CN108508588A - A kind of multiple constraint information without lens holographic microphotography phase recovery method and its device - Google Patents
A kind of multiple constraint information without lens holographic microphotography phase recovery method and its device Download PDFInfo
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- CN108508588A CN108508588A CN201810368146.XA CN201810368146A CN108508588A CN 108508588 A CN108508588 A CN 108508588A CN 201810368146 A CN201810368146 A CN 201810368146A CN 108508588 A CN108508588 A CN 108508588A
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- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000011084 recovery Methods 0.000 title claims abstract description 17
- 238000003384 imaging method Methods 0.000 claims abstract description 23
- 238000003705 background correction Methods 0.000 claims abstract description 14
- 230000001427 coherent effect Effects 0.000 claims description 11
- 238000005286 illumination Methods 0.000 claims description 7
- 230000003287 optical effect Effects 0.000 claims description 6
- 238000001228 spectrum Methods 0.000 claims description 3
- 230000005540 biological transmission Effects 0.000 claims description 2
- 230000004907 flux Effects 0.000 claims description 2
- 238000012546 transfer Methods 0.000 claims description 2
- 238000004800 variational method Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims 1
- 239000000523 sample Substances 0.000 description 32
- 230000000007 visual effect Effects 0.000 description 8
- 230000001575 pathological effect Effects 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 2
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- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 238000001093 holography Methods 0.000 description 1
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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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- 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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Abstract
The invention discloses a kind of multiple constraint information without lens holographic microphotography phase recovery method and its device.This method comprises the following steps：S1：Light source is closed, darkfield image is acquired using no lens holographic microphotography device；S2：Light source is opened, the bright field image under light source uniform irradiation is acquired；S3：On the image sensor side place sample, ensure sample to imaging sensor distance be much smaller than sample to light source distance；Light source is opened, hologram image sequence is acquired；S4：Flat field correction is carried out to calculative any one hologram image；S5：Auto-focusing algorithm is carried out to the image after flat field correction, acquires the position of sample in space；S6：The Phase Retrieve Algorithm that multiple constraint information is used to the hologram image after auto-focusing, reconstructs the accurate amplitude and phase information of sample.The present invention can not increase system complexity and acquire the quantity of image, and accurate phase information is recovered from the hologram image taken.
Description
Technical field
The invention belongs to the extensive without lens holographic microphotography phase of the micro- field of no lens more particularly to a kind of multiple constraint information
Compound method and its device.
Background technology
The optical check of pathological section is the goldstandard in medical diagnosis on disease field.Traditional light microscope is in order to obtain high score
Resolution, it is necessary to use the object lens of enlargement ratio.In order to obtain high-resolution, the image of wide visual field and the big depth of field, it is necessary to depend on
Mechanical scanning, image mosaic and focus adjustment expand visual field and the depth of field, this not only makes imaging process complicate, but also significantly increase
The overall cost of system is added.Even so, the depth of field and temporal resolution still can be affected.
No lens holographic microscope has become new imaging technique in recent years.With the conventional photographic microscope based on lens
It compares, no lens holographic microscope directly samples the light through object, without being used between object and sensor plane
Any imaging len, therefore space-bandwidth product is no longer related to spatial resolution.Traditional automatic focusing is all by machinery side
Formula focuses to find the image planes for wishing to focus.Unlike, in holographic imaging, individual hologram contains whole pictures
The information in face allows for choosing the image planes for needing to focus automatically on computers by numerical algorithm in this way, therefore is referred to as to count
Word focuses automatically.The different piece of usual large area pathology sample tends to occur on different focal planes, therefore virologist
Just have the degree of freedom of bigger when checking sample.(sample and sensor plane apart from minimum, almost with single times of magnifying power
There is no amplification) without lens holographic microscope have size consistent with imaging sensor visual field, without any lens
With other intermediate optical elements.This further allows the simplification of imaging device, simultaneously effective avoids traditional based on lens
Imaging system in inevitably optical aberration and dispersion.In addition, whole system is compact-sized, cost-effectiveness is high, is resource
In limited scene, under big visual field the optical check of pathological section provide one kind and possible solution.
In previous research, using error-reduction algorithms (Mudanyali, Onur, et al. " Compact,
light-weight and cost-effective microscope based on lensless incoherent
holography for telemedicine applications."Lab on a Chip 10.11(2010):1417-
1428.) image taken to no lens holographic microphotography carries out phase recovery, and iterations are more, and convergence rate is slow, be easy by
To the influence of twin image noise, the reconstructed results of high quality can not be obtained.
Invention content
In order to overcome the defect of the above prior art, the purpose of the present invention is to provide a kind of multiple constraint information without lens
Holographic microphotography phase recovery method carries out Dynamic High-accuracy three-dimensional imaging under wide visual field to realize to biological sample.The present invention
Another purpose be to provide realize this method device
Realize that the technical solution of the object of the invention is：
A kind of multiple constraint information without lens holographic microphotography phase recovery method, include the following steps：
S1：Light source is closed, darkfield image is acquired using no lens holographic microphotography device；
S2：Light source is opened, the bright field image under light source uniform irradiation is acquired；
S3：Sample is placed by side on the image sensor, ensures that sample is much smaller than sample to light to the distance of imaging sensor
The distance in source；Light source is opened, hologram image sequence is acquired；
S4：Flat field correction is carried out to calculative any one hologram image；
S5：Auto-focusing algorithm is carried out to the image after flat field correction, acquires the position of sample in space；
S6：The Phase Retrieve Algorithm that multiple constraint information is used to the hologram image after auto-focusing, reconstructs the standard of sample
True amplitude and phase information.
A kind of multiple constraint information of the present invention is passed without lens holographic microphotography phase recovery device, including coherent source, image
Sensor and computer, the coherent light source illumination range cover whole image sensor plane, and sample is positioned over imaging sensor
The top of plane, and sample is much larger than sample at a distance from image sensor plane at a distance from coherent source；The coherent light
Emission lines polarized laser beam in source is incident on sample plane, sample scatter incident light, in described image sensor plane incident light
It is interfered with scattering light, interference figure i.e. hologram image is recorded in described image sensor, and the computer is to hologram image
Flat field correction and phase recovery are carried out, 3-D view is rebuild.
Compared with prior art, the present invention its remarkable advantage is：No lens holographic microphotography can meet big visual field and height simultaneously
The requirement of resolution ratio only passes through collected normal pictures in the case where not increasing system complexity and acquisition amount of images
Sequence is recovered hologram image accurately from the hologram image taken using gradient algorithm using a variety of constraint informations
Phase information, reconstruction image.Existing no lens holographic microscopy is in order to obtain the visual field of preferable resolution ratio and bigger, generally
Using coaxial configuration, reference wave propagates to imaging surface in the same direction with Object light wave, and forms inseparable original when rebuilding
Picture and conjugate image, referred to as twin image.Twin image both reduces picture quality, has also seriously affected the segmentation detection of target in image
With the extraction of other quantitative informations.Using the Phase Build Out method of multiple constraint information proposed by the present invention, can effectively inhibit
The influence of twin image noise on image quality in in-line holographic.
Description of the drawings
Fig. 1 is a kind of flow chart without lens holographic microphotography phase recovery method of multiple constraint information of the present invention.
Fig. 2 is a kind of structural schematic diagram without lens holographic microphotography of multiple constraint information of the present invention.
Fig. 3 is the hologram using device proposed by the present invention shooting.
Fig. 4 is calculated using the Phase Retrieve Algorithm and tradition error reduction of multiple constraint information proposed by the present invention
The comparing result of method, (a) original hologram, (b) hologram rebuild using tradition error reduction algorithms (c) are used
The hologram that the method for the present invention is rebuild.
Specific implementation mode
Referring to Fig. 1, the phase recovery method of multiple constraint information of the present invention is shot semi-transparent using no lens holographic microphotography device
The hologram of bright biological sample, and the amplitude and phase information of birth object sample are rebuild, steps are as follows：
S1：Light source is closed, the darkfield image of (the case where no environment stray light) acquisition sensor shooting under dark room conditions.
S2：Light source is opened, the light field under dark room conditions under (the case where no environment stray light) acquisition light source uniform irradiation
Image.
S3：Referring to Fig. 2, no lens holographic microphotography device of the invention includes coherent illumination source, (the pathology magic lantern of sample 1
Piece), imaging sensor 2, coherent illumination source includes LED light source 3, colour filter 4 and aperture 5, can enhance incident light by aperture 5
Coherence.And visual field is in the same size with sensor chip.Light source range of exposures covers entire sensor plane, and sample 1 is put
Be placed in above imaging sensor 2 plane, and sample 1 and light source distance much larger than sample 1 and 2 plane of imaging sensor at a distance from.
The light in coherent illumination source is incident on 1 plane of sample, and sample 1 scatters incident light, in 2 plane incident light of imaging sensor and scattering light
It interferes.Coherent illumination source, which is placed on, to be arrived away from pathological glass slide 7 at 15cm, and imaging sensor 2 is located at sample
(Z2) at 100 to 600 millimeters of lower section.Therefore, the image of sample 1 is not shot directly, and practical recorded by imaging sensor 2 is done
Pattern is related to, 3-D view is then rebuild.The hologram of shooting is referring to Fig. 3.
S4：Flat field correction is carried out to calculative any one hologram；
In the step, any one hologram of holographic graphic sequence is selected, it will be by the dark of the acquisition described in step S1
Field picture is expressed as Id, I will be expressed as by the bright field image of the acquisition described in step S2O, will be by described in step S3
Calculative any one hologram is expressed as I in the holographic graphic sequence of acquisition, then the image Ic after flat field correction is carried out to I
It is expressed as：
Flat field correction is done to hologram image and obtains a width relative value image, harmful effect can't be generated to image procossing,
And the case where each pixel response differs can be eliminated, the even non-uniform problem of the image value brought of uneven illumination is alleviated.
S5：Hologram backpropagation after the flat field correction for being obtained step S4 using the angular spectrum method of scalar diffraction theory
To different height plane, backpropagation it is opposite be light propagation direction, reversely refer to light from sensor plane toward sample plane
It propagates.The propagation phenomenon of the light spatial filter linear as one, transmission function are expressed as by angular spectrum method：
The light distribution of different height plane in space is obtained with this.Wherein, fXAnd fYRepresentation space frequency, λ indicate light
Wavelength, z indicate the distance propagated.It is (logical by the derivative for calculating brightness of image using auto-focusing (Autofocusing) algorithm
Often to ask single order to lead with sobel operators) variance judge the clarity for the image for propagating back to different height, variance is maximum
Image be considered as most clearly image, its corresponding height be considered as the sample of frame shooting it is practical arrive sensor away from
From.The level where each frame, the i.e. distance of sample plane to sensor plane are obtained in this way；When the big face of shooting
When long-pending pathology sample, when being also at different depth such as sample different zones, then by sample piecemeal calculate separately from
Dynamic focus algorithm, finds out the actual grade residing for each region.
S6：The Phase Retrieve Algorithm of multiple constraint information, object function are carried out to the picture after auto-focusing in step S5
As follows：
Wherein M indicates that collected sample is located at the hologram image of different depth.Imaging model y- | Hx | it indicates, H tables
Show holographic operator,It indicatesA norm, that is, its sum of absolute value.Indicate two-dimensional First-order Gradient operator.
This can also be referred to as full variation regular terms, it can be punished in the case where not influencing image border occurs in neighborhood
Pixel value saltus step is the active constraint condition in image denoising.α is the weight of full variational methods item, is generally taken between 0.5-1.This to image it is considered that be applied with a physical constraint (optical transfer system of sample is not more than 1).L be by
The set that all pixels of image are constituted is collected, it is 1 to take the luminous flux in the not region of sample, and any there are the regions of sample
Pixel value all should be less than 1.In dead beat setting-out this in the case of, one is first shot only to the picture of light source.Specific in each step
In iteration, all limit the image that the step acquires to meet this condition, i.e., image value should be less than shooting in advance only to light source
Imaging, to being unsatisfactory for the pixel of this condition, is projected into this feasible zone, that is, will be greater than background luminance
Value is adjusted to background value.In actually solving, the present embodiment is called alternating proximal gradient (Alternating using one kind
Proximal gradient method) algorithm.The hologram rebuild using traditional algorithm is referring to Fig. 4 (b), using the present invention
The hologram of a kind of multiple constraint information proposed rebuild without lens holographic microphotography phase recovery method is referring to Fig. 4 (c).From Fig. 4
As can be seen that using the Phase Build Out method of multiple constraint information proposed by the present invention, compared to conventional method, in image by twin
Life has obtained effective inhibition as noise reasons for its use diffraction ring, improves image quality.
Claims (5)
1. a kind of multiple constraint information without lens holographic microphotography phase recovery method, which is characterized in that include the following steps：
S1：Light source is closed, darkfield image is acquired using no lens holographic microphotography device；
S2：Light source is opened, the bright field image under light source uniform irradiation is acquired；
S3：Sample is placed by side on the image sensor, ensures that the distance of sample to imaging sensor arrives light source much smaller than sample
Distance；Light source is opened, hologram image sequence is acquired；
S4：Flat field correction is carried out to calculative any one hologram image；
S5：Auto-focusing algorithm is carried out to the image after flat field correction, acquires the position of sample in space；
S6：The Phase Retrieve Algorithm that multiple constraint information is used to the hologram image after auto-focusing, reconstructs the accurate width of sample
Degree and phase information.
2. a kind of multiple constraint information according to claim 1 without lens holographic microphotography phase recovery method, feature exists
In in step S4, the method for carrying out flat field correction is：The darkfield image acquired in step S1 is expressed as Id, will be in step S2
The bright field image of acquisition is expressed as I0, by calculative any one hologram in the hologram image sequence acquired in step S3
As being expressed as I, the image Ic after flat field correction is carried out to image I and is expressed as：
3. a kind of multiple constraint information according to claim 1 without lens holographic microphotography phase recovery method, feature exists
In, in step S5, using scalar diffraction theory angular spectrum transmission method by hologram image sequence each image reversal pass
Cast to different height plane, wherein backpropagation refers to opposite with the direction of light propagation；Then it is obtained using auto-focusing algorithm
The height relative to imaging sensor plane in sample where each particle.
4. a kind of multiple constraint information according to claim 1 without lens holographic microphotography phase recovery method, feature exists
In in step S6, the object function of the Phase Retrieve Algorithm of multiple constraint information is as follows：
Wherein M indicates that collected sample is located at the hologram image of different depth；Imaging model y- | Hx | it indicates, H indicates complete
Operator is ceased, α is the weight of full variational methods item,It indicatesNorm, that is, its sum of absolute value；Indicate two
The First-order Gradient operator of dimension；To be applied with a physical constraint to image, i.e. the optical transfer system of sample is not more than
1, wherein L is the set being made of all pixels of the image collected, and it is 1 to take the luminous flux in the not region of sample, any
There are the pixel values in the region of sample all should be less than 1.
5. a kind of multiple constraint information without lens holographic microphotography phase recovery device, which is characterized in that including coherent source, image
Sensor and computer, the coherent light source illumination range cover whole image sensor plane, and sample is positioned over image sensing
The top of device plane, and sample is much larger than sample at a distance from image sensor plane at a distance from coherent source；It is described relevant
The polarized laser beam of light source emission lines is incident on sample plane, and sample scatter incident light is incident in described image sensor plane
Light is interfered with scattering light, and interference figure i.e. hologram image is recorded in described image sensor, and the computer is to hologram
As carrying out flat field correction and phase recovery, 3-D view is rebuild.
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| CN113168131A (en) * | 2018-12-04 | 2021-07-23 | 加利福尼亚大学董事会 | Portable ultraviolet holographic microscope for high-contrast protein crystal imaging |
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