US20100208339A1 - Microscope and method for operating a microscope - Google Patents

Microscope and method for operating a microscope Download PDF

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US20100208339A1
US20100208339A1 US12/679,967 US67996708A US2010208339A1 US 20100208339 A1 US20100208339 A1 US 20100208339A1 US 67996708 A US67996708 A US 67996708A US 2010208339 A1 US2010208339 A1 US 2010208339A1
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intensity
microscope
specimen
point
light
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Ingo Kleppe
Mirko Liedtke
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Carl Zeiss Microscopy GmbH
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Carl Zeiss MicroImaging GmbH
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence

Definitions

  • the present invention relates, in a first aspect, to a method for operating a microscope as defined in the pre-characterizing clause of claim 1 and to a microscope as defined in the pre-characterizing clause of claim 22 .
  • the invention relates to a method for operating a microscope as defined in the pre-characterizing clause of claim 2 and to a microscope as defined in the pre-characterizing clause of claim 23 .
  • the invention relates to a method for operating a microscope as defined in the pre-characterizing clause of claim 3 and to a microscope as defined in the pre-characterizing clause of claim 24 .
  • the invention relates to a method for operating a microscope as defined in the pre-characterizing clause of claim 4 and to a microscope as defined in the pre-characterizing clause of claim 25 .
  • excitation light is focused on, or beamed to, different points of a specimen, and an intensity of the excitation light is varied point-specifically, and an intensity of light reflected by the specimen is measured in at least one spectral range point-specifically and quantitatively.
  • a generic microscope comprises the following components: a light source for emitting excitation light for the microscopic examination of a specimen, an intensity modulator for varying an intensity of the excitation light, a microscope optics for guiding the excitation light to different points of the specimen to be examined and for guiding light reflected by said different points of the specimen to a detector, and said detector for point-specific and quantitative detection of an intensity of the light reflected by the specimen in at least one spectral range.
  • the dynamic range of present-day photodetectors is, particularly in laser-scanning microscopy, frequently insufficient for simultaneous resolution, with equal sensitivity, of very fine and dark structures on the one hand and very light image areas on the other hand.
  • fluorescence microscopy parts of the image are therefore frequently overdriven and/or other parts can no longer be distinguished from background noise.
  • the problem of insufficient dynamic range can additionally be avoided relatively easily by creating a number of images that are recorded at different degrees of illumination and then computed.
  • the disadvantage of this method is not only the significantly greater stress on the specimen but also the recording time required. This method is either not possible or at least not optimal for many applications, particularly those involving measurements performed on living cells.
  • controlled light exposure microscopy abbreviated to CLEM
  • the exposure time is regulated pixel by pixel during scanned image-recording using a rapid feed-back regulating process in which the illumination for the respective pixel is switched off and the exposure thus stopped once a predetermined threshold value has been reached in the detector.
  • Photodamage caused during measurements performed on living cells can, firstly, occur by excitation of molecules other than those of the dye, cf. Koester H. J., Baur, D. Uhl, R. and Hell, S. W. (1999), Biophys. J., 77(4): 2226-2236. Secondly, photodamage also takes place as a result of excitation of the dye itself, which decomposes to toxic products after a certain number of excitation cycles.
  • improvements can be achieved by changing or optimizing the dyes.
  • optimization can be achieved during the preparation of the object.
  • T-REX illumination is a method for achieving pulsed laser illumination, the pulse rate for the excitation being adapted to a relaxation time of triplet states of the dyes, cf.: Donnert, G., Eggeling, C. and Hell, S. W. (2007), Nat. Methods, 4(1); 81-86.
  • This object is achieved in a first aspect by the method having the features of claim 1 and by means of the microscope having the features of claim 22 .
  • the method of the type mentioned above is developed, according to the invention, in that the intensity and/or a spectral composition of the excitation light beamed to a specific point of the specimen is adjusted by a regulating device dependent on an information previously gained from measured data of the specimen concerning an estimated or actual intensity of the light reflected in the spectral range by said point such that an integral of the intensity of the light reflected in the spectral range by said point during a pixel dwell time is within a predefined value interval.
  • the microscope of the type mentioned above is further developed, according to the invention, in that a regulating device is provided which cooperates with the intensity modulator and the detector and adjusts the intensity and/or a spectral composition of the excitation light beamed to a point of the specimen on the basis of an information previously gained from measured data of the specimen concerning an estimated or actual intensity of the light reflected in the spectral range by said point such that an integral of the intensity, detected for said point by the detector, of the light reflected in the spectral range during a pixel dwell time is within a predefined value interval.
  • the object is achieved by the method having the features of claim 2 and by means of the microscope having the features of claim 23 .
  • the method of the type mentioned above is further developed, according to the invention, in that the intensity and/or a spectral composition of the excitation light beamed to a specific point of the specimen is adjusted by a regulating device on the basis of an information previously gained from measured data of the specimen concerning an estimated or actual intensity of the light reflected in the spectral region from said point such that an integral of the intensity of the light reflected in the spectral region by said point during a pixel dwell time is within a predetermined value interval, which regulating device adjusts the intensity of the excitation light for a specific point such that the integral of the intensity of the light reflected by said point during a pixel dwell time is within a value interval only when a signal criterion for this point is satisfied.
  • a regulating device which cooperates with the intensity modulator and the detector and adjusts the intensity and/or a spectral composition of the excitation light beamed to a point of the specimen on the basis of an information previously gained from measured data of the specimen concerning an estimated or actual intensity of the light reflected in the spectral range by said point such that an integral of the intensity, detected for said point by the detector, of the light reflected in the spectral range during a pixel dwell time is within a predetermined value interval, which regulating device adjusts the intensity of the excitation light for a specific point such that the integral of the intensity of the light reflected by said point during a pixel dwell time is within a value interval only when a signal criterion for this point is satisfied.
  • the method of the type mentioned above is further developed, according to the invention, in that the intensity and/or a spectral composition of the excitation light beamed to a specific point of the specimen is automatically adjusted by a regulating device on the basis of an information previously gained from measured data of the specimen concerning an estimated or actual intensity of the light reflected in the spectral range by said point such that an integral of the intensity of the light reflected in the spectral range by said point during a pixel dwell time is within a predefined value interval, that the excitation light includes a plurality of wavelengths for exciting a plurality of different dyes and that the intensity of the light reflected by the specimen is measured in a plurality of different spectral ranges.
  • the microscope of the type mentioned above is further developed, according to the invention, in that a regulating device is provided which cooperates with the intensity modulator and the detector and automatically adjusts the intensity and/or a spectral composition of the excitation light beamed to a point of the specimen on the basis of an information previously gained from measurement data of the specimen concerning an estimated or actual intensity of the light reflected in the spectral range by said point such that an integral of the intensity, detected for said point by the detector, of the light reflected in the spectral range during a pixel dwell time is within a predefined value interval, that the light source for exciting a plurality of different dyes emits excitation light having a plurality of wavelengths and that a plurality of detectors is present for quantitative and point-specific detection of the light reflected by the specimen in a plurality of spectral ranges.
  • the object mentioned above is achieved by the method having the features of claim 4 and by means of the microscope having the features of claim 25 .
  • the method of the type mentioned above is further developed, according to the invention, in that the intensity and/or a spectral composition of the excitation light beamed to a specific point of the specimen is automatically adjusted by a regulating device on the basis of an information previously gained from measured data of the specimen concerning an estimated or actual intensity of the light reflected in the spectral range by said point such that an integral of the intensity of the light reflected in the spectral range by said point during a pixel dwell time is within a predefined value interval, and that the regulating device adjusts the intensity of the excitation light beamed to a specific point on the basis of an intensity of the reflected light determined for said point in a previous image.
  • a regulating device which cooperates with the intensity modulator and the detector and automatically adjusts the intensity and/or a spectral composition of the excitation light beamed to a point of the specimen on the basis of an information previously gained from measured data of the specimen concerning an estimated or actual intensity of the light reflected in the spectral range by said point such that an integral of the intensity, detected for said point by the detector, of the light reflected in the spectral range during a pixel dwell time is within a predefined value interval, and that the regulating device adjusts the excitation light intensity beamed to a specific point on the basis of a reflected light intensity determined for said point in a previous image.
  • the central idea of the invention differs from that of the controlled light exposure microscopy method in that the intensity of the illumination of the specimen is adapted spatially to the optical properties of the specimen instead of working with substantially one and the same intensity over the entire specimen.
  • One essential finding of the invention resides, due to the nonlinear dependence of photodamaging processes on intensity, in the fact that significant improvements with respect to damage to the specimen can be achieved by specific adaptation of the excitation intensity.
  • a second essential finding of the invention is that by shifting the dynamic variation to the excitation side, a possibly insufficient dynamic depth of the detector used becomes basically of no importance and can be ignored. Therefore, detectors having particularly good signal to noise ratios can be specifically selected.
  • the present invention therefore provides an imaging method which provides considerable improvements in the dynamic depth of the images achieved and additionally significant reduction of photodamaging and photobleaching processes, which constitute limiting factors when examining living specimens.
  • excitation light described herein is to be understood to mean any kind of electromagnetic radiation used for microscopy. This radiation can, but need not, lie in the visible range.
  • the light source used is basically any type of radiation source for electromagnetic radiation in the desired spectral range. It is preferred to use suitable lasers for this purpose.
  • the light reflected by the specimen can basically be any type of electromagnetic response of the specimen to previous excitation by the excitation radiation.
  • different contrast-enhancing principles can be implemented.
  • these can include reflected or scattered radiation.
  • these principles can include fluorescent radiation, dual photon fluorescence or Raman scattering, for example radiation from a CARS process.
  • point refers to a focal volume in the order of magnitude achievable using typical microscope optics.
  • the dye molecules present in the focal volume in question are excited by the excitation light and they emit the typical fluorescence photons following relaxation.
  • the detectors used can be any of those capable of effecting detection in the respective spectral ranges.
  • photo multipliers are used due to their extremely good signal to noise ratios.
  • These may be multiple detectors of the multi-channel plate type.
  • other spatial detectors such as CCD or other semiconductor arrays can be used.
  • the value interval relating to the integral of the intensity of the light reflected by a point during the pixel dwell time is set such that a detector used is able to operate with best possible sensitivity and at a best possible signal to noise ratio.
  • the shift to the excitation side removes the restriction to a reduced dynamic depth of the detector used, and the said interval between values can be carefully adjusted such that the detector is able to operate in an advantageous range.
  • the said value intervals can have a width adapted to a usable dynamic depth of the detector.
  • the regulating process is carried out such that the integral of the intensity of the light reflected by a point during a pixel dwell time is constant, this being particularly true for all points satisfying a signal criterion.
  • Back-computation, for example, of a dye concentration of interest in the focal volume observed is then comparatively simple.
  • the regulating device can adjust the intensity of the excitation light beamed to a specific point for all points such that the intensity integral of the reflected light is within the specified value interval.
  • a variant of the method is particularly preferred in which the regulating device adjusts the intensity of the excitation light for a specific point such that the integral of the intensity of the light reflected by said point during a pixel dwell time is within a predetermined value interval only if a signal criterion for this point is satisfied. It is thus possible to prevent the intensity of the excitation light from being regulated to an extremely high value at points in which, for example, the intensity of the reflected light is very low, and thus from triggering photodamaging processes in adjacent specimen areas, for example.
  • the same pixel dwell time is used for all points. Evaluation, for example, with respect to a dye concentration of interest is again comparatively easy.
  • different pixel dwell times can be used also for different points. It may be advantageous, for example, to use the same pixel dwell time only for all points satisfying a signal criterion. In particular, the pixel dwell time can be reduced for points that do not satisfy the signal criterion. The image-recording time for an image or a scan is thus likewise reduced and the image-recording rate can be increased.
  • the signal criterion can be fed in externally as a point-specific signal.
  • the signal criterion for a specific point is satisfied when an externally supplied point-specific signal has a predefined value.
  • measured information of the point in question is used as the signal criterion.
  • the signal criterion for a specific point is satisfied when the estimated or actual intensity of the light reflected by said point is above a specifiable background threshold.
  • the excitation intensities are adjusted upwardly only in the case of points in which actually significant intensities are reflected, for example, from the dyes present at said points. Photodamage to the specimen can be further reduced in this way.
  • Additional variants of the method of the invention can be distinguished basically in terms of the point in time at which, or the time interval within which, the illumination regulation is carried out.
  • this regulating process can be carried out during the exposure of an individual pixel, that is to say, within a pixel dwell time.
  • a regulating process is possible during a scan process.
  • the required information concerning the estimated reflected light can be gained from measurements on directly or indirectly adjacent points. This is explained in more detail below.
  • the regulating process in question can be carried out between individual images when recording time series, wherein information concerning the specimen already acquired from the previously recorded images is used, in the simplest case in the manner of a negative image.
  • a test pattern or test scan can be recorded.
  • This provides the information required by the invention concerning an estimated intensity of the light reflected by a point in question.
  • Such a test scan or test pattern can be dispensed with if the information concerning the estimated intensity of the light reflected by a point is provided by an initial measurement of the intensity of the light reflected by said point.
  • the intensity of the reflected light is measured at the start of a pixel dwell time.
  • the prerequisite involved is that the regulation process must work sufficiently rapidly and be able to adjust to the correct intensity within a pixel dwell time.
  • the intensity of the excitation light beamed to this point during the dwell time of the excitation beam on this point is monitored accordingly.
  • the illumination for each pixel is regulated dynamically by a fast feedback on the basis of the information already acquired, that is to say, measured data of the specimen.
  • the image is thus constructed not only with the aid of the detected intensity, as is common practice, but also with the aid of the illuminating power used and the exposure time or a combination thereof.
  • the differences in the illumination between conventional imaging, the CLEM method, and the invention presented here, also referred to as the DIM method, are described in detail below.
  • the measured intensity in the detector is proportional to the product of the dye concentration and the illuminating power and, in the case of dual photon excitation, to the square of the illuminating power.
  • the illuminating power is constant in terms of space and time for all pixels in the image:
  • l is the detected fluorescence
  • x is the position vector
  • t is the time
  • p is the beamed illuminating power.
  • the integration limit is varied spatially, but the illuminating power as such is kept spatially constant.
  • the integration limit can be kept constant.
  • the illuminating power is arbitrarily varied in terms of space and time.
  • the DIM method can thus be considered as a further development of the CLEM method, which provides considerable advantages.
  • the functions for illumination described with reference to the patent cited above are step functions, as shown in equation (2), since the illumination is set from a constant to zero when a threshold level is reached.
  • the DIM method of the invention considers arbitrary illumination functions within an exposure time, particularly a fixed exposure time.
  • the special advantage achieved over the CLEM method resides, firstly, in the substantially greater reduction in photobleaching and photodamage due to the dependence of photodamage involving an exponential factor of a that is significantly greater than 1, cf. Hopt A. and Neher E., Biophys., J. 80(4): 2029-2036, Dixit and Cyr, The Plant Journal (2003) 36, 280-290.
  • halving the laser power reduces photodamage by more than a factor of 5 when the factor ⁇ is about 2.5, as determined in a series of experiments for dual photon excitation.
  • the reduction of the dynamic range in a shift to the excitation side in dual photon microscopy is considerably larger than in the CLEM method, in which the constant laser power causes the dynamic range of about 5 orders of magnitude to be imaged 1:1 during the exposure time.
  • the dynamic range is reduced on the excitation side due to nonlinearity problems involving the square root. That is to say, only three orders of magnitude have to be encompassed on the excitation side in order to control six orders of magnitude in the fluorescence signal.
  • the constant laser power used for the entire image must be optimized for the darkest portions, which results in the light portions of the image experiencing extremely short exposure times at a high illumination intensity. However, this results in significantly greater photodamage.
  • the regulating device can be formed by a real-time computer, for example. This solution permits a high degree of variability. When a particularly rapid regulating process is required, for example when the regulating process is required to be carried out within a pixel dwell time, it may be advantageous if an analog control circuit forms the regulating device. Interim solutions are possible in which part of the regulating process is carried out by the computer and other functions are provided by special analog circuits.
  • information concerning the estimated intensity of the light reflected by a point is extracted from a previous measurement, particularly one carried out in the same scan process, of the intensity of the light reflected by an adjacent point.
  • the regulating device adjusts the intensity of the excitation light beamed to a specific point on the basis of the intensity of the reflected light determined for this point in a previous image.
  • This is particularly advantageous when time sequences are in any case tracked and accordingly a plurality of images is recorded sequentially.
  • the illumination is in this case regulated accordingly between two exposures times in a time series of images. Information is extracted from previous image recordings, and an illumination profile containing the necessary information concerning the optical properties of the specimen is prepared.
  • the specimen is. in particular preferably subjected to pulsed illumination with the pulse rate of the excitation light being adjusted to a relaxation time of triplet-states of the dyes with which the specimen has been prepared.
  • the invention can be used in scanning microscopes, in particular. In order to achieve specific image rates, these microscopes operate with relatively high intensities since only little time is available for each point. Accordingly, the advantages gained by the invention are achieved particularly well in connection with point-scanning microscopes and also with line-scanning microscopes.
  • the method of the invention can be used equally advantageously when the microscope is a wide field microscope, since it is here again possible to achieve images having a higher dynamic depth while employing lower illuminating power, and thus causing reduced less specimen damage.
  • the invention can be applied to fluorescence microscopy to particular advantage.
  • Such microscopes are used, in particular, in the field of the biosciences and the problem of photodamaging processes is of particular relevance in this case.
  • the invention enables higher observation times to be achieved in “live cell imaging”, that is, the observation of living cells. This gives rise to completely new research possibilities.
  • the invention can also be applied to total internal reflection fluorescence microscopy.
  • the intensity modulator can have an AOTF, AOM, a Pockels cell, a Faraday cell and/or a Kerr cell.
  • AOTFs or AOMs are preferably used for applications requiring high speeds.
  • the illumination within the line can be regulated by means of a spatial light modulator.
  • the illumination profile can be readjusted for each line when scanning an entire image.
  • the light source itself prefferably be adjustable in terms of intensity and/or for the light modulator to be an integral part of the light source.
  • a spatial light modulator can be used for optimization of the illumination on the specimen, which can be effected in the simplest case in the manner of a negative image.
  • those exemplary embodiments are preferred in which the spatial light modulator is disposed away from a detection beam path.
  • the method of the invention and the microscope of the invention can be used to particular advantage when the specimen is illuminated by light of substantially one wavelength and the reflected light is detected substantially in one wavelength range, which may be very narrow, if desired.
  • Another large field of application open to the present invention is in the field of multicolor fluorescence microscopy.
  • the excitation light has several wavelengths for exciting a plurality of different dyes, and the intensity of the light reflected by the specimen is measured in a plurality of spectral ranges.
  • the spectral range can be of a variable widths.
  • the different spectral ranges can be juxtaposed or can overlap or be spaced by a specific range in which no measurements are carried out.
  • the intensities measured for the different spectral ranges are separated into components pertaining to the respective dyes, and this separation is based on known information concerning the emission spectra of the different dyes and takes into account the position and width of the spectral ranges, and a weighting factor is then determined for each of the different dyes from at least one dye-intensity component pertaining to a spectral range.
  • the optimum channel position of the detection channels for subsequent spectral unmixing depends not only on the spectra of the fluorophores but also on their concentration distribution or the contribution of the individual fluorophores or dyes to the signal in the different detection channels. This, in turn, is influenced by the beamed excitation intensity of different wavelengths.
  • the prior art constitutes the optimization of excitation and detection for the entire image on the basis of the spectral properties of the dyes. This has been described, for example, in DE 102 22 359 B4. But since the contribution of individual dyes to the brightness in the different channels fluctuates strongly due to the concentration distributions between the individual pixels in many applications, the adjustment of the detection channels is not optimal for most pixels. This results in a larger signal to noise ratio of the individual pixels of the spectrally unmixed images.
  • the application of the method of the invention to multicolor fluorescence microscopy substantially consists in carrying out a pixel-accurate mixing of the intensities of different excitation wavelengths in order to optimize the contribution of the individual dyes to the detected fluorescence and thus achieve a predetermined signal to noise ratio in the unmixed images.
  • the regulating process is carried out, depending on the system, by rapidly switching the intensities in the different wavelengths.
  • the intensity modulators described above such as those of electro-optical or acousto-optical types, particularly Pockels cells or AOMs or AOTFs, or optionally a plurality of spatial light modulators can be used.
  • Readjustment can again be carried out during a pixel dwell time or between two images using a so called “pre-scan”, in the manner described above. Readjustment is thus carried out, as in the method described above, but the algorithm for readjustment is more complicated since the intensity regulation of a wavelength does not exclusively influence the contribution of a single dye but possibly the contributions of several dyes to different extents.
  • the intensity and/or spectral composition of the excitation light beamed to a specific point of the specimen is preferably adjusted automatically by means of the regulating device in response to information previously gained from measured data of the specimen concerning estimated or actual intensities of the light reflected by said point in the different spectral regions such that an integral of the intensities of the light reflected by said point in the different spectral ranges during the pixel dwell time are within value intervals individually specifiable for the different spectral ranges.
  • the weighting factors are variables that are easy to view and manipulate, it is particularly advantageous with respect to evaluation when the value intervals for the different spectral ranges are determined such that the weighting factors for each dye are within a value interval or have a predefined value, which value intervals or values in question for the different dyes are determined individually in each case.
  • the value intervals or values for the weighting factors for the individual dyes should not be mistaken for the value intervals for the intensity integral in the different spectral ranges.
  • these variables are generally interdependent so that the values for the intensity integral in the individual spectral ranges are basically determined, too, with the determination of the weighting factors, that is to say, the intensity contributions of the individual dyes. These values are accordingly interchangeable with respect to the regulating process.
  • the light source emits excitation light of a plurality of wavelengths for exciting a plurality of different dyes. Furthermore, a plurality of detectors is available for multicolor fluorescence microscopy for quantitative and point-specific detection of the light reflected by the specimen in a plurality of spectral ranges.
  • FIG. 1 is a diagrammatic representation of a microscope of the invention
  • FIG. 2 is a diagrammatic representation of a line scanner
  • FIG. 3 is a diagrammatic representation of a wide field microscope
  • FIG. 4 is a diagrammatic representation of a control circuit comprising a real-time computer
  • FIG. 5 is a diagrammatic representation showing a combined electronic modulator and detector circuit
  • FIG. 6 is a diagrammatic representation of a regulating device consisting of an analog switching circuit
  • FIG. 7 is a flowchart comprising parts of the sequences of the method of the invention.
  • FIG. 8 is a diagrammatic representation of the beam path in a TIR microscope.
  • the microscope 100 of the invention shown diagrammatically in FIG. 1 is a point-scanning microscope. Its essential components include a light source 10 such as a laser, an intensity modulator 20 , a scanning device 70 , a microscope optics 30 , a detector 50 and a regulating device 60 .
  • the light source 10 emits excitation light 22 for the microscopic examination of a specimen 40 .
  • An intensity of the excitation light 22 is selectively adjusted, according to the invention, with the aid of the intensity modulator 20 .
  • the excitation light 22 reaches a point 41 of the specimen by way of the scanning device 70 , a main beam splitter 80 and the diagrammatically illustrated microscope optics 30 .
  • the excitation beam of the excitation light 22 can be rastered or scanned with the aid of the scanning device 70 , the intensity being adjusted in each case point-specifically, according to the invention, as described below in more detail.
  • a focal volume surrounding the point 41 is excited by the excitation light 22 .
  • dye molecules present in such surroundings can be raised to an electronically excited state. Consequently, the focal volume radiates electromagnetic radiation 42 such as fluorescent light.
  • This light 42 radiated from point 41 passes through the microscope optics 30 , the main beam splitter 80 and other optical components not shown in detail to reach a detector 50 in which the intensity of the reflected light 42 is measured quantitatively.
  • the measured data provided by the detector 50 are fed to the regulating device 60 .
  • the regulating device 60 adjusts the intensity of the excitation light 22 , according to the invention, with the aid of the intensity modulator 20 in such a way that the intensity integral of the reflected light is constant during a pixel dwell time.
  • a point scanner offers the possibility of providing pointwise illumination.
  • the diameter of the beam can, while passing through the intensity modulator 20 , be reduced using techniques known per se.
  • the logic unit that controls the detector can be an FPGA, for example, evaluates the counting rate and determines whether the light intensity reaching the specimen is too high or too low. Limiting values can be defined for this purpose.
  • the logic unit can generate a real-time transmission to an appropriate logic unit regulating the illumination, for example, a control unit of an AOTF.
  • the process of controlling the illumination appropriately adjusts the intensity of illumination with the least possible delay.
  • a higher-level system is informed of the manipulation of the illumination so that the detector values recorded thereinafter can be recalculated. This is described below with reference to FIG. 7 .
  • Step S 10 involves a “system set-up”, wherein, when observed as modules, the behavior of the specimen in relation to optical excitation with a laser is detected and evaluated in Step 11 .
  • Step 12 a working range is then located and described in detail.
  • Steps S 20 to S 25 contain details of the regulating process.
  • Step S 21 an online examination of the excitation takes place in Step S 21 .
  • Step 22 then involves a query as to whether a counting rate can be measured. If so, the photons entering the detector are counted in Step 23 .
  • Step S 24 the regulating device changes the settings of the intensity modulator 20 in Step S 24 .
  • Step S 25 the process is repeated beginning with Step S 21 .
  • the procedure for a pixel is finalized in Steps S 30 and S 31 , a photoncounting rate being computed in S 31 on the basis of the settings of the intensity modulator 20 .
  • the specimen is subjected to point-specific non-linear bleaching effects.
  • the latter can be computed point-specifically since the type of influence is known.
  • FIG. 2 diagrammatically shows a line scanner.
  • excitation light 22 from a light source 10 is focused via a slit diaphragm (not illustrated) and a lens 23 to form a line on the scanner 72 .
  • the lens 24 which is also referred to as a scanning objective
  • the excitation light 22 is scanned via a spatial light modulator 25 .
  • a tube lens 26 then focuses the beam onto the main beam splitter 80 which is a dichroitic beam splitter. This reflects the excitation light, which is imaged through the objective 32 as a line on the specimen 40 .
  • Fluorescent light 42 emitted from a point 41 of the specimen 40 is in turn imaged by the objective 32 and then with the aid of a tube lens 34 onto a point 51 of a spatially resolving detector 50 . Due to a wavelength shift of the fluorescent light, the latter can pass through the main beam splitter 80 .
  • the spatially resolving detector 50 is read out through a virtual aperture. Thus, for example, only one detector element in the region of point 51 is read out in order to provide confocality.
  • the specimen is positioned in the right-side focal plane of the objective 32 and a center of the main beam splitter 80 is positioned in a left-side focal plane of the objective 32 .
  • the paths of the excitation beam and detection beam are separate.
  • the detrimental optical properties such as high light losses in the spatial light modulator therefore do not have any adverse effect on the detection side. Scanning of the two-dimensional spatial light modulator allows for only relatively low switching speeds so that readjustments can be carried out in general only from image to image, but not within a line in most cases.
  • the least possible number of optical components in the detection beam path provides maximum sensitivity and thus leads to reduced photodamage in the specimen.
  • Line scanners are advantageous, for a given frame rate, over point scanners since the pixel dwell time is longer and the intensity of the excitation radiation can thus be lower. As a result, photodamaging processes are diminished.
  • the use of the spatial light modulator additionally allows for a combination of this method with other techniques for structured illumination, for example, for resolution enhancement.
  • the spatial light modulator 25 is readjusted for each image in a feedback loop comprising a camera, for example, and using a real-time computer.
  • a detector 50 with two-dimensional spatial resolution is used.
  • the conditions in the detection beam path largely correspond to the line scanner described with reference to FIG. 2 .
  • the arrangement in the excitation beam path in which a spatial light modulator 25 is likewise used, is considerably simpler, since the entire specimen area is illuminated.
  • the wide field arrangement allows for minimum excitation intensity per pixel, since the exposure time can be appropriately longer in this case.
  • the spatial light modulator in FIG. 3 is again not situated in the detection beam path so that the properties of the spatial light modulator that are detrimental in this respect become irrelevant.
  • the use of the spatial light modulator in a wide field allows for readjustment between the images and for a combination of this method with other methods for structured illumination in order to carry out high-resolution microscopy with dynamic imaging.
  • FIG. 4 shows an arrangement based on an existing modular laser-scanning microscope in which an existing data-processing path is used in order to acquire the necessary control parameters and to transfer the same to the final controlling element.
  • Significant components of the regulating system shown in FIG. 4 include an intensity modulator 20 disposed in the beam path 12 and comprising an electronic modulator circuit 28 and a detector 50 having an electronic detector circuit 58 .
  • these components operatively interact with a controller 92 that can, in particular, be a real-time computer.
  • the electronic modulator circuit 28 is controlled on the basis of the data acquired in the detector 50 and transferred through the data-processing path 95 to the controller 92 .
  • FIG. 5 shows a combined electronic modulator and detector circuit 62 , indicated by a double arrow 97 , that operatively interacts with the controller 92 .
  • a special analog electronic system 64 that performs the regulating process can be developed as shown diagrammatically in FIG. 6 .
  • the regulating parameters are then adjusted externally, for example, by the controller 92 .
  • the reaction time of the regulating process then depends only on the final controlling elements and detectors used.
  • FIG. 8 shows the main components of the excitation beam path in such a microscope in which the intensity of the excitation light is modulated spatially, according to the invention.
  • the excitation light 22 is focused, as a laser beam that has been expanded in a manner known per se or a beam of another light source, onto the conjugate, image-side focal plane 27 by way of a lens 23 that, like the beam of excitation light 22 , is slightly offset perpendicularly to an optical axis 29 of an objective 33 so that, following reflection by a spatial light modulator 25 and imaging through a lens 26 , the excitation light 22 is focused onto a point in the focal plane 31 that is situated at a maximum distance from the optical axis 29 . This enables the excitation light 22 to leave the objective 33 at a small angle of emergence, thus causing a small penetration depth of the evanescent waves across the total reflection.
  • a specimen is then positioned in the region of the object-side focal plane 45 of the objective 33 .
  • the spatial light modulator 25 is formed in the intermediate image formed by the objective 33 and the lens 26 so that the inventive spatial modulation of the excitation light 22 with the aid of the spatial light modulator 25 is imaged in or on the specimen 40 .
  • the same advantages can be achieved with this arrangement as are obtained with the wide field arrangement described with reference to FIG. 3 .
  • the present invention relates to a novel microscope and a novel method in which the intensity of illumination is specifically adjusted in a spatially differentiated manner to suit specifically the optical properties of the object or the specimen for the purpose of imaging an object.
  • This results in advantages with regard to the extension of the dynamic range and the reduction of photodamage in the cells being examined and additionally to reduction of bleaching of the dyes used.
  • the arrangement proposed by the invention requires at least one light source, an intensity modulator, which can be an amplitude modulator and/or a polarization modulator, a detector and feedback regulation from the detector to the intensity modulator.
  • a guide value such as an upper barrier must then be established with regard to the intensity to be achieved.
  • the regulating process of the invention is effective only on the radiation actually coming from the specimen to be examined.
  • the intensity to be achieved is an upper barrier that the illumination regulating process attempts to reach but need not exceed.
  • the background criterion that is to say, the decision as to whether a pixel pertains to the background or is a signal, can be established by external means and need not be determined from measured light ensuing, for example, from previously measured data.

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  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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US20140029091A1 (en) 2014-01-30
DE102008034137A1 (de) 2009-04-02
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EP2156235B1 (de) 2017-08-30
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US9389403B2 (en) 2016-07-12

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