CN104677830A - Spectrophotometric pupil confocal-photoacoustic microimaging device and method - Google Patents
Spectrophotometric pupil confocal-photoacoustic microimaging device and method Download PDFInfo
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Abstract
The invention relates to a spectrophotometric pupil confocal-photoacoustic microimaging device and method. The device and the method are based on a spectrophotometric pupil confocal microimaging system with long working distance, high axial resolution and stray light interference resistance, structures and functions of the spectrophotometric pupil differential confocal microimaging system and a photoacoustic imaging system are organically combined, the spectrophotometric pupil confocal microimaging system is utilized to detect spatial structure information of a biological sample, the photoacoustic imaging system is utilized to detect function information of the biological sample, and then simultaneous detection for the spatial structure information and the function information of the biological sample is realized, so that in-site and non-invasive imaging is performed on a living biological body in real time. With the adoption of the spectrophotometric pupil confocal technology, the axial resolution and the working distance of the spectrophotometric pupil confocal-photoacoustic microimaging device are effectively considered, interference of stray light of a high scattering sample focal plane on the imaging quality can be retrained, the system signal noise ratio is high, and the integrated design of the spectrophotometric pupil confocal-photoacoustic microimaging device is facilitated.
Description
Technical field
The present invention relates to a kind of point of pupil confocal-opto-acoustic microscopic imaging apparatus and method, belong to confocal microscopic imaging technology and opto-acoustic microscopic imaging technical field.
Technical background
Confocal microscopic imaging technology owing to having tomography ability and the high-space resolution ability of optical field uniqueness, and is widely used in fields such as biomedical research, physio-chemical study, testing of materials, micro-nano tests by as important technological means.But, existing confocal microscopic imaging technology mainly adopts the scattered light of object or fluorescence to carry out imaging, what its obtained is only the structural images of observed object, cannot obtain the function image of cell or biomacromolecule, and the chromatography degree of depth of confocal microscopic imaging only reaches submillimeter magnitude simultaneously.
Photoacoustic imaging be based on photoacoustic signal with the optical absorption characteristic of measurand for contrast mechanism carries out imaging, due to the optics of photoacoustic signal and biological tissue, calorifics is relevant with acoustic characteristic, thus the quantity of information entrained by photoacoustic signal is far more than the quantity of information entrained by scattered light in confocal microscopic imaging and fluorescence, therefore, photoacoustic imaging can realize the imaging of biological function, the chemical composition of biological tissue and biological behaviour can be obtained (as growth, apoptosis, metabolism, pathology, sudden change etc.) characteristic information, the chromatography degree of depth of photoacoustic imaging can reach centimetres simultaneously.But the resolving power of photoacoustic imaging only reaches 10 microns, and it cannot realize the structural images of cellular level.
If above-mentioned two kinds of imaging techniques are combined, just confocal microscopic imaging and photoacoustic imaging technological merit separately can be utilized to carry out optoacoustic and confocal complementary imaging to biological tissue, the more information of tissue or cell can be obtained, improve transverse resolution by confocal focusing simultaneously, expand the tomography degree of depth further by ultrasonic time detection.
Based on above-mentioned situation, confocal-opto-acoustic microscopic imaging apparatus and method that the present invention proposes a kind of point pupil with large working distance and high azimuthal resolution, carry out original position, noninvasive real time imagery to utilizing biosome backscattered photons to biological living.
The structure and fuction of point pupil confocal micro imaging system and photoacoustic imaging system organically blends by the present invention, confocal micro imaging system is utilized to detect the component space information of measurand, utilize the composition function information of opto-acoustic microscopic imaging system looks measurand, detect while then realizing measurand component space information and function information.
The employing of pupil confocal micro imaging system is divided to make confocal-opto-acoustic microscopic imaging device significantly increase working distance under the prerequisite not reducing system axial resolving power, be convenient to the integrated of optics and photoacoustic imaging system, suppress high scattering measurand focal plane parasitic light to the interference of confocal microscopic imaging quality.
Summary of the invention
The object of the invention is design a kind of point of pupil confocal-opto-acoustic microscopic imaging device and method, to obtaining measurand component space information and function information simultaneously.
The object of the invention is to be achieved through the following technical solutions.
One of the present invention divide pupil confocal-opto-acoustic microscopic imaging device, comprise point light source system, be placed on successively pointolite exit direction from axle place collimation lens, symmetrical optical axis place optics-optoacoustic confocal some coupled system and measurand, also comprise be placed on measurand backscattering direction to detect photoacoustic signal ultrasonic transducer, be placed on measurand backscattering direction to collect the light collecting lens of backscattering light intensity signal from axle, and the some detection system of detection light collecting lens focal plane Airy disk light intensity signal.
Of the present invention point of pupil be confocal-opto-acoustic microscopic imaging device in, optics-optoacoustic confocal some coupled system comprises the hollow measurement object lens that symmetrical optical axis is placed, the acoustic lens being embedded in hollow measurement objective aperture lower end, and hollow measures the confocal point of object lens and acoustic lens.
Of the present invention point of pupil be confocal-opto-acoustic microscopic imaging device in, optics-optoacoustic confocal some coupled system also comprises measurement object lens, the ultrasound transducer array placed successively along optical axis direction and has the waveguide of cylinder acoustic lens, and measures object lens and the confocal point of waveguide with cylinder acoustic lens.
Of the present invention point of pupil be confocal-opto-acoustic microscopic imaging device in, some detection system comprises the detecting pinhole be placed in light collecting lens focus, and for detecting the light intensity detector of Airy disk microcell light intensity after being placed on detecting pinhole.
Of the present invention point of pupil be confocal-opto-acoustic microscopic imaging device in, some detection system can also be placed on light collecting lens focus place to detect the fiber optic point detection system of Airy disk microcell light intensity.
The present invention divide pupil confocal-opto-acoustic microscopic imaging device in, some detection system can also comprising relaying amplifying lens for amplifying light collecting lens focal spot, being positioned at ccd detector relaying amplifying lens focal plane detecting Airy disk microcell light intensity.
The present invention divide pupil confocal-opto-acoustic microscopic imaging device in, point light source system can be made up of pulsed laser, the condenser lens being placed on laser emitting direction and the pin hole being placed on condenser lens focus place; Wherein, the wavelength of pulsed laser (15), pulsewidth and repetition frequency can be selected as required.
One of the present invention divide pupil confocal-opto-acoustic microscopic imaging method, comprise the following steps:
Step one, open point light source system, the light beam of pointolite outgoing is focused in measurand after collimation lens and optics-optoacoustic confocal some coupled system and inspires photoacoustic signal and light intensity signal.
Step 2, acoustic lens and ultrasonic transducer is utilized to collect and detect photoacoustic signal, for characterizing the biological function information of measurand;
Step 3, utilize optics-optoacoustic confocal some coupled system, the scattered light of measurand focuses on the detecting pinhole that is positioned on light collecting lens focal plane by light collecting lens, be positioned at point confocal signal of pupil of the Airy disk microcell light intensity of the light intensity detector detecting pinhole outgoing after detecting pinhole, for characterizing the structural images information of measurand;
Step 4, computing machine control focused beam by two-dimensional scanning mirrors and axial object lens scanning system and carry out three-dimensional computed tomography scanning to measurand, and the photoacoustic signal inspire measurand and light intensity signal carry out three-dimensional chromatography detection imaging in real time;
Step 5, computing machine carry out fusion treatment to the photoacoustic signal of the measurand of detection and light intensity signal data, can realize the original position of measurand component space information and function information, without wound imaging.
Of the present invention point of pupil be confocal-opto-acoustic microscopic imaging method in, step 3 also can be: utilize optics-optoacoustic confocal some coupled system, the scattered light of measurand focuses on the fiber optic point detection system being positioned at light collecting lens focus place by light collecting lens, fiber optic point detection system for detecting point confocal signal of pupil of Airy disk microcell light intensity, for characterizing the structural images information of measurand.
The present invention divide pupil confocal-opto-acoustic microscopic imaging method in, step 3 also can be: on the ccd detector utilizing optics-optoacoustic confocal some coupled system, light collecting lens, relaying amplifying lens to be focused on by the scattered light of measurand to be positioned on relaying amplifying lens focal plane, amplification Airy disk on ccd detector test surface is divided into Airy disk microcell and carries out strength investigation and obtains the confocal signal of point pupil, for characterizing the structural images information of measurand by computing machine.
Beneficial effect
The present invention contrasts prior art, has the following advantages:
1) the present invention is based on the confocal laser micro imaging system of oblique incidence, exciting light irradiation area and ultrasonic probe surveyed area are coincided, inspire optoacoustic and fluorescence signal by oblique incidence radiation modality with same excitation source simultaneously, to imaging two while realizing optoacoustic and fluorescence, the information that monotype mode is difficult to obtain can be obtained;
2) the confocal laser imaging technique of oblique incidence, under the prerequisite not reducing azimuthal resolution, significantly can increase the working distance of imaging system, confocal micro imaging system and photoacoustic imaging system are structurally easier to integrated, are convenient to the original position of biological living constituent structure information and function information, without wound, real time imagery;
3) under the condition of object lens not introducing high-NA, utilize the object lens of low numerical aperture can improve the azimuthal resolution of confocal system, extend the operating distance of confocal microscope system, what be easier to measurement mechanism realizes miniaturization;
4) the confocal laser imaging technique of oblique incidence can to suppress on focal plane biological measurand parasitic light to the interference etc. of confocal microscopic imaging quality.
Accompanying drawing explanation
Fig. 1. point pupil is confocal-opto-acoustic microscopic imaging device;
Fig. 2. point pupil is confocal-opto-acoustic microscopic imaging device;
Fig. 3. point pupil is confocal-opto-acoustic microscopic imaging device;
Fig. 4. point pupil is confocal-opto-acoustic microscopic imaging device embodiment;
Fig. 5. point pupil confocal microscopy structural representation;
Fig. 6. a point pupil confocal microscopic imaging compares with confocal microscopic imaging azimuthal resolution.
Wherein: 1-point light source system, 2-collimation lens, 3-optics-optoacoustic confocal some coupled system, 4-measurand, 5-hollow measures object lens, 6-acoustic lens, 7-point detection system, 8-ultrasonic transducer, 9-light collecting lens, 10-Airy disk, 11-detecting pinhole, 12-Airy disk microcell, 13-divides pupil confocal signal, 14-light intensity detector, 15-pulsed laser, 16-condenser lens, 17-pin hole, 18-fiber exit pointolite, 19-fiber optic point detection system, 20-relaying amplifying lens, 21-amplifies Airy disk, 22-CCD detector, 23-measures object lens, 24-ultrasound transducer array, 25-has the waveguide of cylinder acoustic lens, 26-ultrasonic signal detection circuit system, 27-computing machine, 28-light intensity signal collection system, 29-two-dimensional scanning mirrors, the axial object lens scanning system of 30-, 31-oblique incidence excitation beam, 32-oblique outgoing measurand scattered light, 33-lamp optical system point spread function (PSF), 34-collects optical system PSF, 35-system synthesis PSF, the 36-emulation point confocal axial characteristic curve of pupil, 37-emulates the confocal axial characteristic curve of common single shaft.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1
The embodiment of the present invention based on point pupil shown in Fig. 4 confocal-opto-acoustic microscopic imaging device, comprise pulsed laser 15, the point light source system 1 that condenser lens 16 and pin hole 17 are formed, be placed on the collimation lens 2 of light beam direct of travel successively, measure object lens 23 and measurand 4, be placed on measurand 4 backscattering direction successively to detect the waveguide 25 with cylinder acoustic lens and the ultrasound transducer array 24 of photoacoustic signal, and for detecting the measurement object lens 23 of measurand 4 back-scattering light or fluorescence signal, light collecting lens 9 and the detecting pinhole 11 be positioned on light collecting lens 9 focal plane, and after being placed on detecting pinhole 11, be used for splitting the light intensity detector 14 of Airy disk microcell 12 of detection Airy disk 10.
The wavelength of pulsed laser 15, pulsewidth and repetition frequency can be selected as required.
Computing machine 27 can carry out 3-D scanning to measurand 4 by control two-dimensional scanning mirrors 29 and axial object lens scanning system 30.
As follows to the process that measurand carries out original position, nothing creates imaging:
The pulsed laser beam line focus lens 16 that pulsed laser 15 sends focus on pin hole 17 and form pointolite, and the light of pin hole 17 outgoing focuses in measurand 4 and inspires photoacoustic signal, scattered light or fluorescence signal after collimation lens 2, two-dimensional scanning mirrors 29, measurement object lens 23.
Photoacoustic signal is detected by ultrasound transducer array 24 after being collected by cylinder acoustic lens 25, then sends computing machine 27 to after the process of ultrasonic signal detection circuit system 26.
The scattered light of the measurand 4 that pulsed laser beam excites after focusing on or fluorescence signal focus on the detecting pinhole 11 on light collecting lens 9 focal plane through measurement object lens 23, light collecting lens 9 again, be positioned at the light intensity detector 14 after detecting pinhole 11 for detecting point confocal signal 13 of pupil of the Airy disk microcell 12 of Airy disk 10, computing machine 27 utilizes this point of confocal signal 13 of pupil can record the structural images information of measurand 4.
Computing machine 27 can carry out 3-D scanning to measurand 4 by control two-dimensional scanning mirrors 29 and axial object lens scanning system 30, thus photoacoustic signal, scattered light or the fluorescence signal that can inspire measurand 4 carry out real-time chromatography detection imaging.
Computing machine 27 controls the three-dimensional position signal (x of two-dimensional scanning mirrors 29 and axial object lens scanning system 30 according to issuing, y, z), light intensity signal collection system 28 gather confocal signal and ultrasonic signal detection circuit system 26 gather photoacoustic signal carry out Data Fusion, component space information and the function information of biological tissue can be obtained simultaneously.
As shown in Figure 5, pupil confocal microscopic imaging technology is divided to utilize the lamp optical system that there is certain angle theta between optical axis and the point spread function (PSF) collecting optical system mutually to restrict, reduce the main lobe width of system point spread function PSF, thus reach the object improving azimuthal resolution and operating distance.
As shown in Figure 4, a point pupil confocal microscopic imaging device utilizes large working distance, the measurement object lens 23 of low numerical aperture constitute illumination optical system and to unify collection optical system, lamp optical system optical axis (z
iaxle) and collect system optical axis (z
caxle) placement into θ angle, (x, y, z) is system coordinate system, (x
i, y
i, z
i) and (x
c, y
c, z
c) be respectively oblique incidence excitation beam 31 and the coordinate of oblique outgoing measurand scattered light 32 in measurand space.
Transformation relation between three coordinate systems is:
x
i＝x cosθ-z sinθ x
c＝x cosθ+z sinθ (1)
y
i＝y y
c＝y (2)
z
i＝x sinθ+z cosθ z
c＝-x sinθ+z cosθ (3)
Point pupil is confocal-and the theoretical model of opto-acoustic microscopic imaging device can be derived by the diffraction theory based on holography.
Pointolite 1, collimation lens 2 and measurement object lens 23 form lamp optical system, and the expression formula of lamp optical system point spread function PSF 33 is:
Measurement object lens 23 and light collecting lens 9 constitute collection optical system, and the expression formula of collecting optical system PSF 34 is:
Wherein, P
i(x
i ρ, y
i ρ) and P
c(x
c ρ, y
c ρ) be measurement object lens 23 for throwing light on and the pupil function for the measurement object lens 23 of collecting light respectively, its equal and opposite in direction; v
ix, v
iyand u
imeasure the normalization optical coordinate of object lens 23 in measurand space, v
cx, v
cyand u
cmeasure the normalization optical coordinate of object lens 23 in measurand space.
Therefore, the expression formula of the system synthesis PSF 35 of point pupil confocal imaging system detected is:
h
cs(x，y，z)＝h
i(x
i，y
i，z
i)×h
c(x
c，y
c，z
c) (6)
Therefore, the intensity response expression formula of point pupil confocal imaging system detected is:
I
cs(x，y，z)＝|h
i(x
i，y
i，z
i)×h
c(x
c，y
c，z
c)|
2(7)
Lamp optical system PSF can be tried to achieve as shown in 33 in Fig. 5 by formula (4), can be tried to achieve by formula (5) and collect optical system PSF as shown in 34 in Fig. 5, the synthesis PSF of point pupil confocal micro imaging system can be tried to achieve as shown in 35 in Fig. 5 by formula (6).
Synthesis PSF35 due to point pupil confocal micro imaging system is lamp optical system PSF and the product collecting optical system PSF, therefore can obtain an axis (z direction) the width Delta z system synthesis PSF35 close with laterally (x direction) width Delta x in point pupil confocal arrangement.
As can be seen from Figure 5: the burnt body of system synthesis PSF35 is less, confocal microscope system just can observe the information of the more fine structure of measurand, thus reaches the object improving confocal system azimuthal resolution.In theory when two optical axises are orthogonal, the burnt body volume of synthesis of system is minimum, can form one and be close to spherical burnt body.
Emulation according to formula (7) the present embodiment divides the confocal axial characteristic curve 36 of pupil as shown in Figure 6, emulates the confocal family curve 37 of common single shaft as shown in Figure 6 under equal numerical aperture NA. condition.
As can be seen from emulation point pupil confocal axial characteristic curve 36 and the confocal axial characteristic curve 37 of the common single shaft of emulation, pupil confocal micro imaging system is divided significantly to improve the azimuthal resolution of confocal microscope system, in other words, reaching under the equal prerequisite axially differentiated, divide the numerical aperture NA. of pupil confocal micro imaging system more much smaller than the NA. value of confocal microscope system, namely divide the working distance of pupil confocal micro imaging system significantly to increase.And the Integrated design being increased to ultrasound transducer array 24 and cylinder acoustic lens 25 and measurement object lens 23 of point pupil confocal micro imaging system working distance provides necessary prerequisite.
Embodiment 2
As shown in Figure 2, point pupil of embodiment 1 confocal-opto-acoustic microscopic imaging device in, replace by pulsed laser 15, condenser lens 16 with optical fiber eye point light source 18 and be positioned at the point light source system 1 that condenser lens 21 focus place pin hole 17 forms; Fiber optic point detection system 19 substitutes the some detection system 7 be made up of the detecting pinhole 11 be positioned on light collecting lens 9 focal plane and the light intensity detector 14 after being positioned at detecting pinhole 11.
All the other measuring methods are identical with embodiment 1.
Embodiment 3
As shown in Figure 3, point pupil of embodiment 1 confocal-opto-acoustic microscopic imaging device in, with amplifying the relaying amplifying lens 20 of light collecting lens 9 focal plane hot spot and being positioned at ccd detector 22 on relaying amplifying lens 20 focal plane and forming a some detection system 7, substitute the some detection system being positioned at the detecting pinhole 11 on light collecting lens 9 focal plane and the light intensity detector 14 after being positioned at detecting pinhole 11 and forming.
All the other measuring methods are identical with embodiment 1.
Below by reference to the accompanying drawings the specific embodiment of the present invention is described, but these explanations can not be understood to limit scope of the present invention.
Protection scope of the present invention is limited by the claims of enclosing, and any change on the claims in the present invention basis is all protection scope of the present invention.
Claims (10)
1. one kind of point of pupil confocal-opto-acoustic microscopic imaging device, it is characterized in that: comprise point light source system (1), be placed on the collimation lens (2) placed from axle of pointolite exit direction successively, optics-optoacoustic confocal some coupled system (3) that symmetrical optical axis is placed and measurand (4), also comprise and be placed on measurand (4) backscattering direction to detect the ultrasonic transducer (8) of photoacoustic signal, measurand (4) backscattering direction is placed on to collect the light collecting lens (9) of backscattering light intensity signal from axle, and the some detection system (7) of detection light collecting lens (9) focal plane Airy disk light intensity signal.
2. one according to claim 1 divide pupil confocal-opto-acoustic microscopic imaging device, it is characterized in that: optics-optoacoustic confocal some coupled system (3) comprises hollow measurement object lens (5) that symmetrical optical axis is placed, the acoustic lens (6) being embedded in hollow measurement object lens (5) lower end, hole, and hollow measures object lens (5) and the confocal point of acoustic lens (6).
3. one according to claim 1 divide pupil confocal-opto-acoustic microscopic imaging device, it is characterized in that: optics-optoacoustic confocal some coupled system (3) also comprises measurement object lens (23), the ultrasound transducer array (24) placed successively along optical axis direction and have the waveguide (25) of cylinder acoustic lens, and measures object lens (23) and the confocal point of waveguide (25) with cylinder acoustic lens.
4. one according to claim 1 divide pupil confocal-opto-acoustic microscopic imaging device, it is characterized in that: some detection system (7) comprises the detecting pinhole (11) be placed in light collecting lens (9) focus, and for detecting the light intensity detector (14) of Airy disk microcell (12) light intensity after being placed on detecting pinhole (11).
5. one according to claim 1 divide pupil confocal-opto-acoustic microscopic imaging device, it is characterized in that: some detection system (7) can also be placed on light collecting lens (9) focus place to detect the fiber optic point detection system (19) of Airy disk microcell (12) light intensity.
6. one according to claim 1 divide pupil confocal-opto-acoustic microscopic imaging device, it is characterized in that: some detection system (7) can also comprising relaying amplifying lens (20) for amplifying light collecting lens (9) focal spot, being positioned at the ccd detector (22) relaying amplifying lens (20) focal plane detecting Airy disk microcell (12) light intensity.
7. one according to claim 1 divide pupil confocal-opto-acoustic microscopic imaging device, it is characterized in that: point light source system (1) is made up of pulsed laser (15), the condenser lens (16) being placed on laser emitting direction and the pin hole (17) that is placed on condenser lens (16) focus place; Wherein, the wavelength of pulsed laser (15), pulsewidth and repetition frequency can be selected as required.
8. point pupil confocal-opto-acoustic microscopic imaging method, it is characterized in that comprising the following steps:
Step one, open point light source system (1), the light beam of point light source system (1) outgoing is focused in measurand (4) after collimation lens (2) and optics-optoacoustic confocal some coupled system (3) and inspires photoacoustic signal and light intensity signal.
Step 2, acoustic lens (6) and ultrasonic transducer (8) is utilized to collect and detect photoacoustic signal, for characterizing the biological function information of measurand (4);
On step 3, the detecting pinhole (11) that utilizes optics-optoacoustic confocal some coupled system (3), light collecting lens (9) to be focused on by the scattered light of measurand (4) to be positioned on light collecting lens (9) focal plane, be positioned at point confocal signal of pupil (13) of Airy disk microcell (12) light intensity of light intensity detector (14) detecting pinhole (11) outgoing after detecting pinhole (11), for characterizing the structural images information of measurand (4);
Step 4, computing machine (27) control focused beam by two-dimensional scanning mirrors (29) and axial object lens scanning system (30) and carry out three-dimensional computed tomography scanning to measurand (4), and the photoacoustic signal inspire measurand (4) and light intensity signal carry out three-dimensional chromatography detection imaging in real time;
Step 5, computing machine (27) carry out fusion treatment to the photoacoustic signal of the measurand (4) of detection and light intensity signal data, can realize the original position of measurand component space information and function information, without wound imaging.
9. one according to claim 8 divide pupil confocal-opto-acoustic microscopic imaging method, it is characterized in that: step 3 can be: utilize optics-optoacoustic confocal some coupled system (3), the scattered light of measurand (4) focuses on the fiber optic point detection system (19) being positioned at light collecting lens (9) focus place by light collecting lens (9), fiber optic point detection system (19) for detecting point confocal signal of pupil (13) of Airy disk microcell (12) light intensity, for characterizing the structural images information of measurand (4).
10. one according to claim 8 divide pupil confocal-opto-acoustic microscopic imaging method, it is characterized in that: step 3 can also be: utilize optics-optoacoustic confocal some coupled system (3), light collecting lens (9), the scattered light of measurand (4) focuses on the ccd detector (22) that is positioned on relaying amplifying lens (20) focal plane by relaying amplifying lens (20), amplification Airy disk (21) on ccd detector (22) test surface is divided into Airy disk microcell (12) and carries out strength investigation and obtains the confocal signal of point pupil (13) by computing machine (27), for characterizing the structural images information of measurand (4).
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