CN109632756B - Real-time fluorescence radiation differential super-resolution microscopy method and device based on parallel light spot scanning - Google Patents
Real-time fluorescence radiation differential super-resolution microscopy method and device based on parallel light spot scanning Download PDFInfo
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Abstract
The invention discloses a real-time fluorescence radiation differential super-resolution microscopy method and a device based on parallel light spot scanning, wherein a laser beam is divided into S polarized light and P polarized light, the S polarized light is modulated into circular polarized solid light spots, and the P polarized light is modulated into vortex polarized light and then is modulated into circular polarized hollow light spots; staggering the solid light spot excitation light and the hollow light spot excitation light on an object plane by at least more than 200 nm; simultaneously carrying out two-dimensional scanning on the fluorescent sample by using the solid light spot excitation light and the hollow light spot excitation light to obtain a positive confocal fluorescence intensity map obtained by modulating the solid light spots and a negative confocal fluorescence intensity map obtained by modulating the hollow light spots; and performing shift matching on the two fluorescence intensity maps. Because two light spots are scanned simultaneously, compared with the traditional method that a fluorescence emission differential microscope system switches the modulated light spots back and forth, the sampling speed is more than twice of the traditional method, the super-resolution dynamic microscope effect under the confocal scanning speed is realized, and the imaging speed can be obviously improved.
Description
Technical Field
The invention belongs to the field of super-resolution microscopy, and particularly relates to a super-resolution microscopy method and a super-resolution microscopy device which can simultaneously obtain a positive confocal image obtained by modulating solid light spots and a negative confocal image obtained by modulating hollow light spots in a far field and realize super-diffraction limit resolution by using a difference method.
Background
The development of fluorescence microscopy has greatly promoted research in fields such as biological cytology, however, conventional far-field optical microscopy has a resolution limit due to optical diffraction, and the diffraction limit can be expressed by the full width at half maximum of the focused spot of the objective lens according to the abbe's diffraction limit theory, i.e., Δ r ═ 0.61 λ/NA, where λ is the wavelength of light and NA is the numerical aperture of the objective lens. In recent decades, many researchers have made a further significant breakthrough in breaking the optical diffraction limit, such as stimulated radiation loss super-resolution microscopy (STED), Fluorescence radiation differential super-resolution microscopy (FED), structured light illumination super-resolution microscopy (SIM), photo-activated positioning super-resolution microscopy (PLAM), and random optical reconstruction super-resolution microscopy (store), among others.
Fluorescence radiation differential super-resolution microscopy is a novel super-resolution microscopy method which is proposed recently, and the method utilizes two excitation light spots of different modes to excite to generate fluorescence images on the basis of confocal, namely, one is a positive confocal microscopic image obtained by modulating solid light spots, and the other is a negative confocal microscopic image obtained by modulating bread-shaped hollow light spots, wherein the center of the hollow light spots is a dark spot with the size smaller than the diffraction limit, and the intensity difference of the two images is utilized to eliminate signals excited by edges, so that super-resolution is realized, and the method is differential imaging.
Compared with other super-resolution microscopy methods, the FED can realize lower fluorescence bleaching characteristic and higher imaging speed and has certain optical chromatography capability. However, due to the principle of FED, it needs a positive confocal image and a negative confocal image when imaging, which causes that if a super-resolution image is to be obtained, it needs to scan twice, so that the imaging speed is reduced.
Disclosure of Invention
Compared with the traditional FED method, the method has the advantages that the imaging speed is increased by two times, and the super-resolution imaging at the confocal imaging speed is realized.
The purpose of the invention is realized by the following technical scheme: a real-time fluorescence radiation differential super-resolution microscopy method based on parallel light spot scanning comprises the following steps:
(1) after laser beams emitted by a laser are collimated, the laser beams are divided into S polarized light and P polarized light by a Polarization Beam Splitter (PBS);
(2) modulating the S polarized light into a circular polarized solid light spot by utilizing a quarter-wave plate;
(3) carrying out phase modulation on the P polarized light to modulate the P polarized light into vortex polarized light;
(4) further modulating the modulated P polarized light into circular polarized hollow light spots by utilizing a quarter-wave plate;
(5) according to a circular hole diffraction limit formula, in order to ensure that the solid light spots and the hollow light spots cannot interfere with each other, the solid light spot excitation light and the hollow light spot excitation light are staggered on an object plane by at least more than 200nm by using a light beam deflection device;
(6) simultaneously carrying out two-dimensional scanning on the fluorescent sample by utilizing the solid light spot excitation light and the hollow light spot excitation light, filtering out stray light and the excitation light, collecting a fluorescent signal, and obtaining a confocal fluorescence intensity diagram I obtained by modulating the solid light spot1(x, y) and negative confocal fluorescence intensity map I obtained by modulating hollow light spot2(x,y)；
(7) Shifting and matching the two fluorescence intensity maps according to the formula I (x, y) ═ I1(x,y)-γI2(x, y) obtaining a super-resolution image I (x, y), wherein
Is a first signal light intensity I1The maximum value of (x, y),
is the second signal light intensity I2A maximum value of (x, y); when I (x, y) is a negative number, I (x, y) is set to 0.
Further, in the step (1), the splitting ratio of the two polarized lights is adjusted by using a half-wave plate, so that the light intensity of the S polarized light is weaker than the P polarized light.
Further, in the step (3), the P polarized light is phase-modulated by using a vortex phase plate, and the modulation function is
Where p is the distance from the optical axis of a point on the beam,
is the included angle between the position polar coordinate vector in the section plane of the light beam vertical to the optical axis and the X axis.
Further, in the step (5), the beam deflecting device may employ a plane mirror, a dichroic mirror, or the like.
Further, in the step (6), the fluorescence signal is collected by an Avalanche Photodiode (APD) or a photomultiplier tube (PMT).
The invention provides two real-time fluorescence radiation differential super-resolution microscopic devices based on parallel light spot scanning, which both comprise a laser, an excitation light modulation optical path submodule, an objective table for bearing a fluorescent sample to be detected, a microscope stand for projecting light to the objective table and a detection optical path submodule.
The excitation light modulation optical path submodule comprises:
the beam expander is used for expanding the light of the point light source emitted by the laser into parallel light;
the half wave plate is used for modulating the polarization direction of emergent light of the beam expander;
the polarization beam splitter is used for splitting emergent light of the half wave plate into P polarized light and S polarized light;
the vortex phase plate is used for carrying out 0-2 pi phase modulation on the P polarized light;
a quarter wave plate for modulating the P-polarized light and the S-polarized light into circularly polarized light;
a beam splitter for combining the P-polarized light and the S-polarized light;
a dichroic mirror for reflecting the S-polarized light and transmitting the fluorescent light;
the first plane reflector is used for staggering two beams of circularly polarized light on an object plane by at least more than 200 nm;
and the scanning lens is used for focusing light emitted by the beam splitter.
The microscope stand comprises:
the second plane mirror is used for deflecting light beams emitted by the scanning lens;
the tube mirror is used for collimating emergent light of the second plane reflector;
the microscope objective is used for converging emergent light of the tube lens to the objective table;
further, the numerical aperture NA of the microscope objective lens is 1.49, the magnification is 100 times, the focal length of the tube lens is 200mm, and the focal length of the scanning lens is 50 mm.
The detection optical path sub-module comprises two schemes:
the first scheme specifically comprises the following steps:
the band-pass filter is used for filtering stray light in emergent light of the dichroic mirror;
the double-cemented lens is used for converging emergent light of the band-pass filter;
the semicircular plane reflector is used for splitting the two paths of emergent light of the double-cemented lens;
the first 4F lens group is used for amplifying the two paths of signal light emitted by the semicircular plane reflector respectively;
the spatial filter is used for carrying out spatial filtering on the light beam emitted by the first 4F lens group; the spatial filter can be realized by a pinhole or a multimode fiber, and the size of the spatial filter is smaller than the diameter of one Airy spot;
a first detector for detecting the outgoing beam of the spatial filter; the first detector may be a photomultiplier tube (PMT) or an Avalanche Photodiode (APD).
The second scheme specifically comprises the following steps:
the band-pass filter is used for filtering stray light in emergent light of the dichroic mirror;
the double-cemented lens is used for converging emergent light of the band-pass filter;
the second 4F lens group is used for amplifying the two paths of emergent light of the double-cemented lens;
the parallel filtering optical fiber is used for respectively carrying out spatial filtering on two paths of light emitted by the second 4F lens group; the spacing between the parallel filter fibers is 375 mu m, and in order to ensure that 60% of energy of the Airy spots is collected, the magnification of the whole system is set to be 800 times, so that the spacing between the two light spots on the object plane is 470nm +/-50 nm.
The second detector is used for detecting the emergent light beam of the parallel filtering optical fiber; the second detector may be a photomultiplier tube (PMT) or an Avalanche Photodiode (APD).
The principle of the invention is as follows: the detection module is improved, one path of staggered signals is reflected to an independent detection system by using a semicircular plane mirror, and the other path of staggered signals is directly detected, or parallel filtering optical fibers are used for detecting the staggered fluorescent signals.
According to the formula of diffraction limit of circular hole
Where k is the wave vector, a is the radius of the circular hole, θ is the aperture angle, I0The central light intensity maximum value, the diameter of the Airy spot can be calculated by the formula
A secondary large relative intensity of I is obtained2≈0.00175I0Therefore, when the distance between two light spots is greater than one Airy spot distance, the influence of the side lobe is less than two thousandth, and therefore the distance between the two light spots is greater than one Airy spot to ensure that the light spots are not influenced mutually.
Compared with the prior art, the invention has the following beneficial technical effects: because two light spots are scanned simultaneously, compared with the traditional method that a fluorescence emission differential microscope system switches the modulated light spots back and forth, the sampling speed is more than twice of the traditional method, the super-resolution dynamic microscope effect under the confocal scanning speed is realized, and the imaging speed can be obviously improved.
Drawings
FIG. 1 is a schematic view of a first embodiment of a microscope arrangement according to the invention;
FIG. 2 is a schematic view of a second embodiment of the microscope apparatus according to the present invention;
FIG. 3 is a schematic diagram of the spacing position of parallel spot scanning on the object plane according to the present invention;
FIG. 4 shows (a) a schematic diagram of a semi-circular planar mirror in the dashed box of FIG. 1 and (b) a schematic diagram of a parallel filter fiber in the dashed box of FIG. 2;
in FIG. 5, (a) and (b) are enlarged schematic diagrams of a solid light spot and a hollow light spot respectively;
FIG. 6 is a graph of solid spot minus hollow spot differential super-resolution.
Detailed Description
The present invention will be described in detail below with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
The real-time fluorescence radiation differential super-resolution microscopy method based on parallel light spot scanning provided by the embodiment comprises the following steps:
(1) after laser beams emitted by a laser are collimated, the laser beams are divided into S polarized light and P polarized light by a Polarization Beam Splitter (PBS);
(2) modulating the S polarized light into a circular polarized solid light spot by utilizing a quarter-wave plate;
(3) carrying out phase modulation on the P polarized light to modulate the P polarized light into vortex polarized light;
(4) further modulating the modulated P polarized light into circular polarized hollow light spots by utilizing a quarter-wave plate;
(5) according to a circular hole diffraction limit formula, in order to ensure that the solid light spots and the hollow light spots cannot interfere with each other, the solid light spot excitation light and the hollow light spot excitation light are staggered on an object plane by at least more than 200nm by using a light beam deflection device;
(6) simultaneously carrying out two-dimensional scanning on the fluorescent sample by utilizing the solid light spot excitation light and the hollow light spot excitation light, filtering out stray light and the excitation light, collecting a fluorescent signal, and obtaining a confocal fluorescence intensity diagram I obtained by modulating the solid light spot1(x, y) and negative confocal fluorescence intensity map I obtained by modulating hollow light spot2(x,y)；
(7) Shifting and matching the two fluorescence intensity maps according to the formula I (x, y) ═ I1(x,y)-γI2(x, y) obtaining a super-resolution image I (x, y), wherein
Is firstSignal light intensity I1The maximum value of (x, y),
is the second signal light intensity I2A maximum value of (x, y); when I (x, y) is a negative number, I (x, y) is set to 0.
Further, in the step (1), the splitting ratio of the two polarized lights is adjusted by using a half-wave plate, so that the light intensity of the S polarized light is weaker than the P polarized light.
Further, in the step (3), the P polarized light is phase-modulated by using a vortex phase plate, and the modulation function is
Where p is the distance from the optical axis of a point on the beam,
is the included angle between the position polar coordinate vector in the section plane of the light beam vertical to the optical axis and the X axis.
Further, in the step (5), the light beam deflection device adopts a plane mirror, a dichroic mirror, and the like.
Further, in the step (6), the fluorescence signal is collected by an Avalanche Photodiode (APD) or a photomultiplier tube (PMT).
Example 2
As shown in fig. 1, the real-time fluorescence radiation differential super-resolution microscopy device based on parallel light spot scanning provided in this embodiment includes a laser 14, an excitation light modulation optical path sub-module, an objective table 1 for bearing a fluorescence sample to be detected, a microscope stand for projecting light to the objective table 1, and a detection optical path sub-module;
the excitation light modulation optical path submodule comprises:
a beam expander 12 for expanding the light of the point light source emitted from the laser 14 into parallel light;
a half wave plate 11 for modulating the polarization direction of the outgoing light from the beam expander 12;
a polarization beam splitter 10 for splitting the light emitted from the half-wave plate 11 into P-polarized light and S-polarized light;
a vortex phase plate 9 for performing 0-2 pi phase modulation on the P polarized light;
a quarter-wave plate 8 for modulating the P-polarized light and the S-polarized light into circularly polarized light;
a beam splitter 6 for combining the P-polarized light and the S-polarized light;
a dichroic mirror 7 for reflecting the S-polarized light and transmitting the fluorescent light;
the first plane reflector 13 is used for staggering two beams of circularly polarized light on an object plane by at least more than 200nm, and the hollow light spots and the solid light spots after beam combination form a certain included angle by adjusting the deflection angle of the first plane reflector 13.
And a scanning lens 5 for focusing the light emitted from the beam splitter 6.
The microscope stand comprises:
a second plane mirror 4 for deflecting the light beam emitted from the scanning lens 5;
the tube mirror 3 is used for collimating the light emitted by the second plane reflector 4;
a microscope objective 2 for converging the emergent light of the tube lens 3 to the objective table 1;
further, the numerical aperture NA of the microscope objective lens 2 is 1.49, the magnification is 100 times, the focal length of the tube mirror 3 is 200mm, and the focal length of the scanning lens 5 is 50 mm.
The detection optical path sub-module includes:
a band-pass filter 15 for filtering out stray light in the light emitted from the dichroic mirror 7;
a double cemented lens 16 for converging the light emitted from the band pass filter 15;
a semicircular plane reflector 17 for splitting the two paths of emergent light of the double-cemented lens 16;
a first 4F lens group 18 for amplifying the two paths of signal light emitted from the semicircular plane reflector 17 respectively;
a spatial filter 19 for spatially filtering the light beam exiting the first 4F lens group 18; the spatial filter 19 can be realized by a pinhole or a multimode fiber, and the size of the spatial filter is smaller than the diameter of one Airy spot;
a first detector 20 for detecting the light beam exiting the spatial filter 19; the first detector 20 may be selected from a photomultiplier tube (PMT) or an Avalanche Photodiode (APD).
The apparatus further comprises a computer 24 for controlling the laser 14 and the first detector 20.
Example 3
As shown in fig. 2, the real-time fluorescence radiation differential super-resolution microscopy device based on parallel light spot scanning provided in this embodiment includes a laser 14, an excitation light modulation optical path sub-module, an objective table 1 for bearing a fluorescence sample to be detected, a microscope stand for projecting light to the objective table 1, and a detection optical path sub-module;
the excitation light modulation optical path submodule comprises:
a beam expander 12 for expanding the light of the point light source emitted from the laser 14 into parallel light;
a half wave plate 11 for modulating the polarization direction of the outgoing light from the beam expander 12;
a polarization beam splitter 10 for splitting the light emitted from the half-wave plate 11 into P-polarized light and S-polarized light;
a vortex phase plate 9 for performing 0-2 pi phase modulation on the P polarized light;
a quarter-wave plate 8 for modulating the P-polarized light and the S-polarized light into circularly polarized light;
a beam splitter 6 for combining the P-polarized light and the S-polarized light;
a dichroic mirror 7 for reflecting the S-polarized light and transmitting the fluorescent light;
a first plane reflector 13 for staggering the two circularly polarized light beams on the object plane by at least more than 200 nm; and a scanning lens 5 for focusing the light emitted from the beam splitter 6.
The microscope stand comprises:
a second plane mirror 4 for deflecting the light beam emitted from the scanning lens 5;
the tube mirror 3 is used for collimating the light emitted by the second plane reflector 4;
a microscope objective 2 for converging the emergent light of the tube lens 3 to the objective table 1;
further, the numerical aperture NA of the microscope objective lens 2 is 1.49, the magnification is 100 times, the focal length of the tube mirror 3 is 200mm, and the focal length of the scanning lens 5 is 50 mm.
The detection optical path sub-module includes:
a band-pass filter 15 for filtering out stray light in the light emitted from the dichroic mirror 7;
a double cemented lens 16 for converging the light emitted from the band pass filter 15;
a second 4F lens group 21 for amplifying the two paths of emergent light of the double-cemented lens 16;
a parallel filter fiber 22 for spatially filtering the two light beams emitted from the second 4F lens group 21; the spacing between the parallel filter fibers 22 is 375um, and in order to ensure that 60% of the energy of the airy spots is collected, the magnification of the whole system is set to be 800 times, so that the spacing between the two light spots on the object plane is 470nm +/-50 nm.
A second detector 23 for detecting the exit beam of the parallel-filtered fiber 22; the second detector 23 may be selected from a photomultiplier tube (PMT) or an Avalanche Photodiode (APD).
The apparatus further comprises a computer 24 for controlling the laser 14 and the second detector 23.
The foregoing detailed description is intended to illustrate and not limit the invention, which is intended to be within the spirit and scope of the appended claims, and any changes and modifications that fall within the true spirit and scope of the invention are intended to be covered by the following claims.
Claims (7)
1. A real-time fluorescence radiation differential super-resolution microscopy device based on parallel light spot scanning is characterized by comprising a laser, an excitation light modulation optical path submodule, an objective table for bearing a fluorescence sample to be detected, a microscope stand for projecting light to the objective table and a detection optical path submodule, wherein the laser is arranged on the laser;
the excitation light modulation optical path submodule comprises:
the beam expander is used for expanding the light of the point light source emitted by the laser into parallel light;
the half wave plate is used for modulating the polarization direction of emergent light of the beam expander;
the polarization beam splitter is used for splitting emergent light of the half wave plate into P polarized light and S polarized light;
the vortex phase plate is used for carrying out 0-2 pi phase modulation on the P polarized light;
a quarter wave plate for modulating the P-polarized light and the S-polarized light into circularly polarized light;
a beam splitter for combining the P-polarized light and the S-polarized light;
a dichroic mirror for reflecting the S-polarized light and transmitting the fluorescent light;
the first plane reflector is used for staggering two beams of circularly polarized light on an object plane by at least more than 200nm, is positioned on the light path of the P polarized light and is positioned behind the quarter-wave plate;
the scanning lens is used for focusing emergent light of the beam splitter;
the microscope stand comprises:
the second plane mirror is used for deflecting light beams emitted by the scanning lens;
the tube mirror is used for collimating emergent light of the second plane reflector;
the microscope objective is used for converging emergent light of the tube lens to the objective table;
the detection optical path sub-module includes:
the band-pass filter is used for filtering stray light in emergent light of the dichroic mirror;
the double-cemented lens is used for converging emergent light of the band-pass filter;
the semicircular plane reflector is used for splitting the two paths of emergent light of the double-cemented lens;
the first 4F lens group is used for amplifying the two paths of signal light emitted by the semicircular plane reflector respectively;
the spatial filter is used for carrying out spatial filtering on the light beam emitted by the first 4F lens group;
a first detector for detecting the outgoing beam of the spatial filter;
the spatial filter is realized by selecting a pinhole or a multimode fiber, and the size of the spatial filter is smaller than the diameter of one Airy spot.
2. A real-time fluorescence radiation differential super-resolution microscopy device based on parallel light spot scanning is characterized by comprising a laser, an excitation light modulation optical path submodule, an objective table for bearing a fluorescence sample to be detected, a microscope stand for projecting light to the objective table and a detection optical path submodule, wherein the laser is arranged on the laser;
the excitation light modulation optical path submodule comprises:
the beam expander is used for expanding the light of the point light source emitted by the laser into parallel light;
the half wave plate is used for modulating the polarization direction of emergent light of the beam expander;
the polarization beam splitter is used for splitting emergent light of the half wave plate into P polarized light and S polarized light;
the vortex phase plate is used for carrying out 0-2 pi phase modulation on the P polarized light;
a quarter wave plate for modulating the P-polarized light and the S-polarized light into circularly polarized light;
a beam splitter for combining the P-polarized light and the S-polarized light;
a dichroic mirror for reflecting the S-polarized light and transmitting the fluorescent light;
the first plane reflector is used for staggering two beams of circularly polarized light on an object plane by at least more than 200nm, is positioned on the light path of the P polarized light and is positioned behind the quarter-wave plate;
the scanning lens is used for focusing emergent light of the beam splitter;
the microscope stand comprises:
the second plane mirror is used for deflecting light beams emitted by the scanning lens;
the tube mirror is used for collimating emergent light of the second plane reflector;
the microscope objective is used for converging emergent light of the tube lens to the objective table;
the detection optical path sub-module includes:
the band-pass filter is used for filtering stray light in emergent light of the dichroic mirror;
the double-cemented lens is used for converging emergent light of the band-pass filter;
the second 4F lens group is used for amplifying the two paths of emergent light of the double-cemented lens;
the parallel filtering optical fiber is used for respectively carrying out spatial filtering on two paths of light emitted by the second 4F lens group;
the second detector is used for detecting the emergent light beam of the parallel filtering optical fiber;
the distance between the parallel filter fibers is 375 mu m, and in order to ensure that 60% of energy of the Airy spots is collected, the magnification of the whole system is set to be 800 times, so that the distance between the two light spots on the object plane is 470nm +/-50 nm.
3. The real-time fluorescence radiation differential super-resolution microscope device based on parallel light spot scanning is characterized in that the numerical aperture NA of the microscope objective is 1.49, the magnification is 100 times, the focal length of the tube mirror is 200mm, and the focal length of the scanning lens is 50 mm.
4. A real-time fluorescence radiation differential super-resolution microscopy method using the real-time fluorescence radiation differential super-resolution microscopy apparatus based on parallel spot scanning according to any one of claims 1 to 3, the method comprising the steps of:
(1) after laser beams emitted by a laser are collimated, the laser beams are divided into S polarized light and P polarized light by a polarization beam splitter;
(2) modulating the S polarized light into a circular polarized solid light spot by utilizing a quarter-wave plate;
(3) carrying out phase modulation on the P polarized light to modulate the P polarized light into vortex polarized light;
(4) further modulating the modulated P polarized light into circular polarized hollow light spots by utilizing a quarter-wave plate;
(5) according to a circular hole diffraction limit formula, in order to ensure that the solid light spots and the hollow light spots cannot interfere with each other, the solid light spot excitation light and the hollow light spot excitation light are staggered on an object plane by at least more than 200nm by using a first plane reflector;
(6) excitation with solid spotsThe light and the hollow light spot exciting light simultaneously perform two-dimensional scanning on the fluorescent sample, stray light and exciting light are filtered out, a fluorescent signal is collected, and a confocal fluorescence intensity graph I obtained by modulating the solid light spot is obtained1(x, y) and negative confocal fluorescence intensity map I obtained by modulating hollow light spot2(x,y)；
(7) Shifting and matching the two fluorescence intensity maps according to the formula I (x, y) ═ I1(x,y)-γI2(x, y) obtaining a super-resolution image I (x, y) in which,
is a first signal light intensity I1The maximum value of (x, y),
is the second signal light intensity I2A maximum value of (x, y); when I (x, y) is a negative number, I (x, y) is set to 0.
5. The real-time differential super-resolution microscopy method for fluorescence radiation as claimed in claim 4, wherein in the step (1), the half-wave plate is used to adjust the splitting ratio of the two polarized lights, so that the intensity of the S polarized light is weaker than that of the P polarized light.
6. The real-time fluorescence radiation differential super-resolution microscopy method as claimed in claim 4, wherein in the step (3), P polarized light is phase-modulated by using a vortex phase plate, and the modulation function is
Where p is the distance from the optical axis of a point on the beam,
is the included angle between the position polar coordinate vector in the section plane of the light beam vertical to the optical axis and the X axis.
7. The real-time fluorescence radiation differential super-resolution microscopy method as claimed in claim 4, wherein in step (6), the fluorescence signal is collected by avalanche photodiode or photomultiplier tube.
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