A method for dark-field scattering microscopy imaging of nanoparticles with high signal-to-noise ratio

Abstract

The invention discloses a nanoparticle high signal-to-noise ratio dark field scattering microscopic imaging method, including: using a reference light with a fixed frequency difference with the scattered light to form beat frequency interference, and selecting the appropriate moment in the time stack to form a constructive interference with the maximum scattering contrast; building a nanoparticle dark field scattering microscope imaging system based on heterodyne interference, performing time-resolved sampling of the image by the sensor of the system to obtain a data cube with system noise, using the discrete Fourier transform analysis of the sampled data along the time superposition to extract the scattered light information at the heterodyne frequency in the frequency superposition, after Fourier filtering, traversing each spatial position, using autocorrelation to calculate the delay between the scattered signal and the reference signal, applying the delay to the phase compensation of the iso-differential interference image, and realizing space-time imaging. The present invention can greatly improve the imaging quality of nanoparticles, and has broad application prospects in many fields such as nanotechnology.

Description

Technical Field

[0001] The invention belongs to the technical field of microscope imaging, and in particular relates to a nanoparticle high signal-to-noise ratio dark field scattering microscope imaging method. Background Art

[0002] In a traditional pure dark-field scattering microscopy system, the irradiance function acquired by the detector is ,in Refers to the far-field light intensity predicted by the Rayleigh scattering model, which is proportional to ; Refers to the background noise of the signal. The scattering contrast C is defined as the ratio of the maximum intensity of the scattered light to the background noise. The contrast of the traditional dark field system is It can be seen that as the size of the scatterer decreases, the scattering contrast of the dark field system decreases in a sixth-order trend, which means that the light signal from the scatterer is easily submerged by the noise signal, greatly limiting the effective acquisition of scattered light information of tiny nanoparticles. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a nanoparticle high signal-to-noise ratio dark field scattering microscopic imaging method, comprising:

[0004] A reference light beam with a fixed frequency difference from the scattered light is used to form beat frequency interference, and the appropriate moment is selected in the time stack to form constructive interference with maximum scattering contrast;

[0005] A nanoparticle dark-field scattering microscope imaging system based on heterodyne interference is built. The sensor of the system performs time-resolved sampling on the image to obtain a data cube with system noise. The time-frequency information of the sampled data is analyzed by discrete Fourier transform superimposed along time, and the scattered light information at the heterodyne frequency in the frequency superposition is extracted. After Fourier filtering, each spatial position is traversed, and the delay between the scattered signal and the reference signal is calculated by autocorrelation. The delay is applied to the phase compensation of the isotropic interference image to achieve spatiotemporal imaging.

[0006] Preferably, the process of using a reference light beam having a fixed frequency difference with the scattered light to form beat frequency interference comprises:

[0007] Obtain the irradiance function of the system when the beat frequency can be resolved by the detector in the time domain;

[0008] The irradiance function of the system is a time-dependent quantity, and the formula is:

[0009]

[0010] in, is the electric field intensity of the incident light, is the far-field light intensity predicted by the Rayleigh scattering model, is the reference optical field, is the phase difference between the reference light and the scattered light, is the background noise of the signal, is the difference frequency of heterodyne interference, is the time variable.

[0011] Preferably, the scattering contrast decreases slowly as the size of the scatterer decreases, and is modulated by the reference light amplitude and the interference term;

[0012] The formula expression of the scattering contrast is:

[0013]

[0014] in, is the electric field intensity of the incident light, is the reference optical field, is the phase difference between the reference light and the scattered light, is the background noise of the signal, d is the scatterer particle size, Represents the wavelength of incident light.

[0015] Preferably, the process of selecting a suitable moment in the time stack to form a constructive interference with maximum scattering contrast comprises:

[0016] The flow of fringes in space is recorded by time stacking, and each spatial position of scattered light on the image plane is The formula for constructive interference with the reference light is:

[0017] .

[0018] Preferably, the process of building a nanoparticle dark-field scattering microscope imaging system based on heterodyne interference includes:

[0019] Connecting light sources, optical path components, sensors and industrial cameras according to optical design principles to ensure accurate light transmission and good matching of parameters of various optical components; wherein the optical path components include lenses and beam splitters;

[0020] Before the experiment begins, the system parameters are calibrated using a calibration instrument; the system parameters include light source intensity, wavelength stability, and collimation accuracy of each optical path.

[0021] Preferably, the process of calculating the delay between the scattered signal and the reference signal by using autocorrelation and applying the delay to the phase compensation of the isotropic interference image comprises:

[0022] The electric field of the spatiotemporal combined imaging is equivalent to introducing a digital phase shift compensation for the complex amplitude of the electric field on the image plane in space. The formula is:

[0023]

[0024] in, represents the real part of the electric field intensity on the phase plane, j represents the imaginary unit, , is the delay between the scattered signal and the reference signal;

[0025] yes The conjugate phase of:

[0026] .

[0027] Preferably, the process of spatiotemporal imaging includes:

[0028] For the heterodyne modulation module, consider the heterodyne frequency generated by the signal generator A matching relationship with the sensor sampling frame rate; wherein the heterodyne frequency is less than 1 / 4 of the sampling frame rate;

[0029] Collect data in the time domain as a data stack, the dimension of which should be [M, N, T], where [M, N] represents the spatial sampling resolution of the industrial camera, and T represents the time sampling frame number of the camera;

[0030] Fourier transform in the time domain along the time axis to extract the heterodyne frequency The frequency component at is then inverse Fourier transformed back to the time domain data stack, and the dimension of the stack is also [M, N, T];

[0031] Traverse each spatial point in the stack, and then traverse each time node at each spatial point, perform autocorrelation calculation with the reference signal and obtain the corresponding delay , the delay Corresponding to the constructive interference formed by the scattered light and the reference light;

[0032] The delay you will get Applied to phase compensation of co-variance interference images to achieve spatiotemporal imaging.

[0033] Preferably, the method further comprises an experimental verification process, wherein the experimental verification process modulates the incident light through a specific filter to control the scattering contrast and energy transfer rate, and performs an imaging test on the gold nanoparticles.

[0034] Preferably, the gold nanoparticles have a diameter of 20 nm.

[0035] Preferably, the process of performing imaging test on gold nanoparticles by modulating incident light through a specific filter to control scattering contrast and energy transfer rate includes:

[0036] The energy transfer rate is defined by Evaluate the effect of reference light energy on the system signal-to-noise ratio, where represents the average value of the system noise;

[0037] Modulating the incident light through a gradient neutral density filter first reduces the system scattering contrast Equal to 1, and then use neutral density filters of different optical densities to attenuate the incident light energy to control the scattering contrast , and the energy transfer rate Take similar measures to first make the reference light energy equal to the noise energy of pure dark field imaging without samples, and then adjust the filter settings to control the reference light energy to adjust .

[0038] Compared with the prior art, the present invention has the following advantages and technical effects:

[0039] The present invention uses reference light with a fixed frequency difference with the scattered light to form beat frequency interference, and uses the time stack to select the appropriate moment to achieve maximum scattering contrast and constructive interference. The imaging process includes time-resolved sampling, discrete Fourier transform, Fourier filtering, autocorrelation calculation delay and phase compensation to achieve spatiotemporal imaging. The experiment modulated the incident light through a specific filter to control the scattering contrast and energy transfer rate, and tested the imaging of gold nanoparticles with a diameter of 20nm. The results show that when the scattered light energy is weaker than the noise energy, the method of the present invention can effectively obtain the scattered light signal when the energy transfer rate meets the conditions, and compared with the traditional system, it has natural anti-vibration characteristics, greatly improving the imaging quality of nanoparticles, and has broad application prospects in many fields such as nanotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0041] Figure 1 A schematic diagram of a method flow of an embodiment of the present invention;

[0042] Figure 2 The transmission electron microscope (TEM) photo and atomic force microscope (AFM) height diagram of the gold nanoparticles according to the embodiment of the present invention;

[0043] Figure 3 This is a diagram of deep sub-wavelength scatterer detection when the scattered light contrast is less than 1 using a spatiotemporal joint imaging framework according to an embodiment of the present invention;

[0044] Figure 4 Schematic diagram of an example of a high signal-to-noise ratio dark-field scattering microscopic imaging framework of nanoparticles based on heterodyne interference according to an embodiment of the present invention;

[0045] Among them, 1. Sample; 2. Translation stage; 3. Objective lens; 4. First beam splitter; 5. Industrial camera; 6. Beam expander; 7. Acousto-optic modulator 1; 8. Acousto-optic modulator 2; 9. Second beam splitter; 10. Laser. DETAILED DESCRIPTION

[0046] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0047] It should be noted that the steps shown in the flowcharts of the accompanying drawings can be executed in a computer system such as a set of computer executable instructions, and that, although a logical order is shown in the flowcharts, in some cases, the steps shown or described can be executed in an order different from that shown here.

[0048] like Figure 1-4 As shown, this embodiment provides a method for dark-field scattering microscopic imaging of nanoparticles with high signal-to-noise ratio, comprising:

[0049] A reference light beam with a fixed frequency difference from the scattered light is used to form beat frequency interference, and the appropriate moment is selected in the time stack to form constructive interference with maximum scattering contrast;

[0050] A nanoparticle dark-field scattering microscope imaging system based on heterodyne interference was built. The system's sensor was used to perform time-resolved sampling of the image to obtain a data cube with system noise. The time-frequency information of the sampled data was analyzed using discrete Fourier transform superimposed along time, and the scattered light information at the heterodyne frequency in the frequency superposition was extracted. After Fourier filtering, each spatial position was traversed, and the delay between the scattered signal and the reference signal was calculated using autocorrelation. The delay was applied to the phase compensation of the isotropic interference image to achieve spatiotemporal imaging.

[0051] Furthermore, in traditional dark field technology, as the size of the scatterer decreases, the scattering contrast of the dark field system decreases to the sixth power. This phenomenon is alleviated after using interferometric illumination, and the irradiance function obtained by the detector is:

[0052]

[0053] in, is the electric field intensity of the incident light field, is the far-field light intensity predicted by the Rayleigh scattering model, is the reference optical field, is the phase difference between the reference light and the scattered light, is the background noise of the signal, is the difference frequency of heterodyne interference, is the time variable.

[0054] The first term is consistent with the pure dark field system; the second term is a known quantity, which is independent of the scatterer and can be easily filtered out; the third term is the interference term between the reference light and the scattered light, which determines the scattering contrast of the dark field interferometer system:

[0055]

[0056] in, is the incident light electric field intensity, is the reference optical field, is the phase difference between the reference light and the scattered light, is the background noise of the signal, d is the scatterer particle size, Represents the wavelength of incident light.

[0057] Comparing equations (1) and (2), it can be seen that the scattering contrast of the dark field interferometer system decreases more slowly as the scale of the scatterer decreases, and will be modulated by the reference light amplitude and the interference term. However, the electric field phase of the scattered light is very complex, so it is difficult to set a reference light with an initial phase to interfere constructively with the scattered light at the position of maximum intensity. For this problem, this embodiment uses a reference light beam with a fixed frequency difference with the scattered light to form beat frequency interference. When the beat frequency can be resolved by the detector in the time domain, the irradiance function of the system will be a time-dependent quantity:

[0058]

[0059] At this point, it is only necessary to select a suitable moment in the time stack to enable the system to form constructive interference with maximum scattering contrast. In addition, the diffraction-limited system disperses the scattered light in space, so that the energy collected by the system at the maximum position is less than the predicted value of the Rayleigh scattering model, and also amplifies the perturbation of the scatterer in space. In other words, if the diffraction spots dispersed in space are not utilized, a lot of potential signal-to-noise ratio will be lost. Here, the advantage of heterodyne interferometry is reflected. Its time stack records the flow of fringes in space, which means that at every spatial position where the scattered light is dispersed on the image plane, , always forms constructive interference with the reference light:

[0060]

[0061] The workflow of spatiotemporal imaging is as follows Figure 1 As shown. The sensor of this system performs time-resolved sampling of the image, first realizing a set of data cubes with system noise. The time-frequency information of the sampled data is analyzed by using discrete Fourier transform superimposed along time, and the scattered light information at the heterodyne frequency in the frequency superposition is extracted. After Fourier filtering, each spatial position is traversed, and the delay between the scattered signal and the reference signal is calculated using autocorrelation. , which corresponds to constructive interference. Finally, we get Applied to phase compensation of co-variance interference images to achieve spatiotemporal imaging.

[0062] From a physical point of view, the electric field of spatiotemporal combined imaging is equivalent to introducing a digital phase shift compensation for the complex amplitude of the electric field in the image plane in space:

[0063]

[0064] in, represents the real part of the electric field on the image plane, j represents the imaginary unit, , is the delay between the scattered signal and the reference signal;

[0065] Since constructive interference is satisfied at every position, the phase is a constant independent of the spatial coordinates, which means yes The conjugate phase of:

[0066]

[0067] Therefore, the core of spatiotemporal combined imaging is still interferometry, which has the ability to recover the amplitude and phase of scattered light. The difference is that compared with traditional interferometry methods, spatiotemporal combined imaging has higher robustness and scalability, such as vibration resistance, extended depth of field, and deep sub-wavelength quantitative measurement.

[0068] Furthermore, the process of building a nanoparticle dark-field scattering microscope imaging system based on heterodyne interferometry includes:

[0069] According to the optical design principle, the light source, optical path components (such as lenses, beam splitters, etc.), sensors and industrial cameras are precisely connected to ensure accurate light transmission and good matching of parameters of various optical components. Before the experiment begins, the system parameters are carefully calibrated using professional calibration instruments, covering key indicators such as light source intensity, wavelength stability, and collimation accuracy of each optical path, to ensure that the initial state of the system is stable and reliable, laying the foundation for subsequent accurate imaging.

[0070] The process of space-time imaging includes:

[0071] For the heterodyne modulation module, consider the heterodyne frequency generated by the signal generator A matching relationship with the sensor sampling frame rate; wherein the heterodyne frequency is less than 1 / 4 of the sampling frame rate;

[0072] Collect data in the time domain as a data stack, the dimension of which should be [M, N, T], where [M, N] represents the spatial sampling resolution of the industrial camera, and T represents the time sampling frame number of the camera;

[0073] Fourier transform in the time domain along the time axis to extract the heterodyne frequency The frequency component at is then inverse Fourier transformed back to the time domain data stack, and the dimension of the stack is also [M, N, T];

[0074] Traverse each spatial point in the stack, and then traverse each time node at each spatial point, perform autocorrelation calculation with the reference signal and obtain the corresponding delay , the delay Corresponding to the constructive interference formed by the scattered light and the reference light;

[0075] The delay you will get Applied to phase compensation of co-variance interference images to achieve spatiotemporal imaging.

[0076] Furthermore, in actual experiments, the non-interference term of a pure interference system can be recorded by recording the image plane irradiance function without a sample and then eliminated by image subtraction, but this requires the system to have high stability. On the other hand, the image subtraction operation potentially requires that the imaging cannot be overexposed, and the increasingly strong reference light makes the system have the risk of overexposure. Therefore, in order to further verify the sensing capability of spatiotemporal joint imaging in deep subwavelength scatterers when the scattered light signal is weaker than the background noise, the proposed imaging framework is used to experimentally measure gold nanoparticles with a diameter of 20 nm, as shown in Figure 2. Figure 2 shown.

[0077] In order to evaluate the influence of reference light energy on the system signal-to-noise ratio, the energy transfer rate is defined as ,in Represents the average value of the system noise.

[0078] The incident light is modulated using a gradient neutral density filter to reduce the scattering contrast of the system. It is approximately equal to 1, that is, the scattered light signal of nanoparticles can be barely detected in pure dark field. On this basis, neutral density filters with different optical densities are further used to attenuate the incident light energy to control the scattering contrast. . Energy transfer rate A similar method is used to control the reference light energy to be equal to the pure dark field imaging noise energy without a sample, and then the reference light energy is controlled by adjusting the setting of the neutral density filter to further adjust It should be pointed out that due to the limited digital depth of the sensor, the reference light energy can only be increased by 3 orders of magnitude at the same gain and exposure time.

[0079] Figure 3 We demonstrate the use of a spatiotemporal joint imaging framework. Figure 4 A schematic diagram of a device built based on the proposed imaging method is shown; Figure 41 represents the sample; 2 represents the translation stage; 3 represents the objective lens; 4 represents the first beam splitter; 5 represents the industrial camera; 6 represents the beam expander; 7 represents the acousto-optic modulator 1; 8 represents the acousto-optic modulator 2; 9 represents the second beam splitter; and 10 represents the laser.

[0080] Detection of deep subwavelength scatterers when the scattered light contrast is less than 1. Figure 3 All images in the image have been normalized, and the digital labels 1, 2, and 3 represent the system corresponding to the current image. For 10 0 , the letter label (a) represents the imaging result of the pure dark field system, (bd) and (eg) represent the results of the traditional dark field interferometric imaging system and the spatiotemporal joint imaging system with three orders of magnitude of reference light energy, respectively. Figure 3 Among them, (b1)(c2)(d3) and (e1)(f2)(g3) are the current system Down The imaging result is exactly 1. It can be seen that Figure 3 As shown in (ad), when the scattered light energy is less than 10 orders of magnitude less than the noise energy, neither the pure dark field system nor the traditional interferometric dark field system can effectively obtain the scattered light information. When it is less than 1, Figure 3 In (e2)(e3)(f3), no scattered light signal can be observed, but when When is greater than or equal to 1, the scattered light signal can be effectively observed, and Figure 3 As shown in (f1)(g1)(g2), The larger the value, the better the imaging SNR. In addition, carefully observe Figure 3 In (d1-d3), it can be found that there are many stripe-distributed noises in the background, which are caused by the inevitable jitter in the system platform or environment. An obvious result of vibration is the introduction of stripe distribution residuals in image subtraction, which worsens with the increase of reference light energy. For the spatiotemporal joint imaging system, even under the condition of a thousand-fold increase in reference light energy, there is no obvious stripe information in the image. This is because the vibration signal of non-target frequency in the time domain is filtered out in the time domain Fourier filtering process. This also shows that the spatiotemporal joint imaging system has a natural anti-vibration property.

[0081] The present invention uses reference light with a fixed frequency difference with the scattered light to form beat frequency interference, and uses the time stack to select the appropriate moment to achieve maximum scattering contrast and constructive interference. The imaging process includes time-resolved sampling, discrete Fourier transform, Fourier filtering, autocorrelation calculation delay and phase compensation to achieve spatiotemporal imaging. The experiment modulated the incident light through a specific filter to control the scattering contrast and energy transfer rate, and tested the imaging of gold nanoparticles with a diameter of 20nm. The results show that when the scattered light energy is weaker than the noise energy, the method of the present invention can effectively obtain the scattered light signal when the energy transfer rate meets the conditions, and compared with the traditional system, it has natural anti-vibration characteristics, greatly improving the imaging quality of nanoparticles, and has broad application prospects in many fields such as nanotechnology.

[0082] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for high signal-to-noise ratio dark-field scattering microscopic imaging of nanoparticles, characterized in that: include: A reference light beam with a fixed frequency difference from the scattered light is used to form beat frequency interference, and the appropriate moment is selected in the time stack to form constructive interference with maximum scattering contrast; A nanoparticle dark-field scattering microscope imaging system based on heterodyne interference is constructed, and a sensor of the system is used to perform time-resolved sampling of images to obtain a data cube with system noise, and a discrete Fourier transform along time superposition is used to analyze the time-frequency information of the sampled data, and the scattered light information at the heterodyne frequency in the frequency superposition is extracted, and after Fourier filtering, each spatial position is traversed, and the delay between the scattered signal and the reference signal is calculated by autocorrelation, and the delay is applied to the phase compensation of the isotropic interference image to achieve spatiotemporal imaging; The scattering contrast decreases slowly as the size of the scatterer decreases, and is modulated by the reference light amplitude and the interference term; The formula expression of the scattering contrast is: in, is the electric field intensity of the incident light field, is the reference optical field, is the phase difference between the reference light and the scattered light, is the background noise of the signal, d is the scatterer particle size, represents the wavelength of incident light; The process of constructive interference to select the appropriate moment in the time stack to achieve maximum scattering contrast involves: The flow of fringes in space is recorded by time stacking, and each spatial position of scattered light on the image plane is The formula for constructive interference with the reference light is: 。 2. The nanoparticle high signal-to-noise ratio dark field scattering microscopic imaging method according to claim 1, characterized in that: The process of using a reference beam with a fixed frequency difference from the scattered light to form beat frequency interference includes: Obtain the irradiance function of the system when the beat frequency can be resolved by the detector in the time domain; The irradiance function of the system is a time-dependent quantity, and the formula is: in, is the electric field intensity of the incident light field, is the far-field light intensity predicted by the Rayleigh scattering model, is the reference optical field, is the phase difference between the reference light and the scattered light, is the background noise of the signal, is the difference frequency of heterodyne interference, is the time variable.

3. The nanoparticle high signal-to-noise ratio dark field scattering microscopic imaging method according to claim 1, characterized in that: The process of building a nanoparticle dark-field scattering microscope imaging system based on heterodyne interferometry includes: Connecting light sources, optical path components, sensors and industrial cameras according to optical design principles to ensure accurate light transmission and good matching of parameters of various optical components; wherein the optical path components include lenses and beam splitters; Before the experiment begins, the system parameters are calibrated using a calibration instrument; the system parameters include light source intensity, wavelength stability, and collimation accuracy of each optical path.

4. The nanoparticle high signal-to-noise ratio dark field scattering microscopic imaging method according to claim 1, characterized in that: The process of calculating the delay between the scattered signal and the reference signal by using autocorrelation and applying the delay to the phase compensation of the isotropic interference image comprises: The electric field of the spatiotemporal combined imaging is equivalent to introducing a digital phase shift compensation for the complex amplitude of the electric field on the image plane in space. The formula is: in, represents the real part of the electric field on the image plane, j represents the imaginary unit, , is the delay between the scattered signal and the reference signal; yes The conjugate phase of: 。 5. The nanoparticle high signal-to-noise ratio dark field scattering microscopic imaging method according to claim 1, characterized in that: The process of space-time imaging includes: For the heterodyne modulation module, consider the heterodyne frequency generated by the signal generator A matching relationship with the sensor sampling frame rate; wherein the heterodyne frequency is less than 1 / 4 of the sampling frame rate; Collect data in the time domain as a data stack, the dimension of which should be [M, N, T], where [M, N] represents the spatial sampling resolution of the industrial camera, and T represents the time sampling frame number of the camera; Fourier transform in the time domain along the time axis to extract the heterodyne frequency The frequency component at is then inverse Fourier transformed back to the time domain data stack, and the dimension of the stack is also [M, N, T]; Traverse each spatial point in the stack, and then traverse each time node at each spatial point, perform autocorrelation calculation with the reference signal and obtain the corresponding delay , the delay Corresponding to the constructive interference formed by the scattered light and the reference light; The delay you will get Applied to phase compensation of co-variance interference images to achieve spatiotemporal imaging.

6. The nanoparticle high signal-to-noise ratio dark field scattering microscopic imaging method according to claim 1, characterized in that: The method also includes an experimental verification process, in which the incident light is modulated by a specific filter to control the scattering contrast and energy transfer rate, and an imaging test is performed on the gold nanoparticles.

7. The nanoparticle high signal-to-noise ratio dark field scattering microscopic imaging method according to claim 6, characterized in that: The diameter of the gold nanoparticles is 20 nm.

8. The method for nanoparticle high signal-to-noise ratio dark field scattering microscopic imaging according to claim 6, characterized in that: The process of performing imaging test on gold nanoparticles by modulating incident light through a specific filter to control scattering contrast and energy transfer rate includes: The energy transfer rate is defined by Evaluate the effect of reference light energy on the system signal-to-noise ratio, where represents the average value of the system noise; Modulating the incident light through a gradient neutral density filter first reduces the system scattering contrast Equal to 1, and then use neutral density filters of different optical densities to attenuate the incident light energy to control the scattering contrast , and the energy transfer rate Take similar measures to first make the reference light energy equal to the noise energy of pure dark field imaging without samples, and then adjust the filter settings to control the reference light energy to adjust .

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