Patent ID: 12259329

DETAILED DESCRIPTION

FIG.1shows a flow chart for a preferred embodiment of the method according to the present disclosure. Here it is presupposed that fluorescence dye molecules3in a fluorescent state22are present in isolated form in a sample2, that a single, spatially isolated fluorescence dye molecule3has been selected for the location determination and that its approximate location1in the sample2is known. For the establishment of these conditions, reference is made to the description of the present disclosure. For the location determination according to the method according to the present disclosure, in the first step a sequence8of scan positions9r1, r2, r3, . . . is determined, wherein the scan positions9are arranged around the assumed location1of the selected fluorescence dye molecule3. Subsequently, the fluorescence dye molecule3is illuminated with an intensity distribution5of excitation light6having a local intensity minimum4, wherein the intensity minimum4is initially positioned at the first scan position9ri, i=1. During illumination, the fluorescence photons emitted by the fluorescence dye molecule3as a result of the illumination are detected and the value of a sensor32indicating a disturbance is read out. After calculating the weighting factor wifrom the measured value of the sensor32, the number nPh,iof detected fluorescence photons, the scan position9and the weighting factor are stored as a tuple44of the form (nPh,i, ri, wi) in a data memory45. The illumination and detection of the fluorescence photons and the measurement of the disturbance is continued for all further scan positions9ri, i=2, 3, . . . of the sequence8. After scanning all scan positions9of the sequence8, the sequence8as a whole can be repeated to improve the signal-to-noise ratio. After data collection, the location of the fluorescence dye molecule3is determined taking into account the weighting factor associated with each data point, resulting in a greatly improved estimate of the location1of the fluorescence dye molecule3compared to the initial estimate. If the desired accuracy of the location determination is not yet achieved, the procedure can be repeated with newly determined scan positions9and possibly an increased total intensity of the excitation light6.

FIG.2shows a flow chart for a further preferred embodiment of the method according to the present disclosure. As before, it is assumed that fluorescence dye molecules3in a fluorescent state22are present in isolated form in the sample2, that a single, spatially isolated fluorescence dye molecule3has been selected for the location determination and that its approximate location1in the sample2is known. In the first step, a sequence8r1, r2, r3, . . . of scan positions is again determined, the scan positions being arranged around the assumed location1of the selected molecule. The intensity minimum4of the excitation light6is positioned at the first of the previously determined scan positions9ri, i=1. Now the value of the sensor32indicating a disturbance is read out. If the value of the disturbance exceeds a first limit value20, the data recording is paused until the value of the disturbance falls below a second limit value21, wherein the second limit value21is preferably set slightly lower than the first limit value20. Subsequently, the illumination of the fluorescence dye molecule3with excitation light6and the detection of the fluorescence photons is continued and a value pair ri) consisting of the number nPh,iof detected fluorescence photons and the scan position9riis stored in a data memory45. The illumination and detection of the fluorescence photons is continued for all further scan positions9ri, i=2, 3, . . . of the sequence8, wherein a check is made before each illumination/detection step as to whether the first limit value20of the disturbance has been exceeded and, if necessary, the data recording is interrupted until the value falls below the second limit value21again. After scanning all scan positions9of the sequence8, the sequence8can be repeated as a whole to improve the signal-to-noise ratio. After data collection, the location of the fluorescence dye molecule3is determined, resulting in a much-improved estimate of the location1of the fluorescence dye molecule3compared to the initial estimate. If the desired accuracy of the location determination is not yet achieved, the method can be repeated with newly determined scan positions9and possibly an increased total intensity of the excitation light.

FIG.3illustrates in detail the sequence of data acquisition in the embodiment of the method according to the present disclosure shown inFIG.2(simulated data). In the sample2there is a structure stained with fluorescence dye molecules3, wherein the area to be scanned comprises only a single dye molecule3in the fluorescent state22, while neighboring dye molecules3are in a dark state23. A sequence8of scan positions9is defined around the assumed location1of the fluorescent molecule3,22. First, the magnitude of the disturbance signal46is determined by means of a measuring device17not shown in this figure, and the disturbance signal46is compared with a first limit value20. At the beginning of the first scanning step #1, the disturbance signal46is below the first limit value20, whereupon the illumination of the dye molecule3with the intensity distribution5of excitation light6having a local intensity minimum4is started at the first scan position9and the fluorescence photons emitted by the fluorescence dye molecule3,22are detected for the duration of the first illumination interval47. The counting events31caused by the fluorescence photons are summed up for the first illumination interval47and assigned to the first scan position9. At the beginning of the second scanning step #2, the disturbance signal46continues to be below the first limit value20, whereupon the intensity minimum4of the intensity distribution5is positioned at the second scan position48and the fluorescence photons emitted by the fluorescence dye molecule3,22are detected for the duration of the second illumination interval47. The counting events31are summed and assigned to the second scan position48. The process is continued up to and including the fourth scanning step #4. After the end of scanning step #4, the disturbance signal46is above the first limit value20and the illumination process is temporarily interrupted. The interruption49is maintained until the time50when the disturbance signal46has fallen below the second limit value21again. Only then is the intensity minimum4of the intensity distribution5positioned at the fifth scan position51and the fluorescence photons emitted by the fluorescence dye molecule3,22are detected for the duration of the fifth illumination interval47. The accumulated counting events31are assigned to the fifth scan position51. At the beginning of the subsequent scanning steps, the disturbance signal46is still below the first limit value20, which is why the illumination and detection of the fluorescence light is continued as described. In the course of illuminating the fluorescent molecule3,22at the sixth scan position, the first limit value20of the disturbance is briefly exceeded again, which is why the illumination and detection of the fluorescence photons is briefly interrupted once more. The illumination interval can be extended by the duration of this interruption49to ensure equally long detection intervals.

FIG.4shows a possible data format42for storing the data. For each of the scan positions9of the local intensity minimum4, the data set contains an entry in the form of a tuple44comprising the x-coordinate52and the y-coordinate53of the intensity minimum4, the number10of detected fluorescence photons and an identifier43in the form of a Boolean truth value18.

Here, TRUE values indicate that the disturbances were below a threshold value during the fluorescence detection of the respective scan position, and FALSE values indicate that the disturbances were above a threshold value during at least one time point50during the fluorescence detection of the respective scan position. A z-coordinate can easily be added to the data format42(not shown) if the coordinates are available in three dimensions. This data format42is particularly suitable if the disturbances are detected with a sensor32whose output only indicates the exceeding of a limit value in digital form, but not the magnitude of the disturbance signal46in quantitative form.

FIG.5shows another possible data format42for storing the data. Deviating from the data format42shown inFIG.4, here not only a Boolean truth value18is stored, which indicates an exceeding of the limit value of the disturbance, but the disturbance is also stored as a quantitative value of the disturbance signal46. Furthermore, each tuple44is assigned a time stamp12that reflects the time of the data recording of the respective tuple44. This data format42is advantageous if the disturbance can be recorded quantitatively in real time, so that the limit value does not have to be determined a priori but can also be determined in the course of a subsequent, separate data evaluation. This also allows a comparative analysis of data with different limit setting. The data format is also suitable for determining the location of the fluorescence dye molecule3taking into account a further disturbance signal46recorded with a sensor32and stored separately, also with a time stamp.

FIG.6shows as an example and schematically the set-up of a laser scanning microscope24for carrying out the method according to the present disclosure with two fluorescence dyes emitting at different emission wavelengths. Two laser light sources54provide excitation light6of different wavelengths55and56, which is combined with a beam coupler58to form a common excitation light beam41. The excitation light beam41now passes through a beam deflection device66, here in the form of two electro-optical deflectors (EODs)59connected in series, for deflecting the excitation light beam41in the horizontal or vertical direction. After passing through the EODs59, the two excitation wavelengths55,56are once again separated by a beam splitter60, and their wave fronts are shaped by two separate phase modulation elements26, here in the form of two liquid crystal modulators61(Spatial Light Modulator, SLM), in such a way that the subsequent focusing by the microscope objective28results in an intensity distribution5of the excitation light6in the sample2, which has a local intensity minimum4. The light beams reflected from the liquid crystal modulators61are coupled into a main beam path63of the laser scanning microscope24by beam couplers62. The beam couplers62are advantageously designed as narrow band reflecting dielectric notch filters64whose reflection range overlaps as little as possible with the emission spectrum of the fluorescence dyes, so that only small portions of the fluorescence light7running in the main beam path63in the opposite direction to the excitation light6are reflected out of the main beam path63. The excitation light6is directed into the rear aperture of the objective28by a scan lens65, a scanner67shown here as an example in a quad configuration for only one scanning direction, and a tube lens68. A further light beam coupled into the main beam path63together with the excitation light for photoactivation of fluorescence dye molecules3into the fluorescent state22is not shown in the figure for simplicity. In the configuration shown, the scanner67serves to provide a comparatively slow coarse positioning of the focused excitation light6on a fluorescence dye molecule3in the fluorescent state22in the sample2, which is possible over a large image field, while the EODs59form a beam deflection device66serving to provide the rapid positioning of the intensity minimum4at several scan positions9arranged closely around the assumed location1of the dye molecule. In this case, the EODs59allow positioning at high speed, but with a positioning range limited to a few micrometers. Alternatively, the beam deflection device66could, for example, also be formed by a fiber bundle with light emission ends of individual fibers, which are positioned in the beam path in such a way that the individual light emission ends are each assigned to different scan positions9, whereby a switching device is additionally provided, which causes individual fibers of the bundle to guide light sequentially in time. Other designs of the beam deflection device66are also possible; the only decisive factor is that the beam deflection device66is configured so that adjacent scan positions9can be sequentially supplied with excitation light. Alternatively, a device integrating the scanner67and the beam deflection device66may be provided; such a device may, for example, be formed with a so-called deformable mirror. In further alternative embodiments, the function of the scanner67could, for example, be performed by a movable sample stage. The fluorescence light7received by the objective28from the sample2propagates along the main beam path63in the opposite direction to the excitation light6, being transmitted by the beam couplers62. The fluorescence light7is focused by a lens69through a confocal pinhole70, collimated by another lens69and split by a dichroic beam splitter71into two wavelength regions comprising the emission range of one and the other fluorescence dye, respectively. The fluorescence light7is separated from scattered light in each case with a band-pass filter72and detected with two detectors30. The optical components of the laser scanning microscope24are connected via a common mechanical carrier73, for example an optical mounting plate74. According to the present disclosure, a measuring device17with a sensor32for a disturbance is coupled to the mechanical carrier73, which is designed here as a vibration sensor75and serves to measure vibrations coupled to the measuring system via the common carrier73or via air movements. The laser scanning microscope24comprises a control unit76with a sequence control for executing the method shown inFIG.1or inFIG.2and functional units for controlling the laser light sources54, the scanner67and the EODs59and for processing the fluorescence light signals detected by the detectors30and storing them in a data memory45, for example in one of the data formats42shown inFIG.4orFIG.5. The control unit76may include further functions, in particular analysis and visualization functions. The functional units may be integrated or designed as separate units. For carrying out the method according to the present disclosure, the control unit76comprises a signal input77for the disturbance registered by the sensor32. The sequence control is configured in such a way that, each time a fluorescence intensity is measured, it also records the current measured value of the sensor32and stores it together with the number of fluorescence photons detected and the current position of the intensity minimum4of the intensity distribution5. If the sensor32only indicates the exceeding of a limit value in digital form, the sequence control stores an identifier indicating the exceeding of the limit value with the fluorescence intensity. Alternatively, the sequence control is configured to suspend the measurement when a first limit value20of the disturbance signal is exceeded until the disturbance signal is again below the first limit value or a second limit value21.

The laser scanning microscope24according to the present disclosure shown inFIG.7corresponds in its construction to the laser scanning microscope24shown inFIG.6, but here it is equipped with only one laser light source54for providing excitation light6of a wavelength55and only one detector30for detecting fluorescence light7. Accordingly, the beam splitters58,60,71and beam couplers62for combining or separating the excitation light6of different wavelengths55,56and for separating the fluorescence light7of different wavelengths are also omitted. Deviating fromFIG.6, the laser scanning microscope shown here is equipped with several sensors32positioned at different locations, wherein one sensor32is designed as a vibration sensor75and two further sensors32are designed as flow sensors78. The vibration sensor75is mechanically connected to the mounting plate74. The flow sensors78are positioned at particularly sensitive or particularly exposed locations of the laser scanning microscope24, for example in the vicinity of the objective28or in the vicinity of open beam paths.

The laser scanning microscope24according to the present disclosure shown inFIG.8corresponds in its construction to the laser scanning microscope24shown inFIG.7, but the measuring device17for detecting a disturbance is formed by an auxiliary light source39, for example a laser emitting in the infrared spectral range, and a light detector34in the form of a segmented detector36. Therein, the measuring light beam40emitted by the auxiliary light source39is coupled into the beam path27with the aid of two dichroic beam splitters71and coupled out again at another point, so that the measuring light beam40runs partially in the beam path27of the excitation light41. Any disturbances that are transmitted to the beam-conducting elements79of the excitation light6and lead to a change in the position of the focused excitation light6in the sample2are thus also transmitted to the measuring light beam40and can be detected by a change of the position of the measuring light beam40on the segmented detector36.

The laser scanning microscope24according to the present disclosure shown inFIG.9corresponds in its construction to the laser scanning microscope24shown inFIG.8, but here the measuring light beam40does not run partially together with the excitation light beam41, but completely separately from it. A position-sensitive detector35, for example a position-sensitive large-area analogue photodiode or a quadrant diode, is provided here as the light detector34. Disturbances which are transmitted to the elements of the laser scanning microscope24via the common mechanical carrier73also reach the measuring light beam40via the beam-conducting elements79of the measuring light beam40as well as the mounting of the auxiliary light source39and lead to a change in position of the measuring light beam40on the position-sensitive detector35. Also, the measuring light beam40, like the excitation light beam41, is influenced by acoustic disturbances or by air currents and can therefore detect them.

The laser scanning microscope24according to the present disclosure shown inFIG.10corresponds in its construction to the laser scanning microscope24shown inFIG.8, but the measuring light beam40is not provided here by an auxiliary light source39but is coupled out of the excitation beam41with a beam splitter80. The measuring device17for measuring the disturbance is designed here as a (Michelson) interferometer38with a beam splitter cube81and two mirrors57for back reflection of the two partial beams82; other interferometer types can also be used, in particular Mach-Zehnder interferometers. The arms of the interferometer38are spatially close to the beam paths of the excitation light6and the fluorescence light7, and the mirror(s)57of the interferometer38are mounted on the same mechanical carrier73as the other components of the laser scanning microscope24. Alternatively, it is also possible to mount the mirror(s)57on one of the other components of the laser scanning microscope24. Behind the exit port of the interferometer38is placed the light detector34, for example a photodiode or a photomultiplier. By its nature, the interferometer38is very sensitive to vibrations, to (turbulent) air currents and sound and translates these disturbances into brightness variations on the light detector34. To further increase the sensitivity, the interferogram can also be recorded with a spatially resolving detector, especially a camera, and the disturbance pattern can be examined for changes with image processing.

InFIG.11, three scan positions9from the sequence8of a method variant according to the present disclosure are shown, in which the fluorescence dye molecule3in the fluorescent state22is illuminated with an intensity distribution5of excitation light6having a local intensity maximum12and an intensity distribution5of fluorescence-preventing light11having a local intensity minimum4. The scan positions9are arranged on the corners of a regular hexagon as inFIG.3, but the illumination of the fluorescence dye molecule3at the first scan position13, the second scan position48and the third scan position is not performed here along an orbital direction of the hexagon, but in an arbitrary order.