the laser scanning microscope is configured to continuously shift the light intensity distribution relative to the object so that the central intensity minimum moves continuously along a track that repeatedly extends around the estimated location of the singularized molecule. The detector of the laser scanning microscope is configured to register with individual photons of fluorescence emitted by the singularized fluorophore upon excitation with the fluorescence excitation light. The controller of the laser scanning microscope is configured to record the intensity minimum position of the central intensity minimum for the excitation of each registered individual photon. The controller is further configured to update the estimated position about which the track will run based on the recorded intensity minimum position in response to each of the plurality of individual photons aligned by the method of the present invention, reduce the size of the track about the estimated position, and / or increase the effectiveness of the fluorescence influence light.

[0038] When any element of a laser scanning microscope is defined using the phrase "configured to," this definition generally includes each component of the laser scanning microscope being configured and positioned within the laser scanning microscope to achieve the defined function.

[0039] The invention can be developed from the claims, the description and the drawings.

[0040] The advantages of the features and combinations thereof mentioned at the beginning of the specification are merely examples, and embodiments according to the present invention do not necessarily have to fulfil these advantages, and they may be substituted or cumulative.

[0041] In the case of the disclosure, and not the scope of protection of the original application or patent, other features may also be gleaned from the drawings, in particular from the illustrated design and the dimensions, relative placement, and functional relationships of several components relative to one another. It is possible, and will be readily apparent to those skilled in the art, to combine features of various embodiments of the invention, or to combine features of different claims independently of the selected references in the claims. This also applies to features shown in different drawings or mentioned in the description. These features may be combined with features of various claims. Furthermore, further embodiments of the invention may not have features recited in the claims, but instead, the features of the independent claims shall not apply.

[0042] When the claims and the specification refer to specific numbers, it should be understood that this covers the exact number as well as any greater number, even without the explicit use of the adverb "at least." For example, when referring to a light source providing a light beam, it should be understood that there may be only one light source providing the light beam, or there may be two or more light sources. Additional features may be in addition to the features recited in the claims, or these features may be the only features of the respective methods or laser scanning microscopes.

[0043] Any reference signs included in the claims do not limit the scope of the matter protected by the claims, but have the sole function of facilitating an understanding of the claimed invention. [Brief explanation of the drawings]

[0044] The present invention will be described in more detail below with reference to preferred embodiments shown in the drawings. [Figure 1] 1 shows a simplified optical setup of a first embodiment of a laser scanning microscope according to the invention, configured for carrying out a first embodiment of a method according to the invention. [Figure 2] The electro-optic deflector subsystem of the optical setup illustrated in Figure 1 [Figure 3] Schematic of the laser source including the fiber amplifier that serves as the light source in the optical setup shown in Figure 1. [Figure 4] (A) Typical lateral donut focus formation used in a first embodiment of the method according to the invention; (B) Typical axial donut focus formation [Figure 5] Variant of the first embodiment of the method according to the invention [Figure 6] A simplified optical setup of a second embodiment of a laser scanning microscope 1 according to the invention, configured to localize fluorescent molecules within its lateral surface, implementing the main parts of the first embodiment of the method according to the invention. [Figure 7] FIG. 1 shows a simplified optical setup of a third embodiment of a laser scanning microscope according to the invention, configured for carrying out the second and third embodiments of the method according to the invention. [Figure 8] A polar track extending around the estimated position of the fluorophore in a first variant of the second embodiment of the method according to the invention, with a ratio of ωxy to ωz of 2:1 [Figure 9] A polar track extending around the estimated position of the fluorophore in a second variant of the second embodiment of the method according to the invention, with a ratio of ωxy to ωz of 5:3 [Figure 10] A polar track extending around the estimated position of the fluorophore in a third variant of the second embodiment of the method according to the invention, with a ratio of ωxy to ωz of 10:7 [Figure 11] A Lissajous curve in which the ratio ωx, ωy, ωz is 4:3:2 in a third embodiment of the method according to the invention, along which a track centred on the estimated position of the fluorophore runs. [Figure 12] Another Lissajous curve in a fourth embodiment of the method according to the invention, in which the ratio of ωx, ωy, ωz is 5:4:3, along which a track centred on the estimated position of the fluorophore runs. [Figure 13] A simplified optical setup of a further embodiment of a laser scanning microscope according to the invention, configured for carrying out a further embodiment of a method according to the invention. [Figure 14] (A) (B) (C) A method for increasing the fluorescence effect effectiveness of fluorescence quenching light by varying the time series of fluorescence quenching light pulses and fluorescence excitation light pulses. [Figure 15] 1 is a flow chart illustrating the steps of one embodiment of the method according to the present invention; Description of the embodiment

[0045] A method for determining the molecular location of a singularized fluorophore in an object includes sampling the object with a light beam containing fluorescence excitation light, providing continuous high frequency sampling on a substantially circular orbit or track, and real-time updating of the estimated location of the fluorophore for each photon of fluorescence from the fluorophore, thereby significantly reducing the signal required for target location accuracy.

[0046] Fixed fluorescent dye molecules →r p Let's place the K sampling positions →r k (k={1,2,…,K}) →n=(n1,n2,…,n K ) photon events can be observed. p} is determined by the probability of observing these detections given the microscope's point spread function PSF(→r). k Applying the quasi-simultaneous sampling condition, the sampling position →r k The proportion of detected values ​​in p k is calculated as follows: TIFF0007749795000001.tif891

[0047] →r0 is the initial estimated position, i.e., the molecular position →r p The sampling pattern is centered at →C0 = →r0 and follows a periodic orbit →S(t) = →C i +R i →s(t), where i is the list of molecular detection values, R i is a scaling coefficient, and →s(t) is the periodic deviation on the orbit. For example, R iis the radius, →s(t)=(cos(ωt),sin(ωt)) is the unit circle with rotation frequency ω=2πf and scanning frequency f.

[0048] If the scanning wave number is much higher than the average detection rate, quasi-simultaneous sampling occurs, and the probability of detecting the next photon at any scanning position is p k Immediately after each photon detection, the center is →r p The scan trajectory approaches the i-th photon's detection position → S i The update is performed by moving the center position by a fraction α in the direction of →C i =(1-α)→C i-1 +α→S i =→C i-1 +αR i-1 →s i-1 Therefore, center → C i is a smoothed proxy for the estimate of the fluorophore's current position. The amplitude |α| ≤ 1 defines the characteristic constant of the exponential filter, and its sign reflects the increase or decrease of the signal as it approaches the PSF center. It increases if the light beam contains additional fluorescence quenching light, and decreases if the light beam contains only fluorescence excitation light. A small |α| updates the center stepwise, resulting in gradual convergence. A large |α| moves the center →r p However, it tends to wander due to dark counts and background. When scanning at about half the maximum PSF, the range 0.1 < |α| < 0.3 typically provides fast convergence without significant overshoot, and the range 0.15 < |α| < 0.2 provides robust convergence even under adverse conditions.

[0049] If the average detection rate approaches or exceeds the scan frequency, the center position will continuously track the scan position, generating a tracking curve that can attenuate the deviation by decreasing |α|. Importantly, the scan trajectory must not be interrupted and reset to the start of the cycle upon photon detection, as this would bias the updates towards that particular scan position.

[0050] →S i is the intensity center of the effective PSF → S(ti ) is understood as a possible origin of the ith detected photon, defined by the effective PSF. If the sensor consists of multiple elements or detectors, each element or detector j has its own trajectory →S defined by the intensity center of its own effective PSF. j (t) is the most sensitive. When the jth element or detector detects the ith photon, the center is the scanning position of this element or detector →S j,i = →S j (t i ) direction, →C i =(1-α)→C i-1 +α→S j,i is established.

[0051] A high-level analysis of all photon detections may be performed recursively. In particular, maximum likelihood estimation of the molecular position can consider the integral scan trajectory →S(t) and its PSF(t) to match the detected photons with the expected signal. Because the scan trajectory is continuously changing, a full analysis would require significant computational resources, but the intermittent blinking and motion of the fluorophore may also be taken into account. A simplified estimator for localizing a stationary fluorophore by quasi-simultaneous sampling of the molecule's neighborhood is presented below.

[0052] The position of the molecule is the weight X along the coordinate axes x, y, and z. i -2 , Y i -2 , Z i -2 Center → C i =(C xi ,C yi ,C zi ) where X i , Y i , Z i are the search ranges R using only fluorescence excitation light, respectively. i , or the FWHM of the PSF with additional fluorescence suppression light along these axes i is. TIFF0007749795000002.tif791

[0053] Instead of weighting the detections, one could discard the first m detections in the spiral in on the fluorophore entirely and average the center position over only the detections in the most confined range. TIFF0007749795000003.tif491

[0054] If approximately balanced sampling occurs in the vicinity of the fluorophore, the molecular position will be i It is estimated by integrating the PSF placed at to obtain a proxy for the probability distribution at that location: fluorophores are most likely to be found at the maxima of this distribution. TIFF0007749795000004.tif791

[0055] In this approach, the probability distribution from the detected values ​​is divided by the sampling probability distribution, which allows for sampling imbalance to be taken into account. TIFF0007749795000005.tif991

[0056] Lateral localization is supported by beam scanners, such as electro-optic deflectors (EODs), which scan over a range of several PSF FWHMs at repetition rates of several hundred kHz. Weber et al.'s "MINSTED fluorescence localization and nanoscopy" (Nat. Phot., 2021, https: / / doi.org / 10.1038 / s41566-021-00774-2) demonstrates this concept for lateral localization by pivoting around the estimated molecule position at a repetition rate of 125 kHz. The beam deflection is smoothed by the EOD driver and its input signal rate limit, resulting in a circular scan trajectory or track, but with a delay in the true beam position relative to the commanded position. This delay is taken into account when aligning the beam position to the detected photons.

[0057] High-speed scanners either lack the ability to arbitrarily position the beam or have a very limited scanning range, making localization along the optical axis difficult. For example, resonant axial scanning at several hundred kHz can be achieved by acousto-optic lenses (AOLs), but this forces the scanning to a sinusoidal trajectory, making it impossible to center the scanning trajectory on the molecular position. Alternatively, electro-optic lenses feature high-speed arbitrary axial scanning, but their range is limited to a very limited range, on the order of the depth of field (DOF).

[0058] Combining the benefits of multiple scanners allows for continuous tracking and localization of fluorophores in three dimensions. For example, an AOL may feature fast axial sampling, an EOD may feature fast lateral sampling, a deformable mirror may characterize the axial and lateral centers of the sampling trajectory within a few PSF FWHM, a galvanometer mirror scanner may provide a wider scan range, and / or a stage scan may allow for a wider range of sample or object positioning. Due to the polarization sensitivity of the EOD, it may be advantageous to perform a backscan only for the central position and collect signals from the current position estimate. This partial backscan reduces detection efficiency during initial coarse localization if the sampling position is on the periphery of the detection PSF. However, because the sampling pattern is confined during localization, the loss in detection efficiency is minimal.

[0059] Fluorophore localization or positioning can be performed using a donut-shaped excitation PSF, i.e., either using fluorescence excitation light alone, as in MINFLUX, or using an excitation PSF that is confined by additional fluorescence quenching light, as in MINSTED. This helps to reduce the ambient background. In the following, confinement refers to a donut-shaped excitation PSF with fluorescence excitation light alone, or a donut-shaped STED PSF with additional fluorescence quenching light sharpening the effective excitation PSF.

[0060] The shape and size of the sampling PSF may vary along the sampling trajectory. To mitigate the effects of misregistration between the sampling PSF and the molecular position in the orthogonal plane during unidirectional localization, it may be advantageous to apply a sampling PSF with limited confinement along the orthogonal direction. For example, lateral localization may rely on a lateral donut characterized by no or weak axial confinement, while axial detection may rely on an axial donut characterized by no or weak lateral confinement. In particular, an axially confined PSF is applied when the top and bottom positions of the fluorophore are sampled, whereas a transversely confined PSF is applied when the left, right, front, and rear positions of the fluorophore are sampled.

[0061] Uncertainty σ in direction j j L j / √N j scaled, where L j is the characteristic length defined by the PSF FWHM and the scan range R, and N j is the number of detected photons from the sampling point in direction j. The uncertainty in the isotropic localization or position is N j ∝L j 2 This can be achieved by adjusting the sampling period and / or the sampling PSF strength in direction j.

[0062] In the following, an optical setup for an embodiment of the method according to the invention as well as for a corresponding embodiment of the laser scanning microscope according to the invention is described. The optical setup illustrates the use of a light beam that includes fluorescence quenching light or STED light in addition to fluorescence excitation light. When a light beam of only fluorescence excitation light is used, the illustrated optical setup can provide a light beam along the beam path of the fluorescence quenching light or STED light.

[0063] Referring now to the drawings in more detail, the embodiment of a laser scanning microscope 1 shown in FIG. 1 comprises a light source 2 providing a light beam 3 comprising fluorescence excitation light 4 and additional STED light as fluorescence quenching light 5. The light source 2 comprises separate excitation light sources 52 and fluorescence quenching light sources 53. Beam shapers 6, 7 and an objective lens 8 are configured to shape and focus the light beam 3 to form a light intensity distribution having a central intensity minimum for the fluorescence quenching light 5. Scanners 9, 10 are intended to continuously shift the light intensity distribution relative to the object 11 so as to move continuously along a track that repeatedly extends around the estimated position of a singularized molecule whose molecular position in the object 11 is to be determined. A detector 12 is individually aligned with a plurality of individual photons of fluorescence 40 emitted by the singularized molecule upon excitation with the fluorescence excitation light 4. A controller 13 records the intensity minimum positions of the central intensity minima for the excitation of the individual photons aligned by the detector.

[0064] More specifically, two pairs of partial beams, the excitation light 4 and the fluorescence-impact light 5, are separately directed laterally by a scanner 9 functioning as an EOD scanner 9 and along the optical axis by a scanner 10 functioning as an AOL scanner in the objective's sample space 8. Beam shapers 6 and 7 functioning as vortex plates and top-hat plates, respectively, imprint the desired phase profile on the partial beams of the fluorescence-impact light 5. Dichroic mirrors 14 and 15 combine the partial beams. A polarizing beam splitter 16 combines the two beam paths. Relay lenses 17 and 18 image the pupils of the EOD scanner 9 and the AOL scanner 10 onto a deformable mirror 19. Another relay system, consisting of relay lenses 20 and 21 and a mirror 22, images the pupils and the deformable mirror 19 into the objective aperture 23. A quarter-wave retarder 24 converts the linear laser beam polarization into circular polarization. Photons of fluorescence 40 emitted from object 11 are reflected by dichroic mirror 25, spatially filtered by lens 26 and pinhole 27, and individually detected by detector 12, which functions as a single-photon detection APD.

[0065] Figure 2 shows the electro-optic deflector subsystem of the scanner 9 of Figure 1, configured for scanning in the transverse x-y plane. An incident linearly polarized laser beam is deflected along a first axis by a first EOD 28. The beam's polarization is then rotated 90° using a half-wave retarder 29 so that the beam can be deflected along a second axis by a second EOD 30. Lenses 31 and 32 image the apparent deflection plane of the first EOD 28 onto the apparent deflection plane of the second EOD 30. In Figures 1, 6, and 7, this subsystem is simplified by sharing a common deflection plane with the EOD scanner 9.

[0066] Figure 3 illustrates a schematic of an inexpensive pulsed fluorescence suppression light source 53 made entirely of standard components, including a fiber amplifier 54. The fiber amplifier 54 comprises a doped single-mode or double-clad fiber 55 positioned between two fiber-coupling lenses 56, 57 and two dichroic mirrors 58, 59. The doped fiber 55 is pumped from its rear end by a laser diode 60 via a dichroic mirror 58, and from its front or output end by two laser diodes 61, 62 via a polarizing beam splitter 63 and a dichroic mirror 59. An additional laser diode 64 serves as a seed laser. A high-frequency driver board 65 (shown diagrammatically) supplies short current pulses to the laser diode 64 to generate laser light with a pulse width of 1–2 ns. These short laser pulses may be too weak for effective fluorescence suppression; in this case, the pulses are amplified within the doped fiber 55. For example, when praseodymium-doped fiber 55 is pumped by laser diodes 60-62 with blue light at an excitation wavelength of approximately 450 nm, it can achieve high gain at wavelengths of approximately 490-492 nm, 604-606 nm, and 636-638 nm. Because laser diodes 61 and 62 emit polarized light, combining their laser beams using a polarizing beam splitter, such as polarizing beam splitter 63, can double the pump power. Dichroic mirrors 58 and 59 reflect the pump light and transmit the seed light and amplified output from laser diode 64, i.e., the fluorescence suppression light 5. The pulsed fluorescence suppression light source 53 shown in Figure 3 can be used in all embodiments of the laser scanning microscope 1. The same design can be used for the excitation light source 52 and the continuous-wave fluorescence suppression light source 53.

[0067] Figure 4A illustrates how a laterally confined PSF is generated by a phase vortex plate beam shaper 6 to create a transverse donut profile at the object 11, i.e., a light intensity distribution 38 with a central intensity minimum 39 extending along the optical axis, or z-axis. This PSF is scanned laterally by an EOD scanner 9 in a periodic orbit near the fluorophore, preferably a roughly circular or elliptical orbit centered on the molecule's estimated location 34 (see Figure 5). Figure 4B illustrates how an axially confined PSF is generated by a phase top-hat plate beam shaper 7 to create an axial donut profile at the object 11, i.e., a light intensity distribution 38 with a central intensity minimum 39 surrounded by intensity maxima extending along the optical axis, or z-direction. This PSF is scanned axially by an AOL scanner 10 in a sinusoidal orbit near the molecule's estimated location 34 (see Figure 5). The two beams are set on separate paths with orthogonal polarizations and combined by a polarizing beam splitter 16 (see Figure 1). The deformable mirror 19 controls the center position of all PSFs within the object 11. A dichroic mirror 25 between the polarizing beam splitter 16 and the deformable mirror 19 extracts the fluorescence 40 photons from the fluorophores and directs them to the detector 12. A confocal pinhole 17 in the detection path rejects out-of-focus photons.

[0068] The axially confined PSF is scanned continuously up and down, but is preferably only illuminated near selected points, e.g., near the edge of the scan range (see FIG. 5). The laterally confined PSF is circulated along a track 31 in the lateral plane, and is preferably only illuminated during the remaining interval of the axial scan. This allows the estimated position 34 of the fluorophore to be updated laterally and axially at alternating intervals. Preferably, the lateral scan ω xy ≫ω zThe beam scans at least a full lateral cycle during the pause in the axial scan. The center position within the track 31 can be sampled during short intervals of the axial scan and used for omnidirectional localization. In this embodiment, the lateral and axial beam scans are decoupled, and all sampling trajectories may be sinusoidal. Thus, both beam scans may be generated resonantly at the highest frequency of the scanner. Above the resonant frequency, damping is strengthened, allowing the scan range to vary in fewer cycles.

[0069] The embodiment of the laser scanning microscope 1 shown in Figure 6 does not include a second pair of partial beams for excitation light 4 and fluorescence influence light 5. Instead, the beam shaper 7 is a top-hat plate, resulting in an axially confined PSF according to Figure 4B. Therefore, there is no option to scan the axially confined PSF up or down. Only a laterally confined PSF according to Figure 4A is generated, which rotates around the track 31 according to Figure 5 for localization by the fluorescence suppression light on the side. Nevertheless, the laser scanning microscope 1 incorporates two excitation paths with orthogonal beam polarizations. In the additional path, the fluorescence excitation light 4 is deflected by the galvanometer mirror of the galvo scanner 66 to scan a large field of view with diffraction-limited resolution. In the other path, which corresponds to the path of the partial beams for excitation light 4 and fluorescence influence light 5 to form the laterally confined PSF in Figure 1, the fluorescence excitation light and suppression light are deflected by the EOD scanner 9 to search for and localize fluorophores within a small field of view. The two paths are merged toward the sample 11 by a polarizing beam splitter 16. In a narrow field of view, the laser scanning microscope 1 detects all polarizations of the fluorescence 40 emitted from the fluorophores and arriving along either of the two paths, maximizing detection efficiency. The fluorescence 40 arriving along the path through the EOD scanner 9 is completely counter-scanned. On the other hand, the fluorescence 40 arriving along the path through the galvanometer scanner 66 can only be counter-scanned for the estimated position of the fluorophores because the galvanometer does not operate at the EOD frequency. Before the detector 12, the two portions of the fluorescence 40 arriving along the two paths are merged by a further polarizing beam splitter 67. The half-wave retarders 68 and 29 in the EOD scanner 9 (see Figure 2) can appropriately rotate the polarization of the portion of the fluorescence 40 between the polarizing beam splitters 16 and 67. For time-gated detection, the optical path lengths of both paths can be matched by an optical delay 69.

[0070] In the embodiment of laser scanning microscope 1 shown in FIG. 7, illumination is rapidly scanned in all three dimensions within the sample by AOL scanner 10 and EOD scanner 9. Relay lenses 17 and 18 image the pupil of EOD scanner 9 onto deformable mirror 19. Another relay system, consisting of relay lenses 21 and 22 and mirror 22, images the pupil and deformable mirror 19 into objective aperture 23. Quarter-wave retarder 24 converts the linear laser beam polarization to circular polarization. Emitted photons are reflected by dichroic mirror 25, spatially filtered by lens 26 and pinhole 27, and detected by APD detector 12. If small variations in beam diameter when scanning along the optical axis cannot be tolerated, an additional relay system (not shown) may image the pupil of AOL scanner 10 onto the pupil of EOD scanner 9.

[0071] The embodiment of the laser scanning microscope 1 shown in Figure 7 can be used to rapidly scan the sampled PSF in three dimensions by combining an EOD scanner 9 for lateral positioning and an AOL scanner 10 for axial positioning. The deformable mirror 19 can position the center of the scan pattern at the estimated location 34 of the fluorophore, while only small deflections need be provided by the high-speed beam scanner.

[0072] The periodic orbit or track 31 around the fluorophore may resemble the orbit of an Earth satellite. Because a typical PSF is nearly isotropic laterally but more elongated along the optical axis, isotropic localization requires more thorough sampling of positions along the optical axis than lateral positions. Therefore, periodic orbits similar to polar satellite orbits are advantageous because they encounter axial points most frequently.

[0073] For example, the frequency f of AOL Scanner 10 AOL is the rotation frequency ω z =2πf AOL Define ω and assume that the lateral deflection is synchronous with the axial deflection. Then, in any plane containing the optical axis, the scan runs on an ellipse. To complete the sampling on the side, the lateral deflection is given by the rotation frequency ω xy=2πf EOD Therefore, the scanning trajectory →s(t) runs on an ellipsoidal surface, with semi-axes X and Y on the sides and semi-axis Z along the optical axis. TIFF0007749795000006.tif1191

[0074] These scan trajectories never sample the interior of the ellipsoid, making them suitable for localization by PSFs characterized by a central maximum, such as when using additional fluorescence suppression light. When using excitation light only, the center can be sampled by switching between two beams, with a dedicated beam bypassing the high-speed scanner.

[0075] To minimize the effect of fluorophore brightness fluctuations, the rotation frequency should be chosen to achieve a balanced sampling of the molecular position from all sides over a short interval. xy ≪ω z The rotation around the axis is too slow, so the orbit samples only one transverse direction at a time, with the orthogonal transverse direction being sampled much later. On the other hand, a small integer ratio of the rotation frequencies produces sampling orbits with a short common period and few distinct sampling points, which can also lead to unbalanced sampling. For example, ω xy =2ω z In this case, a permanently tilted sampling of molecular positions occurs (see Figure 8).

[0076] It is desirable to choose a small non-integer ratio of rotation frequencies so that the sampling orbit or track 31 repeats approximately every few axial and lateral cycles without skipping the region around the fluorophore for long periods of time, e.g., 3ω xy =5ω z samples the lateral position every 1.5 axial cycles, but the sampling of the ellipsoid surface every 3 axial cycles remains coarse (see Figure 9). xy =10ω zsamples only the lateral position approximately every 2.3 axial cycles, but covers the ellipsoid surface much more densely, every 7 axial cycles (see Figure 10).

[0077] 6 to 8, the track 31 has been normalized for its lateral and axial deviations, and the track start 32 and axial intersection 33 are marked with dots.

[0078] Another embodiment of the method according to the present invention can be implemented using the optical setup of Figure 7, where the light beam consists solely of fluorescence excitation light 4. The sampling PSF therefore relies exclusively on the axial donut shape. The sampling PSF is scanned in three dimensions on a sinusoidal trajectory by a high-speed scanner. A deformable mirror can position the center of the scan pattern at the estimated location of the fluorescent dye, while only small deflections need to be provided by the high-speed beam scanner.

[0079] Using frequencies specific to each scan direction, molecular positions can be sampled on a three-dimensional track 31 that runs along the Lissajous curve. For example, the next scan trajectory starts at the center and returns to the center every least common multiple of half periods along the three scan axes. TIFF0007749795000007.tif1191

[0080] A Lissajous curve samples the inside of a 2X x 2Y x 2Z box. A small non-integer ratio between scan frequencies results in dense sampling at the expense of a long repetition period. A reasonable ratio between scan frequencies shortens the repetition period while still maintaining a good balance with the sampling density. For example, 2ω x =3ω y =4ω z The ω repeats its pattern every 6 cycles of x-scan (see Figure 11). x =2ω y =3ω z It is the same as , but has better balance of direction. x =4ω y =5ω zis repeated only every 20 cycles of x-scan (see FIG. 12). The Lissajous curve shaped track 31 is more advantageous when using only excitation light, since the internal points are also sampled.

[0081] In Figures 9-10, the track 31 is also normalized for its lateral and axial deflections.

[0082] In the embodiment of the laser scanning microscope 1 shown in FIG. 13, a deformable mirror 19 is used as a scanner to shift the focal point within the object 11 for scanning. A relay system consisting of lenses 20, 21, and mirror 22 images the deformable mirror 19 within the objective aperture 23. Photons emitted by singularized fluorophores in the sample are reflected by a dichroic mirror 25, spatially filtered by a lens 26 and a pinhole 27, and detected by an APD single-photon detector 12. A further lens 35 collimates a partial beam of the light beam 3. A color vortex plate 36 imprints a helical phase onto a partial beam of excitation light 4 while leaving a partial beam of STED or fluorescence quenching light 5 unchanged. A spatial light modulator 37 imprints a top-hat phase onto a linearly polarized partial beam of the fluorescence quenching light 5, which is converted to radial polarization by a polarization converter 36. The wavefronts of the orthogonally polarized partial beam of fluorescence quenching light 5 and the partial beam of excitation light 4 are not changed by spatial light modulator 37, and both beams are converted to tangential polarization by color vortex plate 36. Color vortex plate 36 may be an S-wave plate.

[0083] In this embodiment, the sampling PSF, i.e., the optical intensity distribution 38 with a central intensity minimum 39, relies on a three-dimensional donut-shaped PSF with an adjustable ratio of axial and lateral confinement according to DE 102018127891 B3. This sampling PSF may be scanned in three dimensions by a deformable mirror 19. This approach features a lean implementation and full inverse scanning detection at the expense of a reduced scanning frequency.

[0084] 14A, 14B, and 14C illustrate a method for enhancing the fluorescence quenching effect of STED or fluorescence quenching light 5 used as fluorescence influencing light by varying the time series of pulses of fluorescence quenching light 5 and fluorescence excitation light 4. This can also be applied to the conventional MINSTED method.

[0085] Locating fluorophores over successively smaller search ranges using both excitation light 4 and fluorescence quenching light 5 requires simultaneously increasing the fluorescence influence efficiency to narrow the effective PSF FWHM. This confinement by the fluorescence quenching light 5 entails significant fluorescence quenching power, which can heat the sample or object 11, cover slide, optics, and immersion liquid. Rapid temperature changes due to changes in power are problematic because they alter the refractive index of optical materials in the illumination path, causing apparent sample displacements. Temperature changes in the immersion liquid are particularly critical. Closed-loop stabilization of the sample position is usually too slow to cancel these apparent sample displacements.

[0086] Fortunately, the fluorescence suppression effect of the STED or fluorescence suppression light 5 can be tuned for a given STED or fluorescence suppression light power by changing the delay between the excitation light and the STED pulse. For example, if the excitation light pulse is timed immediately after the STED or fluorescence suppression light pulse (see Figure 14A), the STED or suppression effect is avoided, resulting in a diffraction-limited effective PSF. On the other hand, if the excitation light pulse is timed immediately before the STED or fluorescence suppression light pulse (see Figure 14C), the suppression effect is maximized, resulting in the most confined effective PSF. By adjusting the timing of the excitation light pulse within the duration of the STED or fluorescence suppression light pulse (see Figure 14B), the suppression efficiency can be tuned, thereby adjusting the effective PSF FWHM. This approach is useful for measuring the time duration τ STED is the fluorescence lifetime τ fl The duration of the excitation light pulse, τ ex This can be implemented by STED or fluorescence quenching light pulses that are much longer than τ ex ≪τ STED <τ flThe relationship holds. Fluorescence detection can be gated to select only photons detected after the STED or fluorescence quenching light pulse, blocking signals from fluorophores that have not yet experienced a full STED or fluorescence quenching light pulse. Maintaining a detection time gate after the STED or fluorescence quenching light pulse and shifting the excitation maximizes fluorescence detection.

[0087] STED fluorescence quenching is typically performed at wavelengths at the far-red end of the fluorophore's emission spectrum to avoid direct excitation of the fluorophore by the STED light and to maximize the detection spectrum bounded by the excitation and STED wavelengths. Due to the small stimulated emission cross-section, STED at the far-red end of the emission spectrum is less efficient and requires higher STEM power, which can lead to the thermal issues mentioned above.

[0088] Fortunately, our method using fluorescence quenching light does not expose the fluorescent dye to high STED intensity. Therefore, the STED wavelength can be closer to the peak emission of the fluorescent dye. In other words, our method can increase the stimulated emission cross-section, thereby lowering the STED power required for a given effective PSF FWHM. A reduction of about one order of magnitude is sufficient to avoid thermal problems.

[0089] The STED wavelength can decrease during fluorophore localization. In this case, the fluorescence suppression effect increases as the wavelength continuously blue-shifts from the red tail of the fluorophore's emission spectrum toward the emission maximum, allowing the STED power to remain constant. Achromatic beam shapers and optics can generate the desired STED focus for all wavelengths. The effects of residual chromatic aberration are minimized by perfecting the STED focus for final localization with the highest fluorescence suppression effect.

[0090] Additionally, temperature variations in the optics and immersion liquid in the illumination path can be minimized by using a separate near-infrared or infrared beam for heating to compensate for heating variations due to variable illumination power. The power of the illumination and heating beams must be balanced so that the overall heating remains constant.

[0091] The embodiment of the method of the present invention shown in the flowchart of FIG. 15 begins with step 42, which singularizes fluorophores from adjacent fluorophores in object 11, so that the singularized molecules are effectively individually excited to emit fluorescence 40, and their photons are aligned in a subsequent step. If the fluorophores in object 11 are already singularized, step 42 is not required. Next, in step 43, which searches for singularized molecules, a beam of fluorescence quenching light is focused to form an excitation-limited light intensity distribution with a central intensity maximum. The excitation-limited light intensity distribution is continuously shifted relative to object 11. A preliminary plurality of individual photons of fluorescence emitted from the singularized fluorophores upon excitation with the fluorescence excitation light is individually aligned using a photodetector array, which spatially resolves the distribution of fluorescence emitted at the central intensity maximum onto the photodetector array. The intensity maximum position of the central intensity maximum for the excitation of each aligned individual photon and the alignment position of the detector array are recorded. In a step 44 of determining an estimated position 34, a starting estimated position 34 is determined from the intensity maximum positions recorded for the preliminary plurality of aligned individual photons and the aligned position of the detector array. In a step 45 of shaping and focusing a light beam 3, a light beam 3 including a fluorescence excitation light 4 and a fluorescence influence light 5 is provided and shaped and focused to form a light intensity distribution having a central intensity minimum of the fluorescence quenching light. In a step 46 of continuously moving the central intensity minimum, the light intensity distribution 38 is continuously shifted relative to the object 11 so that the central intensity minimum 39 moves continuously along a track 31 that repeatedly extends around the estimated position 34 of the singularized fluorophore. In a step 47 of performing alignment, alignment is performed separately for the plurality of individual photons of fluorescence 40 emitted by the singularized molecule upon excitation with the fluorescence excitation light 4. Furthermore, the intensity minimum position of the central intensity minimum 39 is recorded for each excitation of the aligned individual photons.In an updating step 48, in response to each of the aligned individual photons, the estimated position 34 about which the track extends is updated based on the recorded position of the intensity minimum, the extension of the track 31 about the estimated position 34 is decreased and / or the effectiveness of the fluorescence influence light is increased. Depending on the result of query 49 as to whether the molecular position has already been determined with the desired accuracy (or whether the fluorophore has stopped emitting fluorescence), steps 47 and 48 are repeated in a loop 50 in order to singularize another fluorophore molecule in the object 11, or the method is restarted in a loop 51 with step 42 (or directly search for another already singularized fluorophore molecule in the object 11 according to steps 43 and 44).

[0092] 1. Laser scanning microscope 2 light source 3 beams 4. Excitation light 5 Suppression light 6 Beam shaper 7 Beam Shaper 8 Objective Lenses 9 EOD scanner 10. AOL Scanner 11 Object 12 Detectors 13 Controller 14 Dichroic mirror 15 Dichroic mirror 16 Polarizing Beam Splitter 17 Relay Lens 18 Relay Lens 19 Deformable mirror 20 relay lens 21 Relay Lens 22 Mirror 23 Objective aperture 24 1 / 4 wavelength retardation plate 25 Dichroic mirror 26 Lens 27 Pinhole 28 EOD 29 Half wavelength retardation plate 30 EOD 31 Tracks 32 Start of track 31 33 Axial intersection 34 Estimated position 35 Lens 36 Color Vortex Plate 37 Spatial Light Modulator 38 Light intensity distribution 39 Central Intensity Minimum 40 Fluorescence 41 Effective excitation point spread function 42 Steps to singularize fluorescent dye molecules 43 Steps to search for singularized fluorescent dye molecules 44. Determining Estimated Location 45 Shaping and Focusing Steps 46. ​​Step of continuously moving the central intensity minimum value 39 47 Steps for aligning individual photons 48 Steps to Update 49 queries 50 Loops 51 Loop 52 Excitation light source 53 Fluorescent light source 54 Fiber Amplifier 55 Fiber 56 Fiber Coupling Lens 57 Fiber Coupling Lens 58 Dichroic Mirror 59 Dichroic Mirror 60 laser diode 61 Laser Diode 62 Laser Diode 63 Beam Splitter 64 Seed Laser 65 Signal generator 66 Galvo Scanner 67 Polarizing Beam Splitter 68 Half wavelength retardation plate 69 Optical Delay

Claims

1. A method for determining the molecular location of singularized fluorophores in an object (11), comprising the steps of: providing a light beam (3) including a fluorescence excitation light (4) and a fluorescence influence light, the fluorescence influence light being a fluorescence quenching light, which is a STED light (5) having a STED wavelength longer than the excitation wavelength of the fluorescence excitation light (4); shaping and focusing the light beam (3) to form a light intensity distribution (38) having a central intensity minimum (39) of the STED light (5); continuously shifting the light intensity distribution (38) relative to the object (11) so that the central intensity minimum (39) moves continuously along a track (31) that moves repeatedly about the estimated position (34) of the singularized fluorophore; Individually aligning with a plurality of individual photons of fluorescence (40) emitted by the singularized fluorescent dye molecule upon excitation with the fluorescence excitation light (4); recording an intensity minimum position of said central intensity minimum (39) for excitation of each individual photon of the aligned plurality of individual photons; Further, in response to each individual photon of the aligned plurality of individual photons, updating the estimated position (34) based on the recorded position of the intensity minimum, and repeatedly moving the track around the updated estimated position (34); reducing the range of movement of the track (31) around the estimated position (34) and / or increasing the fluorescence, which affects the effectiveness of the STED light; the STED wavelength is set to or continuously reduced to a wavelength at which the fluorescence emission peak of the singularized fluorophore still has at least 25% of its maximum peak intensity.

2. 2. The method of claim 1, wherein the STED wavelength is set to or continuously reduced to a wavelength at which the fluorescence emission peak of the singularized fluorophore still has at least 30%, preferably at least 35%, of its maximum peak intensity.

3. 3. The method of claim 1, further comprising notch- or edge-filtering the fluorescence (40) to suppress light at the STED wavelength before aligning the individual photons, and / or pulsing the STED light to gate the alignment of the individual photons, thereby selecting the photons emitted after each pulse of the STED light.

4. The fluorescent influence effectiveness of the fluorescent influence light is: when the intensity of the STED light is increased, and / or when the effective fluorescence influence cross section of the STED light increases, preferably with a decrease in its STED wavelength, and / or 4. The method according to claim 1, wherein the time series of pulses of the STED light and of the fluorescence excitation light (4) is increased when varying.

5. A method for determining the molecular location of singularized fluorophores in an object (11), comprising the steps of: providing a light beam (3) including a fluorescence excitation light (4) and a fluorescence influence light, the fluorescence influence light being the same as the fluorescence excitation light (4) or the fluorescence influence light being a fluorescence quenching light (5) provided in addition to the fluorescence excitation light (4); The light beam (3) is shaped and focused to form a light intensity distribution (38) having a central intensity minimum value (39) of the fluorescent light; continuously shifting the light intensity distribution (38) relative to the object (11) so that the central intensity minimum (39) moves continuously along a track (31) that moves repeatedly about the estimated position (34) of the singularized fluorophore; Separately aligning with a plurality of individual photons of fluorescence (40) emitted by the singularized fluorescent dye molecule upon excitation with the fluorescence excitation light (4); recording an intensity minimum position of said central intensity minimum (39) for excitation of each individual photon of the aligned plurality of individual photons; Further, in response to each individual photon of the aligned plurality of individual photons, updating the estimated position (34) based on the recorded position of the intensity minimum, and the truck repeatedly moving around the updated estimated position (34); decreasing the range of movement of the truck (31) around the estimated position (34) and / or increasing the fluorescence, which affects the effectiveness of the fluorescence-influenced light; A method characterized in that the fluorescence influence effectiveness of said fluorescence influence light is increased by varying the time sequence of said pulses of said fluorescence influence light and said pulses of said fluorescence excitation light (4).

6. 6. A method according to claim 5, characterized in that the intensities of the fluorescence influence light and the fluorescence excitation light (4) are kept constant.

7. 7. The method according to claim 5 or 6, characterized in that the fluorescence-influencing light is fluorescence quenching light (5), in particular STED light, and the pulse of the fluorescence-influencing light is shorter than the fluorescence lifetime of the singularized fluorophore and longer than the pulse of the fluorescence excitation light.

8. 8. The method of claim 7, wherein the alignment of the fluorescence (40) photons is gated to select photons emitted after each fluorescence-influence light pulse and to block photons from the singularized fluorophores that have not yet experienced their respective complete fluorescence-influence light pulse.

9. 9. The method according to any one of claims 5 to 8, characterized in that the track (31) moves in all three spatial dimensions around the estimated position (34) of the singularized fluorophore.

10. A method for determining the molecular location of singularized fluorophores in an object (11), comprising the steps of: providing a light beam (3) including a fluorescence excitation light (4) and a fluorescence influence light, the fluorescence influence light being the same as the fluorescence excitation light (4) or the fluorescence influence light being a fluorescence quenching light (5) provided in addition to the fluorescence excitation light (4); The light beam (3) is shaped and focused to form a light intensity distribution (38) having a central intensity minimum value (39) of the fluorescent light; continuously shifting the light intensity distribution (38) relative to the object (11) so that the central intensity minimum (39) moves continuously along a track (31) that moves repeatedly about the estimated position (34) of the singularized fluorophore; Separately aligning with a plurality of individual photons of fluorescence (40) emitted by the singularized fluorescent dye molecule upon excitation with the fluorescence excitation light (4); recording an intensity minimum position of said central intensity minimum (39) for excitation of each individual photon of the aligned plurality of individual photons; In response to each individual photon of the aligned plurality of individual photons, updating the estimated position (34) based on the recorded position of the intensity minimum, and the truck repeatedly moving around the updated estimated position (34); A method characterized by decreasing the range of movement of the track (31) around the estimated position (34) and / or increasing the fluorescence which affects the effectiveness of the fluorescence-influenced light.

11. The fluorescent influence effectiveness of the fluorescent influence light is: when the intensity of the fluorescent light increases, and / or the effective fluorescence influence cross section of said fluorescence influence light is increased, preferably by changing its wavelength composition, and / or 11. A method according to claim 10, characterized in that the time sequence of the pulses of the fluorescence influence light and the pulses of the fluorescence excitation light (4) is increased when varied.

12. The estimated position is updated based on the recorded position of the intensity minimum, and the truck is repeatedly moved around the updated estimated position (34); if the fluorescence influence light is the same as the fluorescence excitation light (4), the estimated position (34) moves away from the recorded intensity minimum position, or When the fluorescence quenching light is provided in addition to the fluorescence excitation light (4), the estimated position (34) moves towards the recorded intensity minimum position:

12. The method according to claim 1, wherein the estimated position (34) is shifted by a predetermined distance portion of the distance between the estimated position (34) and the recorded position of the intensity minimum, said distance portion being in the range of 3% to 33%, preferably in the range of 10% to 20%.

13. 12. The method according to claim 4, 5 or 11, characterized in that the range of movement of the track (31) around the estimated position (34) is reduced by a portion in the range of 1% to 10%, preferably 2% to 5%, and the increase in the fluorescence influence effectiveness of the fluorescence influence light causes the expansion of the effective excitation point spread function of the light intensity distribution to be reduced accordingly, preferably by said portion.

14. 12. A method according to claim 4, 5 or 11, characterized in that the range of movement of the track (31) centred on the estimated position (34) is reduced until a predetermined minimum extension is reached and / or the fluorescence influence effectiveness of the fluorescence influence light is increased until a predetermined maximum fluorescence influence effectiveness is reached.

15. 12. The method of claim 4, 5 or 11, characterized in that the movement of the central intensity minimum (39) along the track (31) continues across aligned individual photons, regardless of the update of the estimated position (34) and the reduction of the movement range of the track (31) and / or the increase of the fluorescence influence effectiveness of the fluorescence influence light.

16. 16. The method according to any one of claims 1 to 15, characterized in that the repetition rate of the movement of the track (31) around the estimated position (34) of the singularized fluorophore is at least 50%, preferably at least 100%, of the photon rate at which the individual photons are aligned.

17. 17. The method according to any one of claims 1 to 16, characterized in that the signals indicative of aligned individual photons are sampled at a sample rate that is at least 10 times, preferably at least 100 times, the repetition rate of the movement of the track (31) around the estimated position (34) of the singularized fluorophore.

18. 18. The method according to any one of claims 1 to 17, characterized in that the track (31) is moved in all three spatial dimensions around the estimated position (34) of the singularized fluorescent dye molecule, and the movement of the track (31) around the estimated position (34) in three spatial dimensions preferably reflects an expansion of the effective excitation point spread function (41) of the light intensity distribution (39) in three spatial dimensions.

19. Between responses to two consecutive individual photons of the plurality of individual photons, the track (31) runs on the surface of an ellipsoid having x and y semi-axes in a focal plane along which the light beam (3) converges, and a z semi-axis in the z direction along which the light beam (3) converges to the focal plane; 19. The method of claim 18, characterized in that the track (31) rotates about the z direction at an xy rotational frequency that is preferably not higher than a z rotational frequency about an axis about which the track (31) rotates in the focal plane, the axis rotating at the z rotational frequency.

20. 19. The method of claim 18, wherein the track (31) runs along a Lissajous curve, the Lissajous curve preferably passing through the estimated position (34) during a response to two consecutive individual photons of the plurality of individual photons.

21. Method according to any one of the preceding claims, characterized in that in the absence of a response to aligned photons, the track (31) is a closed loop.

22. Prior to forming a light intensity distribution having a central intensity minimum of the fluorescent light by shaping and focusing the light beam (3), focusing the beam of fluorescence quenching light to form an excitation-limited light intensity distribution having a central intensity maximum; continuously shifting the excitation-limited light intensity distribution relative to the object; performing separate registration of preliminary individual photons of fluorescence emitted from the singularized fluorophores upon excitation with the fluorescence excitation light using a photodetector array, the photodetector array performing spatial resolution of a distribution of the fluorescence emitted at the central intensity maximum imaged onto the photodetector array; recording an intensity maximum position of the central intensity maximum for each individual photon excitation of the aligned preliminary plurality of individual photons and an aligned position of the detector array; 22. The method according to any one of claims 1 to 21, characterized in that the estimated position (34) to start from is determined from the position of intensity maximum recorded for a preliminary plurality of aligned individual photons and the aligned position of the detector array.

23. 23. A method according to any one of claims 1 to 22, characterized in that once the molecular location of the singularised fluorophore has been determined, another fluorophore is singularised to further determine the molecular locations of other singularised fluorophores in the object.

24. A laser scanning microscope (1) for determining the molecular location of singularized fluorophores in an object (11), comprising: a light source (2) configured to provide a light beam (3) including a fluorescence excitation light (4) and a fluorescence influence light, said fluorescence influence light being the same as said fluorescence excitation light (4) or being provided in addition to said fluorescence excitation light; a beam shaper (6, 7) and an objective lens (8) configured to shape and focus the light beam (3) to form a light intensity distribution having a central minimum value of the intensity of the fluorescence influence suppression light; a scanner (9, 10) configured to continuously shift the light intensity distribution relative to the object such that the central intensity minimum moves continuously along a repeating moving track centered on the estimated position (34) of the singularized fluorophore; a detector (12) configured to separately register with a plurality of individual photons of fluorescence (40) emitted by the singularized fluorophore upon excitation with the fluorescence excitation light (4); a controller (13) configured to record an intensity minimum position of the central intensity minimum (39) for excitation of each individual photon of the aligned plurality of individual photons; The controller (13) is further configured to, in response to each individual photon of the aligned plurality of individual photons: updating the estimated position (34) based on the recorded position of the intensity minimum, and the truck repeatedly moving around the updated estimated position (34); configured to decrease the range of movement of the track (31) around the estimated position (34) and / or increase the fluorescence, which affects the effectiveness of the fluorescence-influenced light; A laser scanning microscope that performs the above by the method according to any one of claims 1 to 23.

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