CN1971333A - Confocal micro imaging system using dummy pinhole - Google Patents
Confocal micro imaging system using dummy pinhole Download PDFInfo
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- CN1971333A CN1971333A CN 200610016134 CN200610016134A CN1971333A CN 1971333 A CN1971333 A CN 1971333A CN 200610016134 CN200610016134 CN 200610016134 CN 200610016134 A CN200610016134 A CN 200610016134A CN 1971333 A CN1971333 A CN 1971333A
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Abstract
The invention relates to a confocal imaging system in fields of optical microimaging, it is a confocal microscopic system which uses virtual confocal pinhole to make the system to obtain the longitudinal chromatography capability. It's widely used in the fluorescence microscopy, optical microscopy and so on which possess the microtechnique of three-dimensional imaging capability. The invention includes: light source, collimation lens, beam splitter, microscope objective, objective table, collective lens and CCD. It characterized in that the photosensitive surface of CCD is directly located at the focal plane of the collimation lens, the computer sets the virtual pinhole at the corresponding position of two-dimensional digital image collected by CCD based on the position of the focal point of collimation lens, the signal values of the pixels in the pinhole are accumulated as the signal intensity of current scanning point to eliminate the effect of stray light in non-focal plane to the image quality. The function of virtual confocal pinhole is significant with the physical confocal pinhole and the position, size can be controlled and adjusted by computer, and it possesses the advantages of convenient gauging adjustment.
Description
Technical field
The present invention relates to the confocal imaging system in a kind of optical microphotograph imaging field, specifically, relate to a kind ofly, can be widely used in fluorescence microscopy, nonlinear optics microscopy etc. and have in the microtechnic of three-dimensional imaging ability by adopting virtual confocal pinhole to make system obtain the confocal microscope system of vertical chromatography ability.
Background technology
Because the confocal microscopic imaging technology not only has the resolution higher than traditional optical microscopy, also have unique vertical chromatography ability simultaneously, therefore be widely used in fields such as biology, biomedicine, industry detection and metrology.Confocal pinhole plays crucial effects to vertical chromatography ability of confocal micro imaging system.In the system, measured point and confocal pinhole are in the position of conjugation, because the singularity of confocal pinhole position, make that converging in the front or rear light of focus can not or can not pass through pin hole fully, receive and only could be detected device by confocal pinhole to greatest extent from the flashlight of focal plane, this makes confocal system can not obtain vertical tomographic map of thick sample by section, that is to say that confocal system has possessed vertical chromatography ability.
But what traditional confocal microscope system adopted is the physics confocal pinhole, as the core diameter of optical fiber.In the practical process of reality, there are following problems usually in the physics confocal pinhole: the size of pin hole is generally in the micron dimension scope, need to adopt accurate, stable 3 D locating device, after carrying out accurate and loaded down with trivial details adjustment, pin hole is adjusted to the focal position of collecting lens, otherwise can be easy to cause degradation problem under optical energy loss and the resolution; The pinhole size size can not be according to practical situation regulated at will of reality and change; After the overlong time, pin hole and focal position are easy to be offset, and need to carry out again three-dimensional regulation and calibration again; In addition, also exist pin hole obstruction and pin hole to clean problems such as trouble.
Charge-coupled device (CCD) is the semiconductor devices that grows up early 1970s, and the signal that obtains by CCD is a digital signal, can directly utilize computing machine that the digital signal that obtains is carried out various special processings; And, CCD is through the development of decades, the performance of each side has all obtained breakthrough, particularly obtained developing by leaps and bounds in the image-position sensor application facet, as can be used for faint fluorescence detection field, pixel dimension only for several micron etc., therefore be widely used in fields such as modern observing and controlling, Image Acquisition.
Summary of the invention
Defective at above-mentioned prior art existence, the present invention is applied to CCD in the confocal system, the digital signal that CCD is collected according to the design feature and the optical characteristics of confocal system self is carried out the corresponding calculated machine and is handled, a kind of technical scheme that adopts the confocal micro imaging system of dummy pinhole has been proposed, this system not only has vertical chromatography ability, also has advantages such as calibration adjustments convenience simultaneously.
Basic thought of the present invention is:
The CCD photosurface is positioned over the focal plane position of systematic collection lens, the digital signal that collects for CCD, utilize computing machine generating an interest window with the corresponding position of collecting lens focus, be equivalent to exist with the measured point relation of conjugation, the signal value on the pixel in the window has been added up is used as the signal intensity of current scan point.The size of interest window and effect etc. are identical with the physics confocal pinhole, and are not that entity exists, and are referred to as virtual confocal pinhole.
Technical scheme of the present invention
The confocal micro imaging system of this employing dummy pinhole uses collimation lens, spectroscope, microcobjective, scanning system, collecting lens, charge-coupled image sensor, computing machine;
Its characteristics are that it is made up of three parts:
Part 1: be arranged in order the illumination path that pointolite, collimation lens, spectroscope and microcobjective constitute this system at central optical axis;
Part 2: the detection light path that is arranged in order microcobjective, spectroscope, collecting lens, charge-coupled image sensor construction system;
The 3rd part: computer control scanning system and charge devices, composition data collection, processing and image reconstruction part.
Beneficial effect of the present invention:
1, regulates the location conveniently.The photosurface of CCD is generally a millimeter magnitude, with size only be micron dimension the physics confocal pinhole comparatively speaking, obviously, adjusting on the vertical light axial plane is much easier, be positioned at the focal plane as long as guarantee the CCD photosurface, promptly only need carry out the meticulous adjustment of optical axis direction, and CCD can receive flashlight, just the interest window can be set according to the position of collecting lens focus, operate very simple;
2, the position of dummy pinhole, size can change at any time according to actual needs, do not need that microscopic system is carried out loaded down with trivial details location again and regulate;
3, there is not the pin hole blockage problem in dummy pinhole, does not need to clean the work of aspect yet.
Description of drawings
Fig. 1: the light path synoptic diagram that adopts the confocal micro imaging system of dummy pinhole.
Fig. 2: the catoptron light distribution that CCD detects during apart from microcobjective focus 13.8 μ m.
Fig. 3: the catoptron light distribution that CCD detects during apart from microcobjective focus 6.8 μ m.
Fig. 4: the light distribution that CCD detected when catoptron was positioned at the microcobjective focal plane.
Fig. 5: the catoptron light distribution that CCD detects during apart from microcobjective focus-6.8 μ m.
Fig. 6: the catoptron light distribution that CCD detects during apart from microcobjective focus-13.8 μ m.
Fig. 7: the synoptic diagram of interest window.
Among the figure: 1. pointolite 2. collimation lenses 3. spectroscopes 4. microcobjectives 5. scanning systems 6. collecting lenses 7. charge-coupled device (CCD)s 8. computing machines
Embodiment
Below in conjunction with drawings and Examples the specific embodiment of the present invention is described further:
The confocal micro imaging system of this employing dummy pinhole uses collimation lens, spectroscope, microcobjective, scanning system, collecting lens, charge-coupled image sensor, computing machine;
Its characteristics are that it is made up of three parts:
Part 1: be arranged in order the illumination path that pointolite 1, collimation lens 2, spectroscope 3 and microcobjective 4 constitute this system at central optical axis;
Part 2: the detection light path that is arranged in order microcobjective 4, spectroscope 3, collecting lens 6, charge-coupled image sensor 7 construction systems;
The 3rd part: computing machine 8 gated sweep systems 5 and charge-coupled image sensor 7, composition data collection, processing and image reconstruction part.
The photosurface of charge-coupled image sensor is located immediately at the focal plane position of collecting lens.
Described light source is LASER Light Source or ordinary light source.
Described spectroscope is plain edition spectroscope or dichroism spectroscope.
The formation method of the confocal micro imaging system of described employing dummy pinhole, its characteristics are that it comprises the steps:
1) carries out bidimensional on perpendicular to the direction of optical axis and regulate and make flashlight be collected on the photosurface that lens converge to CCD, vertically regulate making the photosurface of CCD be positioned at the focal plane position of collecting lens then;
2) position of hot spot on the two dimensional image that CCD collects, corresponding to the position of collecting lens focus at the CCD photosurface, the corresponding position of computing machine on two-dimensional digital image is provided with dummy pinhole, and only the signal value on the pixel in the pin hole having been added up is used as the signal intensity of current scan point;
3) the scanning system driving objective table is made three-dimensional translating, and opposed sample thereon carries out 3-D scanning, can obtain the signal intensity of different scanning point, and computing machine finally reconstructs the tomographic map of sample according to these signals that collect.
Changing a kind of describing method is:
Adopt the confocal micro imaging system of dummy pinhole, it is included in the pointolite that central optical axis is arranged in order, collimation lens, spectroscope, microcobjective, objective table, collecting lens and CCD; Pointolite forms directional light by collimation lens and incides on the spectroscope, is focused sample through microcobjective; Surveyed by same microcobjective from reflection or scattered light that sample produces, return,, arrive CCD at last, signal is gathered by CCD through being reflected to behind the spectroscope on the collecting lens along original optical path; The photosurface of CCD is located immediately at the focal plane position of collecting lens, any physical pin is not adopted in the place ahead, position according to the collecting lens focus, the interest window is set on the two-dimensional digital image that CCD collects, signal value on the pixel in the window added up is used as the signal intensity of current scan point, and position of window, big I are by the computing machine adjustment; The one D translation platform driving objective table that is higher than the micron order precision is made three-dimensional translating, and opposed sample thereon carries out 3-D scanning, obtains the signal intensity of different scanning point, finally reconstructs the tomographic map of sample.
Although in this system, do not adopt the physics confocal pinhole, light and parasitic light before or after the focus are all received by CCD, but the CCD photosurface is in a specific position of system, promptly be positioned at the focal plane of collecting lens, a kind of like this optical characteristics of feasible existence: along with the increase of defocusing amount, present phenomenon from the distribution of light on the CCD photosurface at non-focus place with respect to the focus of collecting lens to external diffusion, correspondingly the contribution to signal intensity in the interest window weakens, and that is to say can be by being provided with the influence that the interest window is got rid of non-focal plane light and parasitic light at place, collecting lens focal position; In addition, the pixel dimension of CCD is generally several microns, can be easy to the size of interest window is controlled at micron dimension.Generally speaking, the interest window is identical with the effect of physics confocal pinhole to the effect that microscopic system plays, and can be called virtual confocal pinhole.
Embodiment
Fig. 1 has provided the light path synoptic diagram that adopts the confocal micro imaging system of dummy pinhole, pointolite 1, collimation lens 2, spectroscope 3 and microcobjective 4, and they become light path to connect successively, constitute the illumination path of this system; Moving of scanning system 5 control samples; Microcobjective 4, spectroscope 3, collecting lens 6, CCD7 become light path to connect the detection light path of construction system successively; Computing machine 8 gated sweep system 5 and CCD7, go forward side by side line data collection, processing and image reconstruction.In the present embodiment, microcobjective 4 adopts the flat field apochromatic objective of Olympus, and numerical aperture is 0.4; The P-611.3S piezoelectric ceramics D translation platform of the German PI of scanning system 5 employings company, precision is 2nm, effective travel is 100 μ m; CCD7 selects the MTV-1881EX CCD of Taiwan MINTRON company for use, and pixel dimension is 8.3 μ m * 8.3 μ m, and the photosurface size is 6.4 * 4.8mm, and minimal illumination 0.02LUX is applicable to micro light detecting.
Increase optical characteristics to external diffusion for further specifying non-focal plane light with defocusing amount, a catoptron perpendicular to optical axis is vertically moved, can obtain catoptron when different axial location, the light distribution that CCD detects is as Fig. 2-shown in Figure 6.Obviously, along with the increase of catoptron out of focus distance, outwards diffusion tendency appears significantly in light intensity.
The choice of location of physics confocal pinhole makes non-focal plane flashlight can not or can not be detected device fully and receives in the focal position of collecting lens.For microscopic system proposed by the invention, can handle the two-dimensional digital image that CCD collects by computing machine, the method that promptly adds virtual confocal pinhole realizes.Detailed process is as follows: the width of cloth two-dimensional digital image that CCD is detected, with the corresponding position of collecting lens focus on the interest window is set, as shown in Figure 7, only the signal value on the pixel that will be marked by stain has added up and has been used as the signal intensity of current scan point, just can get rid of the influence of the flashlight that is received by pixel beyond the interest window fully, that is to say the influence of having got rid of non-focal plane flashlight and parasitic light.Pixel size according to MTV-1881EX type CCD is calculated, and the diameter of window is approximately 41.5 μ m among Fig. 7.Simultaneously because CCD is positioned at the focal plane of collecting lens, choosing of interest the window's position is focal position according to collecting lens, there are conjugate relation in it and measured point, and be identical with the effect of physics confocal pinhole to the microscopic system role, it can be called virtual confocal pinhole.Virtual confocal pinhole is generated by computing machine, and its size, position also can be adjusted according to the practical situation of reality with computing machine, and be very convenient.
Claims (5)
1. a confocal micro imaging system that adopts dummy pinhole uses collimation lens, spectroscope, microcobjective, scanning system, collecting lens, charge-coupled image sensor, computing machine;
It is characterized in that it is made up of three parts:
Part 1: be arranged in order the illumination path that pointolite (1), collimation lens (2), spectroscope (3) and microcobjective (4) constitute this system at central optical axis;
Part 2: the detection light path that is arranged in order microcobjective (4), spectroscope (3), collecting lens (6), charge coupled device ccd (7) construction system;
The 3rd part: computing machine (8) gated sweep system (5) and charge-coupled image sensor (7), composition data collection, processing and image reconstruction part.
2, the confocal micro imaging system of employing dummy pinhole according to claim 1 is characterized in that: the photosurface of CCD is located immediately at the focal plane position of collecting lens.
3, the confocal micro imaging system of employing dummy pinhole according to claim 1 is characterized in that: described light source is LASER Light Source or ordinary light source.
4, the confocal micro imaging system of employing dummy pinhole according to claim 1 is characterized in that: described spectroscope is plain edition spectroscope or dichroism spectroscope.
5. the formation method of the confocal micro imaging system of employing dummy pinhole according to claim 1 is characterized in that it comprises the steps:
1) carries out bidimensional on perpendicular to the direction of optical axis and regulate and make flashlight be collected on the photosurface that lens converge to charge-coupled image sensor, vertically regulate making the photosurface of CCD be positioned at the focal plane position of collecting lens then;
2) position of hot spot on the two dimensional image that CCD collects, corresponding to the position of collecting lens focus at the CCD photosurface, the corresponding position of computing machine on two-dimensional digital image is provided with dummy pinhole, and only the signal value on the pixel in the pin hole having been added up is used as the signal intensity of current scan point;
3) the scanning system driving objective table is made three-dimensional translating, and opposed sample thereon carries out 3-D scanning, can obtain the signal intensity of different scanning point, and computing machine finally reconstructs the tomographic map of sample according to these signals that collect.
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