EP3997505A1 - Verfahren und mikroskop mit einer korrekturvorrichtung zur korrektur von aberrationsinduzierten abbildungsfehlern - Google Patents
Verfahren und mikroskop mit einer korrekturvorrichtung zur korrektur von aberrationsinduzierten abbildungsfehlernInfo
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- EP3997505A1 EP3997505A1 EP20739894.2A EP20739894A EP3997505A1 EP 3997505 A1 EP3997505 A1 EP 3997505A1 EP 20739894 A EP20739894 A EP 20739894A EP 3997505 A1 EP3997505 A1 EP 3997505A1
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Classifications
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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Definitions
- the invention relates to a method for correcting aberration-induced imaging errors of an optical system which has an objective and an adaptive optics in the beam path through the objective.
- the present invention also relates to a laser scanning microscope with a first light source for excitation light, a second light source for stimulation light, an objective and a correction device for correcting aberration-induced aberrations, which includes adaptive optics in the beam path through the objective.
- the aberration-induced imaging errors can on the one hand be attributed to the optical system itself if it is not sufficiently achromatic, for example.
- an area of a sample in front of a focus area of the optical system can also cause the underlying aberration.
- the aberration-induced imaging errors of the optical system to be corrected have an effect both when projecting a light intensity distribution with the objective into the respective sample and when imaging the sample with the objective.
- An SLM Spatial Light Modulator
- SLM Spatial Light Modulator
- the phase distortion through the sample is measured by applying a phase pattern to a beam of stimulation light with the SLM, in which the phase shift is modulated in different areas with different frequencies, with the intensity of a spot on which the beam of stimulation light is then focused , registered time-resolved and analyzed by Fourier transformation.
- a search is made for the phase offsets in the individual areas of the phase pattern impressed with the SLM, which compensate for the aberration-induced imaging errors.
- the intensity of the spot is detected by scanning a gold nanoparticle with the spot and by registering the stimulation light scattered by it.
- the respective STED microscope has to be operated in a scattered light mode and the gold nanoparticle has to be arranged at the location of the respective sample for which the aberration-induced imaging errors are to be compensated.
- the gold nanoparticles were only placed on the top and bottom of a sample to demonstrate how the method worked.
- a method for correcting aberrations in STED microscopy with the aid of adaptive optics is known from WO 2014/029978 A1, in which a metric is used that combines image sharpness and image brightness.
- this metric By driving a light modulator, which is an SLM or a deformable mirror, this metric is maximized or minimized.
- the image sharpness can only be determined after the sample has been mapped into an image. Image brightness depends on the concentration and density of fluorescent markers used to mark a structure of interest in the sample. The respective maximum or minimum of the metric combining the image brightness and the image sharpness therefore only has local expressiveness and cannot be compared with other maxima or minima in other areas of the sample.
- the invention is based on the object of showing methods for correcting aberration-induced aberrations of an optical system and a laser scanning microscope with an objective and a correction device for correcting aberration-induced aberrations that correct the aberrations for different areas of a sample enable during the ongoing use of the imaging system or the laser scanning microscope for measuring or imaging the respective sample.
- the present invention uses a metric which represents a measure of the quality of the focus when illuminating a sample with light.
- a metric which represents a measure of the quality of the focus when illuminating a sample with light.
- aberrations such as those that can occur in microscopy, can be corrected.
- the sample is first illuminated with the light with the given setting of the adaptive optics, whereby a measurement signal is influenced by the sample in that the light leads, for example, to the emission of fluorescent radiation.
- the measurement signal is then detected in a time-resolved manner and divided into at least an early and a late period for the subsequent calculation.
- a measure is calculated which represents a measure of the quality of the focus. This measure is determined by repeated adjustment of the adaptive optics, e.g. B. an adaptive mirror optimized, whereby the aberrations are corrected.
- An equivalent measure can be determined in the frequency domain.
- a method for correcting aberration-induced imaging errors of an optical system which has an objective and an adaptive optics arranged in the beam path through the objective, light and a sample are selected so that the light when acting on the sample a measurement signal from the sample either reduced or approaching a saturation value from below, a relative change in the measurement signal depending on an intensity of the light.
- the measurement signal originating from a focal area of the optical system in the sample is recorded over a first time period in order to determine a first measurement value and over a second time period in order to determine a second measurement value.
- the selected light is focused with the optical system in the focus area in the sample.
- the first period is at least partly earlier than the second period and / or the second period is at least partly later than the first period, and the third period at least partly overlaps in time with the first period and / or the second period and / or an intermediate period Intermediate period.
- a measure that is a strictly monotonically increasing or decreasing function of the relative change in the measurement signal is determined and is used as a metric when controlling the adaptive optics, which is to be optimized by changing the control.
- the aberration-induced aberrations to be corrected have the effect, if the selected light is focused with the optical system in the focal area in the sample in the third time period, that the intensity of the selected light compared to its maximum value without the aberration-induced aberrations to be corrected, i.e. . H. the so-called Strehl number is reduced.
- the relative change in the measurement signal from the sample caused by the selected light is correspondingly reduced.
- the above relative definitions of the first, second and third time period have the consequence that the first measured value is less strongly influenced by the selected light focused in the focus area in the third time period than the second measured value. Due to the above selection of the light and the sample and the above relative definitions of the first, second and third time periods to one another, the first and the second measured value depend in the same way on local properties of the sample in the respective focus area. Therefore, for example, the determination of the measure by forming a quotient from the second measured value determined as the integral of the measurement signal over the second period and the first measured value determined as the integral of the measurement signal over the first period leads to a normalization to an initial value of the measurement signal, i.e. H. to independence from the absolute height, d. H. the level of the measurement signal. However, this measure remains dependent on the intensity of the selected light in the focus area.
- the measure is thus comparable across the fluctuating level of the measurement signal.
- the dimension figure can be optimized over adjacent areas of a sample, even if the level of the measurement signal fluctuates over these adjacent areas of the sample.
- the dimension figure can be continuously optimized, particularly when the sample is scanned with the focus area.
- the optimization of the dimension figure is not only constant over the changes in the control of the adaptive optics Position of the focus area in the sample possible.
- the metric to be optimized in accordance with the invention ie the measurement figure for all positions of the focus area in a sample, has the same value.
- the sample itself is the cause of aberrations that will vary at least in the axial direction along the optical axis of the objective, ie over the depth of the sample.
- the optimized index will fluctuate only slightly, so that an optimization for a directly adjacent position of the focus area in the sample can begin with the control of the adaptive optics, which is the case with the earlier position of the focus area has led to the optimization of the metric according to the invention.
- the figure either increases or decreases with the intensity of the selected light in the focus area.
- the dimension figure is to be maximized or minimized as a metric to be optimized by controlling the adaptive optics in order to maximize the intensity of the light in the focus area. This maximum intensity is reached exactly when the aberration-induced imaging errors have been eliminated. Therefore, in the method according to the invention, the measure is suitable as a metric to be optimized by changing the control of the adaptive optics in order to achieve the goal of correcting the aberration-induced imaging errors.
- the measure determined from the first measured value and the second measured value which is used as a metric to be optimized by changing the control of the adaptive optics, does not have to be a quotient, in particular a direct quotient of these two measured values. It is basically harmless if the denominator or numerator of the quotient also has a further factor that is multiplied by the measured value, or an offset that is added to the measured value. Furthermore, the denominator can have a difference and / or the numerator of the quotient can have a sum of the measured values or vice versa. In general, any measure is suitable that is a strictly monotonically increasing or decreasing function of the relative change in the measurement signal. It goes without saying that this function only needs to have these properties in the relevant range of the measured values.
- each such measure reaches an extreme as the direct quotient of the second measured value determined as the integral of the measured signal over the second period and the first measured value determined as the integral of the measured signal over the first period. It is fundamentally advantageous if the measure is a constant and thus injective function of the relative change in the measurement signal. But this is by no means necessary.
- the following procedure can be used.
- An old measure is determined from the first measured value and the second measured value.
- the control of the adaptive optics is changed in one direction of change.
- the measurement signal is recorded again over the same first time period and the same second time period, the light being focused into the focal area in the sample over the same third time period in order to determine a new first measured value and a new second measured value.
- a new measure is determined from the new first measured value and the new second measured value, and a difference between the new measure and the old measure is determined.
- the control of the adaptive optics is changed again in the previous or another, ie in particular special in a change direction opposite to the previous change direction.
- the metric according to the invention is to be maximized due to the underlying dependence of the change in the measurement signal on the intensity of the selected light, then, if the new measure is greater than the old measure, the control of the adaptive optics must be changed again in the previous direction of change, and if the new dimension is smaller than the old dimension, the change direction must be changed when the control of the adaptive optics is changed again. In the case of a metric according to the invention to be minimized, the situation is reversed.
- the steps of re-acquiring the measurement signal over an identical first period of time and an identical second period of time, the light being focused into the focus area in the sample over an identical third period of time in order to determine a new first measured value and a new second measured value, the determination a new measure from the new first measured value and the new second measured value, the determination of a difference between the new measure and the old measure and the renewed change of the control of the adaptive optics in the previous or another change direction depending on a direction of the difference can at least can be carried out a further time, the new measure formed during a previous implementation of the steps being used as the old measure in the current implementation of the steps.
- the steps mentioned can be repeated until the adaptive optics are controlled in the respective change direction, at which point the dimension figure reaches an extremum, ie either a minimum or a maximum.
- an extremum ie either a minimum or a maximum.
- it can be used, for example, that the difference between the new measure and the old measure falls below a limit value related to the last change in the control of the adaptive optics or that the difference between the new measure and the old measure is above the last repetitions of the steps repeatedly changes direction.
- the size of the change in the control of the adaptive optics for each repetition of the steps can be dependent on the size of the difference between the new measure and the old measure in at least one of the preceding steps. In this way, overshooting of the sought extremum of the respective metric by changing the control of the adaptive optics too much can be avoided.
- the measurement signal which is influenced with the selected light in the method according to the invention and is integrated to obtain the first and the second measurement value, can in particular be measurement light emitted from the sample.
- This measuring light can be imaged with the optical system on a detector that detects the measuring light and, if necessary, integrates it directly.
- the aberration-induced imaging errors of the optical system to be corrected have an effect. However, depending on the type of detection of the measuring light, this additional effect can be insignificant. This applies, for example, in all cases in which the detector registers the measurement light from the focus area without spatial resolution.
- the direction of change in which the control of the adaptive optics is changed when the method according to the invention is carried out can be selected from various directions of change. These include in particular the directions of change in which a spherical aberration, a defocus, an astigmatism or a coma of the optical system can be compensated.
- Which change in the control of the adaptive optics makes a particularly large contribution to the approach to the respective extreme of the metric according to the invention depends on the respective optical system and in particular on the respective sample. In many cases, it is sufficient to compensate for just one of the aberrations mentioned in order to keep the aberrations of the optical system very small. In particular, this can be spherical aberration.
- the light can be selected from light that leads to an exponential decrease in the measurement signal towards zero over time or that leads to an exponential approximation of the measurement signal from below keeps the saturation value over time.
- the selected light can be excitation light that excites the fluorescent dyes in the focus area into a fluorescent state until the intensity of the resulting fluorescent light from the fluorescent dyes approaches the saturation value.
- the selected light can be stimulation light that stimulates the fluorescent dyes to stimulate emission before the fluorescent dyes emit the fluorescent light emit. Then the fluorescence light remaining after the action of the stimulation light depends non-linearly on the intensity of the stimulation light.
- the additional excitation light can be focused together with the stimulation light from the optical system in the focus area in the sample. If an intensity distribution of the selected light has a central intensity minimum that coincides with a central intensity maximum of the excitation light in the focus area, there are intensity distributions of the excitation light and the stimulation light, as they are also used in STED microscopy.
- the method according to the invention thus leads to the correction of the aberration-induced imaging errors of the imaging system, which have a negative effect on the spatially narrowly limited effective point smear function when the respective sample is excited to fluorescence.
- the method according to the invention corrects the aberration-induced aberrations for precisely this environment of the intensity minimum.
- this environment surrounds the minimum intensity, which corresponds to the respective point of the sample measured in STED microscopy, but does not itself include it, this also means a correction of the aberration-induced imaging errors for the respectively measured point of the sample. This correction is even highly specific for the respective measured point on the sample because the dimensions of the surroundings are already smaller than the diffraction limit for wavelengths of light and fluorescent light.
- the method according to the invention can be used while a STED microscopic measurement or also a STED measurement is being carried out.
- microscopic image acquisition Specifically, the photons of the fluorescent light registered for the respective measurement or image recording can be used to carry out the method according to the invention if they are timed Resolution are registered so that they can be assigned to the first and the second period.
- the method according to the invention can also be carried out before the optical system is used for the actual imaging of the sample.
- the quotient whose denominator contains the first measured value and whose numerator contains the second measured value is used as the metric to be minimized. This means that if the respective new measure determined as a quotient is smaller than the respective old measure determined as a quotient, the control of the adaptive optics must be changed again in the previous direction of change because the decreasing metric indicates decreasing aberration-induced imaging errors.
- the method according to the invention aims instead at the fact that the stimulation light prevents the emission of fluorescent light as far as possible in the entire focus area. This requires the maximum intensity of the stimulation light in the focus area, as can only be achieved without aberration-induced aberrations.
- the remaining emission of fluorescent light from the intensity minimum of the intensity distribution of the stimulation light which can even increase with decreasing aberration-induced imaging errors, does not interfere with the function of the method according to the invention.
- the respective first period of time, the respective second period of time and also the respective third period of time can each be a closed period of time independently of one another or be made up of partial periods of time separated from one another.
- the fact that the third time period is made up of partial time periods can mean, for example, that the light is directed into the focus area in several very rapidly successive pulses that are not resolved when the measurement signal is recorded.
- the fact that the first time period and / or the second time period is made up of partial time periods can mean that the measurement signal from the sample is recorded in several successive partial time periods, between which there are dead times in the registration of the measurement signal.
- the first, the second and the third time period are composed of partial time periods, that the focus area is shifted somewhat between a partial period of the first, the second and the third time period in the sample before each second and then possibly further partial periods of the first period, the second period and the third period follow.
- averaging is carried out over a spatial area of the sample when the measurement signal is recorded and, if necessary, integrated. This has proven to be particularly advantageous in the embodiment of the method according to the invention, which is close to STED microscopy, in order to average out effects of a concentration of the fluorescent dyes in the sample that fluctuates with high spatial frequency on the measure used as the metric.
- the acquisition and, if necessary, integration of the measurement signal can be carried out over the same first and the same second time periods, with the light being focused over the same third time period in the focal area in the sample, for several image points around the respective first Measured value and the respective second measured value.
- the correction device for performing the following steps of the method according to the invention are formed: Detecting the measurement signal over the first time period and the second time period, the stimulation light as the light that reduces the measurement signal when it acts on the sample, being focused into the focus area in the sample over the third time period, to determine the first measurement value and the second measurement value; Determining the measure from the first measured value and the second measured value; and using the measure when controlling the adaptive optics as a metric to be optimized by changing the control.
- the adaptive optics of the laser scanning microscope according to the invention can for example have an adaptive mirror and / or a controllable micromirror array and / or an SLM (spatial light modulator).
- the SLM in particular can also be used for wavefront shaping in the case of the Stimulation light can be used in such a way that the stimulation light forms the intensity distribution with the central intensity minimum in the focus area.
- first light, second light and a sample are selected so that the first light is applied when acting on the sample excites first measurement signal from components of the sample with a first transition probability, which depends with a first power on an intensity of the first light, and that the second light when acting on the sample, the first or a second measurement signal from the same components of the sample with a second transition probability excites, which depends with a second power on an intensity of the second light, wherein the first and the second power are different by at least one.
- the first light is then focused with the optical system into a focal area of the optical system in the sample, the first measurement signal excited by the first light being recorded from the focal area over a first period in order to determine a first measured value.
- the second light with the optical signal is focused into the focal area in the sample, the first or second measurement signal excited by the second light from the focal area in the sample being recorded over a second period in order to determine a second measured value.
- a measure that is a strictly monotonically increasing or decreasing function of a relative change in the first measurement signal or a relative difference between the first and the second measurement signal is determined and used as a metric when driving the adaptive optics which is to be optimized by changing the control.
- the different dependencies of the first and second transition probabilities on the intensity of the first and second light when the respective measurement signal is excited are used to determine a measure that is used as a metric to be optimized when driving the adaptive optics.
- the measure which is a strictly monotonically increasing or decreasing function of the relative change in the first measurement signal or the relative difference between the first and the second measurement signal, by forming a quotient between the first and the second measurement value certainly.
- the first and second transition probabilities depend on the intensity of the light to a first and second power, which differ by at least one, while the intensities of the first and second light both depend in the same way on the aberration-induced aberrations, this quotient is dependent on the aberrations. All other influences on the two measured values, on the other hand, are included in a constant factor of the quotient, which does not change when determining the first and second measured value for the same focus area with the aberration-induced imaging errors. This basically applies even when the first light excites other components of the sample in the same or even a different focus area to emit the first measurement signal than the second light.
- the measurement signal excited with the second light can be the same measurement signal as the measurement signal excited with the first light, which can simplify the implementation of the method according to the invention, but it can also be a second measurement signal, for example measurement light of a different wavelength.
- the first light can excite the first measurement signal from the constituents of the sample by a one-photon process, while the second light excites the first or the second measurement signal from the same constituents of the sample by a multiphoton process.
- a phase shift between the light modulation and a signal modulation of the measurement signal is used as a measure, which is a strictly monotonically increasing or decreasing one
- the function of the relative change in the measurement signal is determined and used as a metric when controlling the adaptive optics, which is to be optimized, ie to be minimized or maximized, by changing the control.
- This method according to the invention is the equivalent of the method according to the invention described first in the frequency domain.
- the phase shift between the light modulation and the signal modulation is similarly dependent on the aberration-induced imaging errors as the quotient of the second measured value and the first measured value in the inventive method explained above.
- FIG. 1 shows a laser scanning microscope according to the invention.
- FIG. 3 is a histogram of photons of fluorescent light, which is recorded as a measurement signal in the laser scanning microscope, over time.
- FIG. 4 shows a plot of the metric according to the invention over various spherical aberrations (black squares) that are specifically caused and over a free-running optimization of the metric according to the invention (white circles).
- Fig. 5 is a plot of a metric according to the invention against a spherical one
- FIG. 6 shows, above the same spherical aberration, the half-width of images of the beads filled with dye.
- FIG. 7 shows, in comparison with FIG. 5, above the same spherical aberration, the intensity of the fluorescent light from the beads filled with dye.
- FIG. 8 shows the course of the metric according to the invention over a defocus of the optical system of the laser scanning microscope according to FIG. 1.
- FIG. 9 shows the course of the metric according to the invention over a spherical one
- FIG. 10 shows the course of the metric according to the invention over an astigmatism of the optical system of the laser scanning microscope according to FIG. 1.
- FIG. 12 shows a bright image of a structure marked with a fluorescent dye and the associated course of the metric according to the invention over a spherical aberration.
- FIG. 13 shows a less bright image of the same structure as in FIG. 12 and the associated course of the metric according to the invention over the same spherical aberration.
- FIGS. 12 and 13 shows a dark image of the same structure as in FIGS. 12 and 13 and the associated course of the metric according to the invention over the same spherical aberration.
- 15 shows an intensity distribution of excitation light and stimulation light in
- 16 shows different time sequences of a first time period in which the measurement signal is integrated into a first measurement value, a second time period in which the measurement signal is integrated into a second measurement value and a third Time period in which selected light is focused in the focus area of the respective optical system.
- 17 is a flow chart of the essential steps of an inventive method
- Fig. 18 is a flow chart of the essential steps of a further inventive method.
- 19 is a flow chart of the essential steps of yet another method according to the invention.
- the laser scanning microscope 1 according to the invention shown in FIG. 1 is based on a STED microscope and accordingly has a first light source 2 for excitation light 3 and a second light source 4 for excitation by excitation light 3 in the form of stimulation light 6 on.
- Each of the two light sources 2 and 4 comprises a laser 7 or 8, a polarization-maintaining optical fiber 9 or 10 and a collimation optics 11 or 12 for the excitation light 3 or 10 emerging from the optical fiber 9 or 10.
- an SLM 13 for wavefront shaping is arranged in the beam path of the stimulation light 6, so that the stimulation light 6 forms a light intensity distribution with a central intensity minimum in the focus area of an objective 14.
- the stimulation light 6 is combined with the excitation light 3 behind the SLM 13 after further optics 15 with the aid of a dichroic beam splitter 16.
- the excitation light 3 and the stimulation light 6 are coupled into the objective 14 via a deformable mirror 17 as adaptive optics 18 and yet another optics 19, which focus the excitation light 3 and the stimulation light 6 together into a focus area in a sample 20.
- the optics 15 and 19 are designed such that the deformable mirror 17 and the SLM 13 are in planes that are conjugate to an entrance pupil of the objective 14.
- the position of the focus area in which the excitation light 3 and the stimulation light 6 are focused can be moved in the sample 20 with the aid of a scanner 21.
- the scanner 21 is indicated as a movable sample table.
- the scanner 21 can, however, also be designed differently, and specifically for this, the excitation light 3 and the stimulation light 6 opposite the objective 14 to shift, in particular to tilt a center of the entrance pupil of the objective 14.
- Fluorescent light 22 emitted from the focus area in the sample 20 due to the excitation of fluorescent dyes located there with the excitation light 3 is decoupled from the beam path of the excitation light 3 with a second dichroic beam splitter 23, focused with an optics 24 in a multimode optical fiber 25 and through this led to a time-resolving detector 26.
- the deformable mirror 17 is controlled as adaptive optics 18 arranged in the beam path through the objective 14 by a controller 27 to which a measurement signal 28 indicating the fluorescent light is fed from the detector 26.
- the adaptive optics 18 could also be formed by an SLM, a micromirror array or in another manner known to the person skilled in the art.
- Fig. 2 shows light intensity distributions 29 and 30 of the excitation light 3 and the stimulation light 6 along a section in the x direction through the center of the focus area.
- the intensity distribution 29 of the excitation light is that of a diffraction-limited spot.
- the spatially donut-shaped intensity distribution 30 of the stimulation light has an intensity minimum in the center 31 in the form of a zero 32, which in the section according to FIG. 2 is delimited by intensity maxima 33.
- intensity maxima 33 the intensity of the stimulation light exceeds a saturation intensity Is, which leads to the excitation of fluorescent dyes being completely returned by the excitation light 3.
- FIG. 3 shows the times at which the individual photons of the fluorescent light 22 arrive at the detector 26 according to FIG. 1 after a pulse 35 of the excitation light and a subsequent pulse 36 of the stimulation light.
- the photons of the fluorescent light 22 registered in a period 37 after the pulse 36 are then those from the environment 34 of the zero 32.
- the number of photons of the fluorescent light 22 registered in the period 37 becomes a number of photons of the fluorescent light 22, which are registered during an earlier time period 38.
- This earlier period 38 is also referred to here as the first period, while the period 37 is referred to as the second period.
- the pulses 35 and 36 fall in the first time period 38.
- the photons registered in the second time period 37 are related to the photons of the fluorescent light 22 registered in the first time period 38 by determining a measure, in particular by forming a Quotients of the photons registered in the second period 37 and the photons registered in the first period 38.
- the invention is based on the knowledge that this measure assumes an extreme when the controller 27 controls the adaptive optics 18 in such a way that aberration-induced imaging errors of the optical system of the laser scanning microscope 1 comprising the lens 14 are precisely compensated by the adaptive optics 18 be sated. Aberration-induced imaging errors of the optical system of the laser scanning microscope 1 according to FIG.
- the intensities of the excitation light 3 and the stimulation light 6 in the focal area in the sample 20 in such a way that the intensities decrease with increasing imaging errors. Due to increasing imaging errors, the excitation of fluorescent dyes in the sample 20 and thus the fluorescent light 22 also decrease with the intensity of the excitation light. However, this decrease is indistinguishable from other decreases in the intensity of the fluorescent light 22, such as, for example, a decrease caused by spatial variations in the concentration of the fluorescent dyes or a decrease caused by bleaching of the fluorescent dyes. However, if one considers the relative effect of the stimulation light 6 on the fluorescent dyes previously excited by the excitation light 3, which is indicated by the number determined from the photons registered in the two time periods 37 and 38, this is only dependent on the intensity of the stimulation light 6.
- the effect of aberration-induced imaging errors can also be explained in such a way that the saturation intensity ls is only exceeded at a greater distance from the zero 32 and correspondingly more fluorescent light from the environment with the intensity distribution 30 of the stimulation light 6 reduced overall by aberration-induced imaging errors 34 of the zero 32 remains.
- the metric 4 is the quotient of the photons of the fluorescent light 22 registered in the first time period 38 divided by the photons of the fluorescent light 22 registered in the second time period 37.
- the metric therefore increases if in the second time period 37 relatively fewer photons are registered than in the first time period 38 because the stimulation light 3 had a stronger reducing effect on the fluorescent light 22 due to an increased intensity.
- the effects of six spherical aberrations (black squares) introduced artificially in the laser scanning microscope 1 according to FIG. 4 with the adaptive optics 18 are shown. The increasing spherical aberrations lead, regardless of their sign, to increasing decreases in the metric compared to its maximum.
- the metric was created for a 2D STED image of a layer filled with 40 nm sized dyestuff-filled beads with the laser scanning microscope 1 according to FIG. 1, the photons of the fluorescent light 22 according to FIG. 3 being recorded in a time-resolved manner .
- FIG. 5 is a plot of the same metric according to the invention as in FIG. 4, with STED images of individual 40 nm large dye-filled spheres for various spherical aberrations introduced in a targeted manner with the adaptive optics 18.
- FIG. 6 shows the corresponding half-widths of the images of the individual spheres and
- FIG. 7 shows the fluorescent light intensity of the individual spheres, each plotted over the aberrations introduced by the same.
- the comparison of FIGS. 5 to 7 shows that the maximum of the metric according to the invention in FIG. 5 is reached within a significantly broader minimum of the half-width in FIG. 6 and within an even broader maximum of the intensity of the fluorescent light in FIG becomes.
- FIGS. 8 to 11 show the influence of different aberrations on the metric according to the invention according to FIGS. 4 and 5.
- the different brightnesses of the images of the structure are based on bleaching of the fluorescent dyes with which the structure is marked.
- the metric always shows its maximum with the same artificially introduced spherical aberration close to zero, and the optimization of the metric according to the invention is successful.
- the elimination of aberration-induced imaging errors according to the invention therefore takes place reliably regardless of the image brightness.
- the stimulation light 6 illustrates an alternative intensity distribution 30 of the stimulation light 6 compared to the intensity distribution 29 of the excitation light 3. Specifically, the stimulation light 6 likewise forms a diffraction-limited spot. Even then, the optimization of a quotient of early and late photons of the fluorescent light by changing the control of the adaptive optics 18 according to FIG. 1 leads to an elimination of aberration-induced aberrations. As with the intensity distribution 30 of the stimulation light 6, this also applies when the stimulation light 6 does not reach the saturation intensity Is.
- FIG. 16 illustrates various sequences of the first time period 38 in which the measurement signal 28 is integrated from the sample 20, of the later second time period 37 in which the measurement signal 28 is reintegrated, and a third time period 39 in which the reference to the Measurement signal 28 acting light 5 is focused in the focus area of the sample 20.
- the third time period 39 corresponds to the pulse 36 according to FIG. 3, in which the stimulation light 6 is directed onto the sample there.
- the period 37 directly followed the period 38, and the period 39 or the pulse 36 overlaps with the later part of the period 38.
- the sequence of the periods 37 and 38 is the same.
- the third period 39 covers the first period 38 Completely.
- the sequence of time periods 37 and 38 is again the same.
- the third time period 39 overlaps both with part of the first time period 38 and with part of the second time period 37.
- the sequence of time periods 37 and 38 is again the same.
- the third time period 39 only overlaps with the second time period 37.
- the third time period 39 is arranged between the time periods 37 and 38 without any overlap.
- the light 5 focused into the sample 20 in the third time period 39 has a stronger effect on the measurement signal integrated over the later time period 37 than on the measurement signal integrated over the time period 38 because the light 5 contains the measurement signal 28, for example in form of the fluorescent light 22 according to FIG. 1, increasingly reduced.
- the measured value obtained by integrating the measurement signal 28 in the first time period 38 would only be influenced by the light 5 no less than the second measurement value obtained by integrating the measurement signal 28 in the second time period 37 if the third time period 39 was completely before the first period 38 would be.
- the flow chart 40 of a method according to the invention shown in FIG. 17 begins with a selection 41 of the light 5 and the sample 20, so that the light 5 when acting on the sample 20 either reduces the measurement signal 28 from the sample 20 or from below to a saturation value approximates, a change in the measurement signal 28 depending on an intensity of the light 5. Then, during acquisition 42, the measurement signal from a focal area of the optical system, whose aberration-induced aberrations are to be corrected, is acquired in the sample 20 over the first period 38 and, for example, integrated in order to determine the first measured value, and over the later second period 37 in order to determine the second measured value, the light 5 being focused over the third time period 39 with the optical system into the focus area in the sample 20.
- a new measure is determined, for example in the form of a new quotient, and then, when determining 44, a difference between the new measure and an earlier measure is determined.
- a direction of an earlier change in the control of the adaptive optics 18 is maintained or changed. Steps 42 to 45 are repeated in a loop 46. During these repetitions, the focus area of the optical system in the sample 20 can be shifted in order to continuously optimally compensate aberration-induced aberrations of the optical system with the adaptive optics 18 for different positions of the focus area in the sample 20.
- the Focus area in the sample 20 are shifted in order to carry out spatial averaging.
- the time periods 37 to 39 are to be divided into corresponding partial time periods, one of which is assigned to the respective location of the focus area in the sample 20.
- FIG. 18 shows a flow chart 40 of a method modified as follows compared to the method explained with reference to FIG. 17.
- first and second light and a sample are selected in such a way that the first light, when acting on the sample 20, excites a first measurement signal from constituents of the sample with a first transition probability that depends on an intensity of the first light to a first power
- the second light when acting on the sample, excites a second measurement signal from the same components, i.e. in particular the same fluorescent dyes, of the sample with a second transition probability that depends on an intensity of the second light to a second power
- the first and the second power are different by at least one.
- the first light is then first focused with the optical system in a focus area of the optical system in the sample, the first measurement signal excited by the first light from the focus area being recorded over a first period of time 38 in order to obtain a first measurement value determine.
- the second light is focused with the optical system in the same focus area in the sample 20, the second measurement signal excited by the second light from the focus area in the sample being recorded over a second time period 37 by a second measurement value to determine.
- the determination 43 in the same way as in the method according to the invention explained with reference to FIG.
- a new measure is determined, for example in the form of a new quotient, and then in the determination 44 a difference between the new measure and an earlier measure is determined and in a change 45, the control of the adaptive optics 18 is changed in the same or a different direction than before.
- the steps repeated in loop 46 here include steps 48, 49 and 43 to 45.
- the selection 41 corresponds to the selection 41 according to FIG. 17.
- the subsequent focusing 50 of the selected light takes place with a temporal light modulation of its intensity and the simultaneous acquisition of the measurement signal 28 takes place over time dissolved.
- determining 51 a phase shift between the light modulation and a signal modulation of the measurement signal 28 is then determined as a new measure.
- determining 44 the difference between this new measure and an old measure is then determined, and the change 45 the control of the adaptive optics 18 takes place depending on this difference.
- the loop 46 here comprises steps 50, 51, 44 and 45.
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DE102019118446.0A DE102019118446A1 (de) | 2019-07-08 | 2019-07-08 | Verfahren und Mikroskop mit einer Korrekturvorrichtung zur Korrektur von aberrationsinduzierten Abbildungsfehlern |
PCT/EP2020/068501 WO2021004850A1 (de) | 2019-07-08 | 2020-07-01 | Verfahren und mikroskop mit einer korrekturvorrichtung zur korrektur von aberrationsinduzierten abbildungsfehlern |
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DE19930532C2 (de) * | 1999-06-30 | 2002-03-28 | Zeiss Carl Jena Gmbh | Anordnung zur Optimierung der Pulsform in einem Laser-Scanning-Mikroskop |
DE10227120A1 (de) * | 2002-06-15 | 2004-03-04 | Carl Zeiss Jena Gmbh | Mikroskop, insbesondere Laserscanningmikroskop mit adaptiver optischer Einrichtung |
FR2967791B1 (fr) * | 2010-11-22 | 2012-11-16 | Ecole Polytech | Procede et systeme de calibration d'un modulateur optique spatial dans un microscope optique |
GB201217171D0 (en) * | 2012-08-23 | 2012-11-07 | Isis Innovation | Stimulated emission depletion microscopy |
CN104769481B (zh) * | 2012-10-12 | 2018-12-18 | 统雷有限公司 | 紧凑、低色散以及低像差自适应光学扫描系统 |
DE102013218795A1 (de) * | 2013-09-19 | 2015-03-19 | Carl Zeiss Microscopy Gmbh | Laserscanningmikroskop und Verfahren zur Korrektur von Abbildungsfehlern insbesondere in der hochauflösenden Scanning-Mikroskopie |
DE102014002328B4 (de) * | 2014-02-12 | 2021-08-05 | Carl Zeiss Microscopy Gmbh | Multifokales Fluoreszenzrastermikroskop |
EP3290983A1 (de) * | 2016-09-05 | 2018-03-07 | Abberior Instruments GmbH | Verfahren zum justieren eines laserscanning-fluoreszenzmikroskops und laserscanning-fluoreszenzmikroskop mit einer automatischen justiervorrichtung |
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WO2021004850A1 (de) | 2021-01-14 |
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