CN112505908B - Pinhole-free scanning type confocal microscope based on heterodyne detection system - Google Patents

Pinhole-free scanning type confocal microscope based on heterodyne detection system Download PDF

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CN112505908B
CN112505908B CN202011277508.8A CN202011277508A CN112505908B CN 112505908 B CN112505908 B CN 112505908B CN 202011277508 A CN202011277508 A CN 202011277508A CN 112505908 B CN112505908 B CN 112505908B
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董洪舟
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Abstract

The invention discloses a pinhole-free scanning type confocal microscope based on a heterodyne detection system, belongs to the field of microscopic imaging, and particularly relates to the field of laser confocal microscopic imaging. The invention aims to overcome the defects of the background technology, and theoretical analysis indicates that in the heterodyne detection system confocal microscopy, the resolution of the system is the same as that of a direct detection system confocal microscopy under the condition of not needing a conjugate pinhole and a focusing lens in front of the pinhole. Based on the theory, a conjugate pinhole and a focusing lens in front of the pinhole are omitted, so that the limitation of the pinhole on the confocal microscopy in the prior art is eliminated, and the development of the confocal microscopy is further promoted.

Description

Pinhole-free scanning type confocal microscope based on heterodyne detection system
Technical Field
The invention belongs to the field of microscopic imaging, in particular to the field of laser confocal microscopic imaging.
Background
In the fields of biomedicine and materials science, confocal microscopy has been widely used for three-dimensional structure measurement of microscopic samples. The confocal microscopy technology at present basically adopts a direct detection system, which is roughly divided into an infinite working distance system and a finite working distance system, as shown in fig. 1 and 2. The direct detection system confocal microscopy has the following two problems which are difficult to overcome:
first, the detection sensitivity is low
As is well known, compared with the heterodyne detection system, the direct detection system has a very low sensitivity and no advantage in detecting weak signals. There are documents: "I Renhorn, O Steinvall. Performance student of a coherent laser radar [ A)]SPIE,1983,415:39-50 ", discloses a minimum detectable power of 10 for heterodyne detection- 13W order of magnitude, and the minimum detectable power for direct detection is 10-8The W magnitude, that is to say, the sensitivity of the direct detection system is 5 magnitudes lower than that of the heterodyne detection system. There are also analyses which consider, for example, the literature "Amyuying, great morning east, von Ji, photodetection and signal processing [ M]Beijing, science publishers, 2010 ", if direct detection is used as a reference, the conversion gain of heterodyne detection can be increased by 10 when the signal light power is weak7～108Magnitude. Therefore, the confocal microscopy of the current direct detection system has no advantage on the detection of weak signals. Especially when the super-resolution imaging is realized by adding the pupil filtering technology to the optical path (as shown in fig. 1 and fig. 2), the signal energy has an exponential decreasing trend with the increase of the resolution, as shown in the document "Tasso r].Rochester,University of Rochester,1997”，In which case the sensitivity of the detection system is further reduced. Therefore, in the direct detection system confocal microscopy technology, a good super-resolution effect is difficult to obtain.
Second, practical difficulties caused by conjugate pinholes
To achieve axial tomographic capabilities, direct detection confocal microscopy must place a conjugate pinhole in front of the detector, as shown in figures 1 and 2. However, conjugate pinholes of micrometer-scale width cause some practical difficulties as follows. First, when aligned with the laser convergence point, the pinhole position will suffer aberration even if there is a submicron deviation, resulting in a decrease in resolution, as described in the literature "Wilson T, carini A.R," Effect of detector displacement in capacitive imaging systems, "appl.opt.27 (18), 3791-.
Shigeharu K., Wilson T, "Effect of axial lipid displacement in capacitive microscopes," appl.Opt.32(13), 2257-; therefore, strict requirements on the accuracy of system installation and debugging and the capability of resisting vibration interference are required; secondly, in practical applications, even if tiny dust falls on the pinhole, aberration is introduced and the signal light intensity is weakened, and even imaging failure is caused. The dust erasure requires disassembling the corresponding module of the confocal microscope system and then repeating the high-precision alignment work, which increases the difficulty of application of the confocal microscope technology. Finally, the larger the size of the pinhole, the more the received signal light power, the lower the resolution, such as the documents "Huayong Ge, Qiushi Ren, bao hua Wang and wangnong Li," fluorescence of pinhole size and fiber-optical imaging system, "spie.5633: 506-. In conclusion, the conjugate pinhole makes great practical application difficulty for practical application.
The above is the main difficulty for restricting the further development of the current confocal microscopy technology. In a research publication published in 2020, e.g., the publications "Hongzhou Dong, Mingwu Ao, Xianming Yang, Yong Liu, Chunping Yang, Superresolution technology based on a hectodene detection system, Applied Optics,59(10), 3132-; the applicant of the present patent has proposed a new confocal microscopy imaging system for improving the sensitivity of the detection signal. Different from the existing direct detection system, the system adopts a heterodyne detection system, and the power of an intermediate frequency signal is used as detection measurement to realize three-dimensional imaging. However, in this system a pinhole and a focusing lens in front of the pinhole are still provided in front of the detector, as shown in fig. 3. Therefore, the system still cannot get rid of the above difficulties of conjugate pinhole in practical application.
Disclosure of Invention
The invention aims to overcome the defects of the background technology, and theoretical analysis indicates that in the heterodyne detection system confocal microscopy, the resolution of the system is the same as that of a direct detection system confocal microscopy under the condition of not needing a conjugate pinhole and a focusing lens in front of the pinhole. Based on the theory, a conjugate pinhole and a focusing lens in front of the pinhole are omitted, so that the limitation of the pinhole on the confocal microscopy in the prior art is eliminated, and the development of the confocal microscopy is further promoted.
The technical scheme of the invention comprises a pinhole-free scanning confocal microscope based on a heterodyne detection system, and the device comprises: the device comprises a laser (1), an optical fiber beam splitter (2), an acousto-optic frequency shifter A (3), an acousto-optic frequency shifter B (4), a collimator (5), a photoelectric detector (6), a beam combining plate A (7), a pupil filter (8), a collimating lens (9), a beam combining plate B (10), a focusing lens (11), an imaging sample (12) and a three-axis high-precision moving platform (13); the laser is characterized in that the frequency f laser emitted by the laser (1) is divided into two beams of laser by the optical fiber beam splitter (2), wherein one beam is subjected to frequency shift by the acousto-optic frequency shifter B (4) and then the frequency is changed into f + f1The light after frequency shift is converted into parallel light as signal light through a collimating lens (9), and then sequentially passes through a beam combining sheet B (10) and a focusing lens (11) and is focused on an imaging sample (12); reflected light of the imaging sample (12) sequentially passes through a focusing lens (11), is reflected by a beam combining sheet B (10), passes through a pupil filter (8), passes through a beam combining sheet A (7) and reaches a photoelectric detector (6); the light with the frequency f emitted by the laser (1) passes through the optical fiber beam splitter (2)The other path of the two signals is transmitted to an acousto-optic frequency shifter A (3), and the frequency of the acousto-optic frequency shifter A is changed into f + f after frequency shift2The light is changed into parallel light after passing through a collimator (5), is reflected by a beam combining sheet A (7) and enters a photoelectric detector (6), and is coherent with incident imaging sample reflected light to generate a frequency delta f1-f2The intermediate frequency signal of (1); for a stationary imaged object, Δ f remains unchanged during imaging; because the reflectivity of the object is different, the intensity of the reflected signal is different, the intensity of the intermediate frequency signal with the frequency delta f generated by the interference of the local oscillator light is also different, and the object is imaged through the analysis of the intensity of the intermediate frequency signal of different image points in the scanning imaging process.
According to the method, Fourier transform is carried out on an intermediate-frequency current signal with the frequency delta f output by a detector, and then the signal-to-noise ratio is determined according to the power spectral density of the signal and noise, namely:
Figure GDA0003303464830000031
wherein the PSDIFPSD being the value of the power spectral density at intermediate frequencynIs the average of the noise in the power spectral density; whether the ratio is higher than a threshold value is taken as a criterion for judging whether a reflected signal exists or not, and the intensity of the signal light is determined by the amplitude exceeding the threshold value; that is, the method provided herein takes the average power of the intermediate frequency current signal as the processing object.
Compared with the traditional direct detection system confocal microscopic system, the pinhole-free heterodyne detection system confocal microscopic system has the same transverse and longitudinal resolution. Therefore, the invention avoids a series of practical application difficulties caused by conjugate pinholes under the condition of improving the sensitivity of detection signals.
Drawings
Fig. 1 shows a conventional infinity confocal microscope system based on a direct detection scheme.
Fig. 2 shows a finite distance confocal microscope system based on a direct detection system.
Fig. 3 shows a confocal microscope system based on the heterodyne detection scheme proposed in document 1.
Fig. 4 is a pinhole-free scanning confocal microscopic imaging system based on a heterodyne system.
Fig. 5 is an equivalent optical path diagram of fig. 1 and 4.
Fig. 6 is a partial imaging optical path of fig. 5, wherein fig. 6(a) is used for lateral resolution analysis and fig. 6(b) is used for longitudinal resolution analysis.
Detailed Description
A pinhole-free scanning type confocal microscopic imaging method based on a heterodyne detection system comprises the following steps:
step 1: the laser (1) is turned on to lead the optical field with the frequency f to be divided into two paths after passing through the optical fiber beam splitter (2), one path is shifted to be f + delta f after passing through the acousto-optic frequency shifter A (3)1The other path of the signal is subjected to frequency shift of f + delta f after passing through an acousto-optic frequency shifter B (4)2；
Step 2: frequency shift of f + Δ f1The light as the local oscillation light is converted into parallel light through the collimator (5), and then is reflected by the beam combining sheet A (7) and then is incident on the photoelectric detector (6);
and step 3: frequency shift of f + Δ f2The light is used as signal light, becomes parallel light after passing through a collimating lens (9), and is focused on an imaging sample (12) placed on a three-axis high-precision moving platform (13) after passing through a beam combining sheet B (10) and a focusing lens (11);
and 4, step 4: light reflected by the imaging sample (12) is reflected by the beam combining sheet B (10), passes through the pupil filter (8) and the beam combining sheet A (7) for the first time, and then is incident on the photoelectric detector (6);
and 5: the signal light incident on the photodetector (6) is coherent with the local oscillator light to generate a signal light having a frequency of Δ f1-Δf2The intermediate frequency signal of (1);
step 6: after detecting the intermediate frequency signal corresponding to the reflecting point of one imaging sample (12), the three-axis high-precision moving platform (13) moves to the next scanning point, so that scanning type imaging is realized. The invention discloses a pinhole-free scanning type confocal microscopic imaging method and device of a heterodyne detection system, belongs to the field of laser confocal microscopic imaging, and particularly can omit a conjugate pinhole in the conventional confocal microscopic system. According to the nature of heterodyne detection, a focusing lens in front of a pinhole can be omitted, so that the application difficulty of the system is reduced.
Compared with the prior direct detection confocal microscope system, the heterodyne detection confocal microscope system provided by the invention still has the same transverse and longitudinal resolution without a conjugate pinhole. The resolution characteristics of the confocal microscope system of the conventional direct detection system shown in fig. 1 and the confocal microscope system of the heterodyne detection system shown in fig. 4 according to the present invention are compared as follows.
For the purpose of comparative analysis, the conventional direct detection system confocal microscope system described in fig. 1 and the heterodyne detection system confocal microscope system of the present invention shown in fig. 4 are all equivalent to the system shown in fig. 5. The equivalents are described as follows:
A. in fig. 5, when no local oscillator light is incident, no beam combiner is present, and the photodetector is located at position 2, fig. 5 is equivalent to the system shown in fig. 1;
B. in fig. 5, when there is local oscillator light incident, there is a beam combiner, there is no lens 4, there is no pinhole, and the detector is placed at position 1 in the figure, fig. 5 is equivalent to the system of the present invention shown in fig. 4.
In fig. 5, lens 1 and lens 2 form a dual lens illumination system, while lens 3 and lens 4 form a dual lens collection system. Wherein (xi, eta) is the imaging sample plane, (x)1,y1) Being the back plane of the pupil filter. f. of1Is the focal length of lens 1 and lens 2, f2It is the focal length of lens 3 and lens 4, and in the actual optical path, lens 1 and lens 2 are the same microscope objective, and lens 3 and lens 4 can be different objectives, but the same objective is still used in the following analysis to form a completely symmetrical optical path. In the figure, d ═ f1+ Δ z, Δ z is the defocus of the sample.
1. Transverse resolution contrast analysis
For direct probing confocal Microscopy systems, the theoretical result of resolution is obtained under the assumption that the object being imaged is a point object and the probe is a point probe, as described in the references "C.R.J. Shepard, T.Wilson," Depth of Field in the Scanning Microcopy, "Opt.Lett, 3(3),115-117 (1978)".When the imaged object is the (xi, eta) plane in FIG. 5, the off-axis distance is rsFor a point with a defocus amount Δ z, as shown in FIG. 5, the light intensity response function of the point detector is, for example, described in "Weiqian Zhao, Lirong Qiu, Shanshan Chen and Zhengde Feng," Image reduction phase-filtering temporal reconstruction microprocessor, "Chin. Phys. Lett.23 (856), and-" 859(2006) "
I(v,u)＝|h1(v,u)|2×|h2(v,u)|2， (1)
Wherein h is1(v, u) and h2(v, u) is the point spread function of the illumination and collection arms, v ═ krsa/f1，u＝kaΔz/f1Where a is the pupil radius of the lens 1, k 2 pi/lambda is the wave number, and
Figure GDA0003303464830000051
Figure GDA0003303464830000052
where P (ρ) and T (ρ) are the illumination arm aperture function and the pupil filter transmittance function, respectively, J0(.) is a zero order Bessel function. The transverse and longitudinal resolution of the system is determined by I (v,0) and I (0, u), which are written as r for easy comparison with subsequent analysissAnd Δ z, i.e.
I(rs,0)＝|h1(rs,0)|2×|h2(rs,0)|2， (4)
I(0,Δz)＝|h1(0,Δz)|2×|h2(0,Δz)|2， (5)
In the heterodyne system confocal microscope system provided by the invention, similarly, an imaging object is assumed to be a point object, and the resolution characteristic of the heterodyne system confocal microscope system is analyzed by researching the intermediate frequency signal power generated by signal light and local oscillation light emitted by the point object. For lateral resolution, the current mapWhen the system in fig. 5 works in the heterodyne detection scheme (which is equivalent to fig. 4, that is, when local oscillation light enters, there is a beam combiner, there is no lens 4, there is no pinhole, and the detector is placed at position 1 in the figure), in order to analyze the lateral resolution alone, Δ z is made equal to 0. At the moment, the off-axis distance on the object plane (xi, eta) to be examined is rsPoint O of (a) is shown in fig. 6 (a). The point becomes a parallel light field with an incident angle γ after passing through the lens 1, and γ ≈ r as can be understood from fig. 6(a)s/f1. Due to the point spread function h of the illumination arm in fig. 51(v, u) is also a light field distribution function of the (xi, eta) plane, and the amplitude of the parallel light generated after the O point passes through the lens 3 is set as E0SThen there is E0S∝|h1(rs0) |. According to the backward-propagating local oscillator light (BPOL) theory, as in document "[ 3 ]]Siegman, "The anti properties of optical heterodyne receivers," Appl Opt.5,1588-1594(1966) ", in The pupil filter rear plane (x1,y1) The virtual local oscillator light transmitted to the beam combiner in the backward direction has the same heterodyne efficiency with the transmitted light of the signal light passing through the pupil filter as the result of the analysis of the two light beams on the photosensitive surface of the detector. Therefore, for ease of analysis, plane (x) is chosen1,y1) As a virtual photosensitive surface, and setting a virtual local oscillator light field on the surface as EL(x2,y2)＝E0Lexp (i φ), wherein E0LPhi is a constant phase term for the local oscillator light amplitude. Then, according to the formula (15) of reference 1 in the background art, the intermediate frequency signal amplitude AIFCan be expressed as a number of times as,
Figure GDA0003303464830000061
where beta is the detector responsivity, fxAnd gamma/lambda is approximately distributed. Considering gamma ≈ rs/f1After the (6) is finished, the product can be obtained
Figure GDA0003303464830000062
Let ρ' ═ a ρ in the above formula, and according to
Figure GDA0003303464830000063
Omission of constants not related to the analysis
Figure GDA0003303464830000064
Wherein v is as defined in formula (1). Will E0S∝|h1(rs0) is substituted and then h is determined according to the formula (3)2(v, u) is defined as followsIF(rs)∝|h1(rs,0)|×h2(rs0), then the heterodyne signal power is PIF∝|AIF|2I.e. by
PIF(rs)∝|h1(rs,0)|2×|h2(rs,0)|2， (9)
In the comparison formula (4), when the current power of the intermediate frequency signal is used as the detection measurement, the lateral resolutions of the confocal microscope systems of the heterodyne detection system and the direct detection system are the same.
2. Longitudinal resolution contrast analysis
For longitudinal resolution, consider the distance d-f from the lens on the optical axis1One object point O' of + Δ z, as shown in FIG. 6(b), then it is at the pupil filter back surface (x)1,y1) The light field generated is
Figure GDA0003303464830000065
Also set to transmit to (x)1,y1) The virtual local oscillator light field of the surface is EL(x1,y1)＝E0Lexp (i phi), because the defocus amount Deltaz is small in practical application, df in the denominator of the (10) formula exponential term can be made1≈f1 2The amplitude of the heterodyne signal generated by the two beams is
Figure GDA0003303464830000066
Figure GDA0003303464830000071
Also has E0S∝|h1(0, Δ z) |, again according to h2(v, u) and ignoring constants irrelevant to the analysis, AIF(Δz)＝|h1(0,Δz)|×h2(0,. DELTA.z). Then by PIF∝|AIF|2Can obtain the product
PIF(Δz)∝|h1(0,Δz)|2×|h2(0,Δz)|2， (12)
As can be seen from comparison of (12) and (5), the longitudinal resolution of the two systems was also the same. That is, the lateral and longitudinal resolutions of both systems shown in fig. 1 and 4 are the same. It should be noted that the above analysis is performed when the pupil filtering technique is applied to the heterodyne system and the direct detection system confocal microscopy, and the conventional case without pupil filter corresponds to the case where T (ρ) ═ 1 in the formula (3), and the same resolution of the two systems is also applicable.
From the analysis results, the transverse and longitudinal resolutions of the system without the conjugate pinhole are the same as those of the conventional direct detection system confocal microscopic system; but solves some practical operation difficulties caused by conjugate pinholes in the background art.

Claims (1)

1. A pinhole-free scanning confocal microscope based on a heterodyne detection system, the apparatus comprising: the device comprises a laser (1), an optical fiber beam splitter (2), an acousto-optic frequency shifter A (3), an acousto-optic frequency shifter B (4), a collimator (5), a photoelectric detector (6), a beam combining plate A (7), a pupil filter (8), a collimating lens (9), a beam combining plate B (10), a focusing lens (11), an imaging sample (12) and a three-axis high-precision moving platform (13); the laser is characterized in that the frequency f laser emitted by the laser (1) is divided into two beams of laser by the optical fiber beam splitter (2), wherein one beam is subjected to frequency shift by the acousto-optic frequency shifter B (4) and then the frequency is changed into f + f1The frequency-shifted light being the signalThe light is changed into parallel light after passing through a collimating lens (9), and then sequentially passes through a beam combining sheet B (10) and a focusing lens (11) and is focused on an imaging sample (12); reflected light of the imaging sample (12) sequentially passes through a focusing lens (11), is reflected by a beam combining sheet B (10), passes through a pupil filter (8), passes through a beam combining sheet A (7) and reaches a photoelectric detector (6); the light with the frequency f emitted by the laser (1) is transmitted to the acousto-optic frequency shifter A (3) through the other path of the optical fiber beam splitter (2), and the frequency of the light is changed into f + f after the frequency shift of the acousto-optic frequency shifter A2The light is changed into parallel light after passing through a collimator (5), is reflected by a beam combining sheet A (7) and enters a photoelectric detector (6), and is coherent with incident imaging sample reflected light to generate a frequency delta f1-f2The intermediate frequency signal of (1); for a stationary imaged object, Δ f remains unchanged during imaging; because the reflectivity of the object is different, the intensity of the reflected signal is different, the intensity of the intermediate frequency signal with the frequency delta f generated by the interference of the local oscillator light is also different, and the object is imaged through the analysis of the intensity of the intermediate frequency signal of different image points in the scanning imaging process.
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CN104634282A (en) * 2015-02-28 2015-05-20 清华大学 Phase error compensation device for phase interferometry by optical probe

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