CN105425377A - Imaging method and system for microscope - Google Patents
Imaging method and system for microscope Download PDFInfo
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- CN105425377A CN105425377A CN201511019416.9A CN201511019416A CN105425377A CN 105425377 A CN105425377 A CN 105425377A CN 201511019416 A CN201511019416 A CN 201511019416A CN 105425377 A CN105425377 A CN 105425377A
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
- G02B21/08—Condensers
- G02B21/12—Condensers affording bright-field illumination
- G02B21/125—Condensers affording bright-field illumination affording both dark- and bright-field illumination
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
- G02B21/08—Condensers
- G02B21/14—Condensers affording illumination for phase-contrast observation
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/361—Optical details, e.g. image relay to the camera or image sensor
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
- G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
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Abstract
The invention belongs to the imaging technical field and relates to an imaging method and system for the microscope. The imaging method and system are used for realizing high-resolution imaging of strong-absorption, weak-absorption and unstained transparent medical or biological samples. The imaging system for the microscope of the invention comprises a microscope. The imaging system is characterized in that the imaging system further comprises a light source device, a photographing device and a processing device; the light source device is used for providing a plurality of lighting modes for the samples in the form of a light-emitting diode array so as to make sample transmission light carry different sample information; the microscope is used for receiving the sample transmission light to obtain observation results; the photographing device is used for acquiring the observation results of the microscope to obtain acquired images; and the processing device is used for obtaining imaged pictures according to the acquired images. The imaging method and system for the microscope of the invention have the advantages of bright-field, dark-field and phase-contrast imaging results, convenience, flexibility and high controllability.
Description
Technical field
The present invention relates to technical field of imaging, particularly one is used for microscopical formation method and system, in particular for realizing strong absorption, weak absorbing, transparent medical science of being unstained or the high-resolution imaging of biological sample.
Background technology
The mainly light field imaging of early stage microscope imaging method, but the contrast of this microscope imaging result is not high.The method improving microscope imaging contrast has a lot, is mainly divided into labelling method and unmarked method two class.Wherein, comparatively conventional unmarked method comprises dark-field imaging, phase contrast imaging etc.Light field imaging can collect sample low frequency part information, is mainly applicable to observe have the strong sample absorbed; Dark-field imaging is the scattered light imaging by capturing wide-angle, can obtain the detail of the high frequency of sample, therefore is applicable to weak absorbing and the sample of clear-cut; Phase contrast imaging can extract the phase information of sample.And for the transparent sample of no dyeing, its transmitted light has uniform intensity distributions, unique change only has its phase information.Therefore, phase-contrast method is very applicable to the observation of this kind of sample.
Light field imaging, dark-field imaging and phase contrast imaging method supplement mutually, can provide comparatively complete sample message, so need these three kinds of imaging patterns comprehensive under many circumstances.But for traditional microscope, the realization of often kind of imaging pattern, all based on different hardware configurations, needs condenser diaphragm, polarizer or special object lens.Realize three kinds of imaging pattern complicated operations.So, a kind of proposition of more simple method that simultaneously can realize light field, details in a play not acted out on stage, but told through dialogues, phase contrast imaging, essential.
Summary of the invention
At present of the present invention, being to solve the problem, proposing a kind ofly to realize light field, the formation method of details in a play not acted out on stage, but told through dialogues and phase contrast Three models and system simultaneously.
Technical scheme of the present invention is: a kind of for microscopical formation method, it is characterized in that, comprises the following steps:
A. adopt light source to irradiate sample, make transmitted light carry the phase place change information caused by sample distribution inequality; Described light source is for generation of controlled multiple lighting system;
B. microscope receives sample transmitted light), be transferred to eyepiece and camera installation respectively after imaging processing;
C. camera installation gathers image that microscope becomes, and it is sent to treatment facility by the collection image of acquisition;
D. the Image Acquisition imaging picture that gathers according to camera installation for the treatment of facility.
Further, described light source is light emitting diode matrix.
In such scheme, light emitting diode matrix can be the array size such as 4 × 4,8 × 8,16 × 16,32 × 32.
Further, described light emitting diode matrix comprises light field region and details in a play not acted out on stage, but told through dialogues region, produces bright field illumination mode and dark ground illumination mode respectively; Then,
In step a, during bright field illumination, sample transmitted light carries sample distribution low-frequency information, claims light field light.During dark ground illumination, sample transmitted light carries sample distribution detail of the high frequency, claims details in a play not acted out on stage, but told through dialogues light;
In step b, microscope receives the light field light of sample transmission, obtains light field observed result, is gathered by camera installation, or microscope receives the details in a play not acted out on stage, but told through dialogues light of sample transmission, obtains details in a play not acted out on stage, but told through dialogues observed result, is gathered by camera installation;
In step c, camera installation gathers microscope gained light field observed result and obtains light field collection image, or camera installation gathers microscope gained details in a play not acted out on stage, but told through dialogues observed result acquisition details in a play not acted out on stage, but told through dialogues and gathers image; The light field of acquisition collection image or details in a play not acted out on stage, but told through dialogues are gathered image and is sent to treatment facility;
In steps d, treatment facility gathers image according to light field and obtains light field imaging picture, or treatment facility gathers image according to details in a play not acted out on stage, but told through dialogues and obtains dark-field imaging picture.
In such scheme, the size in light emitting diode matrix light field region and details in a play not acted out on stage, but told through dialogues region is determined by microscope objective numerical apertures (NA); When only lighting light emitting diode in light field region, sample transmission light is light field light; When only lighting the light emitting diode in details in a play not acted out on stage, but told through dialogues region, sample transmission light is details in a play not acted out on stage, but told through dialogues light.
Further, the light field region of described light emitting diode matrix also can be divided into two complementary regions, is respectively light field first area and light field second area; Then,
The light of light field described in step a also comprises the first light field light and the second light field light;
The observed result of light field described in step b also comprises the first light field observed result and the second light field observed result;
Light field described in step c gathers image and also comprises the first light field collection image and the second light field collection image;
In steps d, treatment facility obtains the method for final light field imaging picture and is:
Supposing that the first light field gathers image is I
1, second light field gather image be I
2, then light field imaging picture I
brightpreparation method be: I
bright=I
1+ I
2.
In such scheme, the dividing mode in the first light field region and the second light field region can be, will be set to the first light field region on the left of light field region, right side is set to the second light field region; Or light field top is set to the first light field region, bottom is set to the second light field region.
In such scheme, the dividing mode in the first light field region and the second light field region is except left and right divides up and down, can also change angularly: crossing light field mid point and with the straight line of the counterclockwise angle α of horizontal direction ° for axis of symmetry, be two regions along this line symmetry by light field Region dividing.
Further, in steps d, treatment facility can also obtain the phase contrast imaging picture on sample different directions, and concrete grammar is:
If the phase contrast imaging picture in sample a direction is I
ph, then I
phpreparation method be:
In such scheme, to obtain the phase contrast direction of phase contrast imaging picture mutually vertical with the division axis of symmetry in light field region.
A kind of for microscopical imaging system, comprise microscope, it is characterized in that, also comprise light source, camera installation and treatment facility; Described light source is used for providing different lighting systems to sample, makes sample transmission light carry different types of information; Described microscope, for receiving the dissimilar transmitted ray of sample, obtains corresponding observed result; Described camera installation, for gathering microscopic result, obtains and gathers image; Described treatment facility is used for according to the final imaging picture of collection Image Acquisition.
In such scheme, camera installation can be with the CCD camera of external trigger, CMOS camera, sCMOS camera and the magazine one of EMCCD.In addition, for preventing Caton phenomenon, suggestion adopts monotype frame frequency >=24Hz
Further, described light source is light emitting diode matrix; Described light emitting diode matrix comprises light field region and details in a play not acted out on stage, but told through dialogues region; Then,
After light source irradiates sample, sample transmission light is light field light or details in a play not acted out on stage, but told through dialogues light;
Microscope receives the light field light of sample transmission, obtains light field observed result and is transferred to camera installation, or microscope receives the details in a play not acted out on stage, but told through dialogues light of sample transmission, obtains details in a play not acted out on stage, but told through dialogues observed result and is transferred to camera installation;
Camera installation gathers microscope light field observed result and obtains light field collection image, or camera installation gathers microscope dark field observed result acquisition details in a play not acted out on stage, but told through dialogues and gathers image; The light field of acquisition collection image or details in a play not acted out on stage, but told through dialogues are gathered image and is sent to treatment facility;
Treatment facility gathers image according to light field and obtains light field imaging picture, or treatment facility gathers image according to details in a play not acted out on stage, but told through dialogues and obtains dark-field imaging picture.
Further, described light field region comprises the first light field region and the second light field region; Then,
Described light field light also comprises the first light field light and the second light field light;
Described light field observed result also comprises the first light field observed result and the second light field observed result;
Described light field gathers image and also comprises the first light field collection image and the second light field collection image;
The method that treatment facility obtains light field imaging picture is:
Supposing that the first light field gathers image is I
1, second light field gather image be I
2, then light field imaging picture I
brightpreparation method be: I
bright=I
1+ I
2.
Further, treatment facility can also obtain the phase contrast imaging picture on sample different directions, and concrete grammar is:
If in sample a direction phase contrast imaging picture be I
ph, then I
phpreparation method be:
In such scheme, to obtain the phase contrast direction of phase contrast imaging picture mutually vertical with the division axis of symmetry in light field region.
Further, also comprise controller, described controller is connected with light emitting diode matrix and camera installation respectively; Described controller is for controlling the switch of light emitting diode matrix and camera installation.
Beneficial effect of the present invention is, can light field simultaneously, details in a play not acted out on stage, but told through dialogues and phase contrast Three models imaging results; Also have convenient, flexible, the advantage that controllability is strong.
Accompanying drawing explanation
Fig. 1 is the System's composition schematic diagram realizing Three models imaging based on light emitting diode matrix of the present invention;
Fig. 2 is numerical aperture of objective schematic diagram;
Fig. 3 is LED array structure and division schematic diagram in imaging system of the present invention; Wherein, (a) is LED array structure schematic diagram; B () is divided into the structural representation of left and right halves for light emitting diode matrix; C () be the structural representation of upper and lower halves for light emitting diode matrix is divided into; D () is divided into the structural representation of tiltedly up and down halves for light emitting diode matrix;
Fig. 4 is present system image acquisition and processing controls schematic diagram;
Fig. 5 controller control flow chart;
Wherein, 1 is that (10 is a light emitting diode in light emitting diode matrix to light emitting diode matrix; 11 is light emitting diode matrix details in a play not acted out on stage, but told through dialogues part; 12 is light emitting diode matrix light field part, and it is made up of 13 and 14 two parts, and 13 is left light field, and 14 is right light field); 2 is testing sample; 4 transmission electron microscopes both deposited for market, 3 and 6 are respectively its object lens part and eyepiece part; 5 is camera; 7 is PC end, for display and the process of image; 8 is storer; 9 is controller; Represent light emitting diode matrix light field radius; Represent light emitting diode matrix and micro objective spacing; Represent the spacing of adjacent two light emitting diodes of light emitting diode matrix; Represent that light field divides translation-angle; A, b represent the control signal that controller inputs to light emitting diode matrix and clock signal respectively; C, d represent the control signal that controller inputs to camera and the feedback signal that camera transmits to controller respectively; E represents storage drive program; T1, T2, T3 represent the transmission link, the jtag interface between controller and camera and the USB annexation between storer and camera that connect camera and computing machine respectively
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in detail
Adopt the imaging system based on light emitting diode matrix as shown in Figure 1, its critical piece comprises a light emitting diode matrix, a transmission electron microscope and a camera.Wherein, microscope used is inverted transmission electron microscope, and testing sample is placed on microscope stage.
Choosing of light emitting diode matrix size is relevant by the numerical aperture NA of selected micro objective, as shown in Figure 2, and the computing formula of known NA:
Wherein, n is the medium refraction index of light process, is taken as 1 in some embodiments of the present invention; θ is the light maximum angle that can enter object lens; R is the radius that emergent ray can enter the maximum magnitude of the light emitting diode matrix of object lens, usually claims this maximum magnitude to be the light field of light emitting diode matrix, claims this radius to be light field radius; D is light emitting diode matrix and micro objective spacing.
From formula, the light field radius of light emitting diode matrix is expressed as:
Will with ensureing that selected light emitting diode matrix can produce light field imaging and dark-field imaging, then its array size is at least
wherein, as shown in Figure 3, Δ is the spacing between adjacent two light emitting diodes of light emitting diode matrix.
The enlargement ratio of selected object lens is larger, and its corresponding numerical aperture also can more greatly, now when constant with light emitting diode matrix spacing, and should suitable expanded matrix size.Such as, in one embodiment, light-emitting diodes tube pitch 4mm is set; Be positioned over 60mm place above sample, because sample is placed on microscope stage, with object lens close together, distance can be ignored, therefore approximate thinks that light emitting diode matrix and object lens spacing are 60mm; Select 10 × microcobjective, the numerical aperture of its correspondence is 0.25.Now, formula is utilized:
Calculating respective leds array light field radius is: R=15.5mm, array sizes is at least: 8 × 8.
After selected object lens determination light emitting diode matrix light field range size, for the imaging realizing Three models also needs light field region Further Division.
In some embodiments of the invention, as shown in Fig. 3 (b), by light field Region dividing in order to two, left and right part, under dark ground illumination, the illumination of light field left part and light field right part light illumination mode, capturing three width image I respectively
dark, I
l, I
r.Process I
land I
rtwo width images, can obtain the phase contrast imaging result in light field imaging and horizontal direction:
In some embodiments of the invention, as shown in Fig. 3 (c), by light field Region dividing in order to upper and lower two parts, under dark ground illumination, light field top lighting and light field bottom light illumination mode, capturing three width image I respectively
dark, I
u, I
d.What its imaging results and left and right divided is differently that its phase contrast result is the phase contrast imaging result on vertical direction.
In other embodiments of the present invention, for obtaining the phase contrast imaging result in all directions, take, along the light field dividing mode of angle [alpha] conversion, as shown in Fig. 3 (d), can obtain along the phase contrast imaging result on α+90 ° of directions.
In addition, camera shown in Fig. 1 is the sCMOS high speed camera of a monotype frame frequency >=24Hz, place it in microscope front end for capture microscope under different lighting system become image.
As shown in Figure 4, for realizing the synchronous of light emitting diode matrix lamp mode conversion and camera exposure, also need to use a controller.While the three kinds of ignition mode circulations of this controller energy control realization light emitting diode matrix are carried out, the imaging results under this several light illumination mode captured by control camera.The display of imaging results and process need be realized by a computing machine, and camera and intercomputer realize the transmission of image information by a video camera LI(link interface).
Fig. 5 is that some embodiments of the invention middle controller 9 realizes synchronous control flow chart, step 100 and step 101 are synchronously carried out, composition graphs 4, controller 9 is to light emitting diode matrix 1 input control signal a and clock signal b, and meanwhile its computer for controlling 7 processes the picture that the last time takes.In step 102,103, after light emitting diode matrix control signal a transmission and image procossing all complete, controller 9 sends the trigger pip c of pictures taken to camera 5, carry out step 104.After step 105 has exposed, the picture of shooting is passed to computing machine by video camera LI(link interface) T1, give controller 9 one feedback signal d simultaneously, reenter step 101,102.
Claims (10)
1. for a microscopical formation method, it is characterized in that, comprise the following steps:
A. adopt light source to irradiate sample, make sample transmission light carry sample message; Described light source is for generation of controlled multiple lighting system, and under different lighting system, sample transmission light carry information is different;
B. microscope receives sample transmission light, and gained observed result is transferred to eyepiece and camera installation respectively;
C. camera installation gathers microscopic result and obtains collection image, and it is sent to treatment facility by the collection image of acquisition;
D. the final imaging picture of Image Acquisition that gathers according to camera installation for the treatment of facility.
2. one according to claim 1 is used for microscopical formation method, and it is characterized in that, described light source is light emitting diode matrix.
3. one according to claim 2 is used for microscopical formation method, and it is characterized in that, described light emitting diode matrix comprises light field region and details in a play not acted out on stage, but told through dialogues region, produces bright field illumination mode and dark ground illumination mode respectively; Then,
In step a, sample transmission light is light field light or details in a play not acted out on stage, but told through dialogues light;
In step b, microscope receives the light field light of sample transmission, obtains light field observed result and is transferred to camera installation, or microscope receives the details in a play not acted out on stage, but told through dialogues light of sample transmission, obtains field observed result secretly and be transferred to camera installation after process;
In step c, camera installation gathers microscope light field observed result and obtains light field collection image, or camera installation gathers microscope dark field observed result acquisition details in a play not acted out on stage, but told through dialogues and gathers image; The light field of acquisition collection image or details in a play not acted out on stage, but told through dialogues are gathered image and is sent to treatment facility;
In steps d, treatment facility gathers image according to light field and obtains light field imaging picture, or treatment facility gathers image according to details in a play not acted out on stage, but told through dialogues and obtains dark-field imaging picture.
4. one according to claim 3 is used for microscopical formation method, and it is characterized in that, the light field region of described light emitting diode matrix also comprises the first light field region and the second light field region; Then,
The light of light field described in step a also comprises the first light field light and the second light field light;
The observed result of light field described in step b) also comprise the first light field observed result and the second light field observed result;
Light field described in step c gathers image and also comprises the first light field collection image and the second light field collection image;
In steps d, the method for the treatment of facility acquisition light field imaging picture is:
Supposing that the first light field gathers image is I
1, second light field gather image be I
2, then light field imaging picture I
brightpreparation method be: I
bright=I
1+ I
2.
5. one according to claim 3 is used for microscopical formation method, and it is characterized in that, in steps d, treatment facility can also obtain the phase contrast imaging picture on sample different directions, and concrete grammar is:
If the phase contrast imaging picture in sample a direction is I
ph, then I
phpreparation method be:
6. for a microscopical imaging system, comprise microscope, it is characterized in that, also comprise light source, camera installation and treatment facility; Described light source is for irradiating sample to make sample generation with the transmitted ray of sample message; Described microscope, for receiving sample transmission light, obtains observed result; Described camera installation obtains collection image for gathering microscopic result; Described treatment facility is used for according to collection Image Acquisition imaging picture.
7. one according to claim 6 is used for microscopical imaging system, and it is characterized in that, described light source is light emitting diode matrix; Described light emitting diode matrix comprises light field region and details in a play not acted out on stage, but told through dialogues region; Then,
After light source irradiates sample, sample transmission light is auspicious is light field light or details in a play not acted out on stage, but told through dialogues light;
Microscope receives sample transmission light field light, obtains light field observed result and is transferred to camera installation, or microscope receives sample transmission details in a play not acted out on stage, but told through dialogues light, obtains details in a play not acted out on stage, but told through dialogues observed result and is transferred to camera installation;
Camera installation gathers microscope light field observed result and obtains light field collection image, or camera installation collection microscope sends details in a play not acted out on stage, but told through dialogues observed result acquisition details in a play not acted out on stage, but told through dialogues and gathers image; The light field of acquisition collection image or details in a play not acted out on stage, but told through dialogues are gathered image and is sent to treatment facility;
Treatment facility gathers image according to light field and obtains light field imaging picture, or treatment facility gathers image according to details in a play not acted out on stage, but told through dialogues and obtains dark-field imaging picture.
8. one according to claim 7 is used for microscopical imaging system, and it is characterized in that, described light field region comprises the first light field region and the second light field region; Then,
Described light field light also comprises the first light field light and the second light field light;
Described light field observed result also comprises the first light field observed result and the second light field observed result;
Described light field gathers image and also comprises the first light field collection image and the second light field collection image;
The method that treatment facility obtains light field imaging picture is:
Supposing that the first light field gathers image is I
1, second light field gather image be I
2, then light field imaging picture I
brightpreparation method be: I
bright=I
1+ I
2.
9. one according to claim 7 is used for microscopical imaging system, and it is characterized in that, treatment facility can also obtain the phase contrast imaging picture on sample different directions, and concrete grammar is:
If the phase contrast imaging picture in sample a direction is I
ph, then I
phpreparation method be:
10. the one according to claim 6 ~ 9 any one is used for microscopical imaging system, and it is characterized in that, also comprise controller, described controller is connected with light emitting diode matrix and camera installation respectively; Described controller is for controlling the switch of light emitting diode matrix and camera installation.
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