Dynamic lock-in detection bandwidth for SRS imaging

Abstract

An electric circuit for a microscope includes a lock-in amplifier. The lock-in amplifier has an input for an input signal, an input for a reference signal, an output for an output signal and a bandwidth filter configured such that a low bandwidth frequency value and/or a high bandwidth frequency value is variably settable. A dynamic bandwidth controller is configured to receive at least one parameter of a current setting of the microscope as an input, and to control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier as a function of the at least one parameter of the current setting of the microscope.

Claims

1. An electric circuit for a microscope comprising: a lock-in amplifier, the lock-in amplifier comprising: an input for an input signal; an input for a reference signal; an output for an output signal; and a bandwidth filter configured such that a low bandwidth frequency value and/or a high bandwidth frequency value is variably settable, and a dynamic bandwidth controller configured to: receive at least one parameter of a current setting of the microscope as an input; and control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier as a function of the at least one parameter of the current setting of the microscope, wherein the at least one parameter of the current setting of the microscope is at least one of the following: a pixel dwell-time, a laser-scan speed, a stage-scan speed, an objective magnification, an objective numerical aperture, an image scan format or a pixel size, an image scan width or a physical image size, or a zoom value.

2. The electric circuit of claim 1, wherein the function of the at least one parameter of the current setting of the microscope is at least one of the following: inversely proportional to the pixel dwell-time, proportional to the laser-scan speed, proportional to the stage-scan speed, inversely proportional to the objective magnification, proportional to the objective numerical aperture, proportional to the image scan format or the pixel size, proportional to the image scan width or the physical image size, or inversely proportional to the zoom value.

3. The electric circuit of claim 1, wherein the dynamic bandwidth controller is adapted to control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier depending on a potential highest temporal frequency in a demodulated signal.

4. The electric circuit of claim 3, wherein the potential highest temporal frequency in the demodulated signal is given by the formula: $f_{\mathrm{High}}=\frac{F_{\mathrm{Sys}}.Math.f_{\mathrm{scan}}.Math.{\mathrm{NA}}_{\mathrm{MO}}}{1.22.Math.\mathrm{Zoom}.Math.M_{\mathrm{MO}}.Math.\mathrm{fill}}.$

5. The electric circuit of claim 1, wherein the dynamic bandwidth controller is adapted to control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier depending on at least one of the following: a wavelength of light being focused onto an object, a numerical aperture NA.sub.MO of a microscope objective, a system dependent length F.sub.Sys which depends on a magnification of the microscope optics, a magnification M.sub.MO of the microscope objective, a value Zoom representing an additional magnification of a beam path of the microscope, a duty cycle fill of the laser scan, or a scan frequency f.sub.scan.

6. The electric circuit of claim 5, wherein the dynamic bandwidth controller is adapted to control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier as a function being at least one of the following: inversely proportional to the wavelength of light being focused onto an object, proportional to the numerical aperture NA.sub.MO of a microscope objective, proportional to the system dependent length F.sub.Sys which depends on a magnification of the microscope optics, inversely proportional to the magnification M.sub.MO of the microscope objective, inversely proportional to the value Zoom representing an additional magnification of a beam path of the microscope, inversely proportional to the duty cycle fill of the laser scan, or proportional to the scan frequency f.sub.scan.

7. The electric circuit of claim 1, wherein the dynamic bandwidth controller comprises a control circuit functionality which calculates the low bandwidth frequency value and/or the high bandwidth frequency value as the function of the at least one parameter of the current setting of the microscope, wherein the calculated low bandwidth frequency value and/or the high bandwidth frequency value is set in the lock-in amplifier such that at least one image acquisition is conductable and a measurement bandwidth spectrum of an acquired signal is analysable based on the calculated and set low bandwidth frequency value and/or the high bandwidth frequency value.

8. The electric circuit of claim 1, wherein the dynamic bandwidth controller is formed as a hardware and/or software module.

9. A microscope comprising the electric circuit according to claim 1.

10. An electric circuit for a microscope comprising: a lock-in amplifier, the lock-in amplifier comprising: an input for an input signal; an input for a reference signal; an output for an output signal; and a bandwidth filter configured such that a low bandwidth frequency value and/or a high bandwidth frequency value is variably settable, and a dynamic bandwidth controller configured to: receive at least one parameter of a current setting of the microscope as an input; and control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier as a function of the at least one parameter of the current setting of the microscope, wherein the dynamic bandwidth controller is adapted to control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier depending on at least one of the following: a wavelength of light being focused onto an object, a numerical aperture NA.sub.MO of a microscope objective, a system dependent length F.sub.Sys which depends on a magnification of the microscope optics, a magnification M.sub.MO of the microscope objective, a value Zoom representing an additional magnification of a beam path of the microscope, a duty cycle fill of the laser scan, or a scan frequency f.sub.scan, and wherein the dynamic bandwidth controller is adapted to control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier as a function being at least one of the following: inversely proportional to the wavelength of light being focused onto an object, proportional to the numerical aperture NA.sub.MO of a microscope objective, proportional to the system dependent length F.sub.Sys which depends on a magnification of the microscope optics, inversely proportional to the magnification M.sub.MO of the microscope objective, inversely proportional to the value Zoom representing an additional magnification of a beam path of the microscope, inversely proportional to the duty cycle fill of the laser scan, or proportional to the scan frequency f.sub.scan.

11. An electric circuit for a microscope comprising: a lock-in amplifier, the lock-in amplifier comprising: an input for an input signal; an input for a reference signal; an output for an output signal; and a bandwidth filter configured such that a low bandwidth frequency value and/or a high bandwidth frequency value is variably settable, and a dynamic bandwidth controller configured to: receive at least one parameter of a current setting of the microscope as an input; and control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier as a function of the at least one parameter of the current setting of the microscope, wherein the dynamic bandwidth controller is adapted to control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier depending on a potential highest temporal frequency in a demodulated signal, and wherein the potential highest temporal frequency in the demodulated signal is given by the formula: $f_{\mathrm{High}}=\frac{F_{\mathrm{Sys}}.Math.f_{\mathrm{scan}}.Math.{\mathrm{NA}}_{\mathrm{MO}}}{1.22.Math.\mathrm{Zoom}.Math.M_{\mathrm{MO}}.Math.\mathrm{fill}}.$

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be described in even greater detail below based on the exemplary figures. The invention is not limited to the exemplary embodiments. All features described and/or illustrated herein can be used alone or combined in different combinations in embodiments of the invention. The features and advantages of various embodiments of the present invention will become apparent by reading the following detailed description with reference to the attached drawings which illustrate the following:

(2) FIG. 1 shows a preferred embodiment of a SRS microscope system usable for the invention.

(3) FIG. 2 shows input and output laser beam intensities in a SRS microscope system usable for the invention.

(4) FIG. 3 shows a preferred embodiment of a lock-in amplifier usable for the invention.

(5) FIG. 4 shows exemplary signals in a lock-in amplifier usable for the invention.

(6) FIG. 5 shows a preferred embodiment of a lock-in amplifier and a dynamic bandwidth controller unit according to the invention.

DETAILED DESCRIPTION

(7) In an embodiment, the present invention provides a suitable setting of the lock-in output filter bandwidth. Preferably, it should be possible to obtain a good image with respect to the signal-to-noise ratio for any given image acquisition parameters.

(8) According to an embodiment of the invention, an electric circuit for a microscope, in particular for a Coherent Raman scattering microscope (CRSM), a Coherent anti-Stokes Raman scattering (CARS) microscope, a coherent Stokes Raman scattering (CSRS) microscope, a Raman-induced Kerr-effect scattering (RIKES) microscope, a stimulated Raman scattering (SRS) microscope or a pump-probe microscope, comprising a lock-in amplifier, wherein the lock-in amplifier comprises an input for an input signal, an input for a reference signal, an output for an output signal, and a bandwidth filter device, wherein the bandwidth filter device is adapted such that a low bandwidth frequency value and/or a high bandwidth frequency value is variably settable, wherein the electric circuit comprises a dynamic bandwidth controller unit into which at least one parameter of a current setting of the microscope is input, wherein the dynamic bandwidth controller unit is adapted to control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier as a function of the at least one parameter of a current setting of the microscope, and a microscope (system), in particular one of the types mentioned, having such an electric circuit are proposed. Advantageous further developments form the subject matter of the dependent claims and of the subsequent description.

(9) An embodiment of the present invention provides the advantage that the bandwidth of the lock-in amplifier can be adapted to the current setting such that a maximal measurement bandwidth spectrum is obtained as the output signal for further signal and/or image processing.

(10) Embodiments of the invention provide a method and an apparatus for implementing e.g. stimulated Raman scattering microscopy (SRS) with an automatic adaptable lock-in detection bandwidth (or time-constant) for optimal imaging for various imaging settings. One aspect of this invention is to provide a device and a method in which the lock-in bandwidth/time-constant is automatically adapted (increased or decreased) to deliver a high quality image and/or the best possible image for a given set of image acquisition parameters. The described apparatus can be directly applied for other lock-in based imaging techniques such as OHD-RIKES [2], and pump-probe microscopy techniques [4].

(11) Advantageously the at least one parameter of a current setting of the microscope is at least one of a pixel dwell-time, a laser-scan speed, a stage-scan speed, an objective magnification, an objective numerical aperture, an image scan format or a pixel size, an image scan width or a physical image size, and a zoom value. According to a preferred embodiment, the function of the at least one parameter of a current setting of the microscope is at least one of inversely proportional to the pixel dwell-time, proportional to the laser-scan speed, proportional to the stage-scan speed, inversely proportional to the objective magnification, proportional to the objective numerical aperture, proportional to the image scan format or the pixel size, proportional to the image scan width or the physical image size, and inversely proportional to the zoom value. Using these parameters allows for easily adapting the measurement bandwidth to the current microscope setting.

(12) It is preferred that the dynamic bandwidth controller unit is adapted to control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier depending on a potential highest temporal frequency in a demodulated signal, i.e. to set the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier to a value depending on said potential highest temporal frequency. Preferably, the value is equal to said potential highest temporal frequency or differs at most 10%, 20%, 30%, 40% or 50% from said potential highest temporal frequency. Thus, a good suitable bandwidth spectrum is obtained.

(13) Advantageously the dynamic bandwidth controller unit is adapted to control the low bandwidth frequency value and/or the high bandwidth frequency value of the lock-in amplifier depending on at least one of a wavelength of light being focused onto an object, a numerical aperture NA.sub.MO of a microscope objective, a system dependent length F.sub.Sys which depends on a magnification of the microscope optics, a magnification M.sub.MO of the microscope objective, a value Zoom representing an additionalpreferably variable settablemagnification of a beam path of the microscope, a duty cycle fill of the laser scan, and a scan frequency f.sub.scan. This allow for precisely tuning the bandwidth.

(14) According to a preferred embodiment the dynamic bandwidth controller unit comprises a control circuit functionality which calculates the low bandwidth frequency value and/or the high bandwidth frequency value as the function of the at least one parameter of a current setting of the microscope, wherein the calculated low bandwidth frequency value and/or the high bandwidth frequency value is set in the lock-in amplifier and at least one image acquisition is conducted and the measurement bandwidth spectrum of the acquired signal is analysed.

(15) The dynamic bandwidth controller unit can be formed as a hardware and/or software module, in particular as an ASIC (Application Specific Integrated Circuit) or in a FPGA (Field Programmable Gated Array) or DSP (Digital Signal Processor).

(16) Further advantages and embodiments of the invention will become apparent from the description and the appended figures.

(17) It should be noted that the previously mentioned features and the features to be further described in the following are usable not only in the respectively indicated combination, but also in further combinations or taken alone, without departing from the scope of the present invention.

(18) In the following, the invention is described exemplarily on basis of a SRS microscope system as shown in FIGS. 1 to 3.

(19) For SRS imaging it is very advantageous to choose the correct bandwidth based on the bandwidth of the modulated SRS signal due to laser-scanning or stage-scanning; choosing a lock-in output filter bandwidth much larger than the signal bandwidth would lead to noisy images and on the other hand setting a narrow filter bandwidth would lead to blurred images.

(20) Firstly, the case of laser scanning system is considered. The bandwidth of the modulated signal is determined by the speed with which the illumination/excitation laser beam is scanned across the object. If w.sub.pixel is defined as the length in the sample object which is represented as a single point or pixel in the acquired image, then the transit speed of the laser beam across the pixel is given by

(21) $v_{\mathrm{transit}}=\frac{w_{\mathrm{pixel}}}{t_{\mathrm{pixel}}}$
where
t.sub.pixel is the pixel dwell-time.

(22) The pixel width w.sub.pixel is determined by the magnification of (a) the objective and (b) the relay optics in the confocal scanner and also on length across which the laser light is scanned, which is given by the Zoom parameter in a typical commercial laser scanning microscope system.

(23) On the other hand, the pixel dwell-time t.sub.pixel is determined by the frequency of the laser scan, the number of pixels in a single line and the time it takes to scan a single line. The pixel dwell-time can be represented as follows:

(24) $t_{\mathrm{pixel}}=\frac{\left(\frac{\mathrm{fill}}{f_{\mathrm{scan}}}\right)}{N_{\mathrm{pixels}}}$
where fill is the duty cycle of the laser scan (the factor of the single scan period during which the laser is on for imaging/excitation), f.sub.scan is the scan frequency, and N.sub.pixels is the number of pixels in a single line of the image.

(25) The pixel size is given by:

(26) $w_{\mathrm{pixel}}=\frac{\left(\frac{F_{\mathrm{Sys}}}{\mathrm{Zoom}.Math.M_{\mathrm{MO}}}\right)}{N_{\mathrm{pixels}}}$
where F.sub.Sys is a system dependent length which depends on the magnification of the relay optics, and the scan lens/tube lens combination, M.sub.MO is the magnification of the objective, and Zoom is a value representing an additionalpreferably variable settablemagnification of a beam path of the microscope.

(27) Based on the above formulae, the transit speed of the laser spot across a pixel is given by:

(28) $v_{\mathrm{transit}}=\frac{F_{\mathrm{Sys}}.Math.f_{\mathrm{scan}}}{\mathrm{Zoom}.Math.M_{\mathrm{MO}}.Math.\mathrm{fill}}$

(29) Since the focused spot size is determined by the Airy diameter D.sub.Airy, the largest temporal frequency that is generated due to the scanning of the laser spot across the sample can be estimated as:

(30) $f_{\mathrm{High}}=\frac{v_{\mathrm{transit}}}{D_{\mathrm{Airy}}}$
where

(31) $D_{\mathrm{Airy}}=\frac{1.22}{{\mathrm{NA}}_{\mathrm{MO}}}$
is the Airy diameter with being the wavelength of light that is being focused onto the sample and
NA.sub.MO is the numerical aperture of the objective.

(32) Hence, the potential highest temporal frequency in the demodulated signal (e.g. output of mixer 301 in FIG. 2) is given by

(33) $f_{\mathrm{High}}=\frac{F_{\mathrm{Sys}}.Math.f_{\mathrm{scan}}.Math.{\mathrm{NA}}_{\mathrm{MO}}}{1.22.Math.\mathrm{Zoom}.Math.M_{\mathrm{MO}}.Math.\mathrm{fill}}$

(34) Thus, the frequency content of the demodulated signal depends on the various system and scan parameters. If any of these parameters change, the frequency content in the signal is bound to change. Hence, the output low-pass filter should have a bandwidth that is broad enough to let f.sub.High through; it should not be too broad otherwise one would have noisy images with the noise coming from the frequency spectrum beyond f.sub.High.

(35) In a typical laser scanning experiment, one would like to change the zoom factor to zoom into a certain portion of the image to visualize a particular region more closely. This zoom could change by a factor of up to 64 in a commercial microscope system. Similarly, the scan frequency f.sub.scan, with all other parameters being constant, could vary up to a factor of 1200 in a commercial microscope system. This implies f.sub.High could change by three orders of magnitude.

(36) A representative example of the dependence of the signal bandwidth on the scan parameters is shown in FIG. 4. FIG. 4 is a screenshot of the signal in frequency domain as measured with an Agilent spectrum analyser (Model E7401A). Line 401 corresponds to the baseline noise curve with no light incident on the detector. Line 402 corresponds to the signal that is generated with the zoom parameter set to 0.75. Line 403 corresponds to the signal with the zoom parameter set to 1.0.

(37) The rest of the scan parameters for generating these three curves were: M.sub.MO=20, NA.sub.MO=0.75, f.sub.scan=600 Hz and N.sub.pixels=512.

(38) One can clearly notice the difference between lines 402 and 403; for larger zoom, the laser traverses each pixel a bit slower. Consequently, this leads to a reduction in the signal bandwidth for larger zooms. Having a fixed bandwidth low-pass filter would lead to deterioration of the image with respect to the signal to noise ratio when the zoom increases. Instead, if the bandwidth of the filter is tuneable according to the invention, then the image quality can be retained irrespective of changes in any of the scan/microscope parameters.

(39) Hence, it is proposed that an electronic circuit, or a software module or a combination of hardware/software module(s) be used which take(s) into account the changes in the scan parameters that might be initiated by a user of the microscope and modify (modifies) the output low-pass filters' bandwidth to let the highest possible signal frequencies through for further processing or display or storage.

(40) A pictorial representation of the concept of the present invention is shown in FIG. 5. In a particular embodiment of the present invention, a commercial lock-in UHFLI from Zurich Instruments AG, Zurich, Switzerland along with a software module 500 was used; the latter determines the scan parameters P, calculates the bandwidth values BW and sets this value in the lock-in amplifier unit 300.

(41) Similar analysis can be performed for a stage scanning microscope while noting that the pixel dwell time t.sub.pixel and the pixel width w.sub.pixel are given by the speed of the stage scanner and the Airy diameter of the focus respectively. But the basic idea described above is still valid and can be readily applied for this case too.

(42) While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. It will be understood that changes and modifications may be made by those of ordinary skill within the scope of the following claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above and below. Additionally, statements made herein characterizing the invention refer to an embodiment of the invention and not necessarily all embodiments.

(43) The terms used in the claims should be construed to have the broadest reasonable interpretation consistent with the foregoing description. For example, the use of the article a or the in introducing an element should not be interpreted as being exclusive of a plurality of elements. Likewise, the recitation of or should be interpreted as being inclusive, such that the recitation of A or B is not exclusive of A and B, unless it is clear from the context or the foregoing description that only one of A and B is intended. Further, the recitation of at least one of A, B and C should be interpreted as one or more of a group of elements consisting of A, B and C, and should not be interpreted as requiring at least one of each of the listed elements A, B and C, regardless of whether A, B and C are related as categories or otherwise. Moreover, the recitation of A, B and/or C or at least one of A, B or C should be interpreted as including any singular entity from the listed elements, e.g., A, any subset from the listed elements, e.g., A and B, or the entire list of elements A, B and C.

REFERENCES

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