CN113985593A - Portable coaxial digital holographic microscope based on 3D printing technology and imaging method - Google Patents
Portable coaxial digital holographic microscope based on 3D printing technology and imaging method Download PDFInfo
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- G02B21/00—Microscopes
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- G02B21/0008—Microscopes having a simple construction, e.g. portable microscopes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/18—Arrangements with more than one light path, e.g. for comparing two specimens
- G02B21/20—Binocular arrangements
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- 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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Abstract
The invention discloses a portable coaxial digital holographic microscope based on a 3D printing technology and an imaging method. The device comprises a base, a supporting plate connected with the upper end of the base, a light source loading groove connected with the upper end of the supporting plate, a light source connected with the inside of the light source loading groove, an objective lens connected with the upper end of the base, a reflector loading table connected with the inside, a reflector and an image sensor, wherein the signal output end of the image sensor is connected with a computer. When light emitted from the LED light source passes through a sample, the light scattered by the sample is used as object light, the light not scattered by the sample is used as reference light, the two beams of light are superposed and amplified by an objective lens, then the two beams of light are received by an image sensor and displayed on a computer to form a holographic image, and then the computer is used for calculating and reconstructing the obtained holographic image to obtain a light intensity image of the three-dimensional position and the focal plane of the sample. In addition, the device is low in cost, convenient to use, and light source and micro objective can change by oneself in order to adapt to the observation of the not unidimensional microcosmic object, are fit for using widely.
Description
Technical Field
The invention relates to the technical field of digital holographic microscopy, in particular to a portable coaxial digital holographic microscope based on a 3D printing technology.
Background
With the deep exploration of the micro world, the microscopic technology is more and more concerned, and people not only realize the observation of objects with smaller size, but also realize the spanning from the recording of two-dimensional information of the objects to the recording of three-dimensional information. Digital holographic microscopy is a non-contact, label-free, real-time imaging technique. The digital holographic microscope reconstructs the recorded light field information to obtain the three-dimensional information of the observed sample, thereby realizing the three-dimensional appearance observation and the three-dimensional motion behavior tracking of small-size objects such as target particles and the like.
Digital holographic microscopes can be classified by structure as on-axis digital holographic microscopes and off-axis digital holographic microscopes. The coaxial digital holographic microscope has a simple structure, a sample to be observed is scattered into object light by light emitted by a light source, the unscattered light is used as reference light, the object light and the reference light have the same optical axis and the same transmission direction, interference fringes with alternate light and shade are formed, and the interference fringes are amplified by a microscope objective and recorded by a camera to obtain a hologram. Finally, three-dimensional information of the observed target fine particles and the like is obtained through computer calculation and reconstruction.
The existing digital holographic microscopes have the defects of large occupied space, heavy weight, difficulty in moving and the like, cannot adapt to the observation requirements in a changing environment, and particularly have special requirements on the observation environment or time. The invention integrally designs and manufactures the coaxial digital holographic microscope by a 3D printing technology, and solves the defects that the existing digital holographic microscope needs to occupy larger space, is difficult to move, has high cost and is complex in light path adjustment. The digital holographic microscope can be carried about, has light weight, small space restriction and flexible design, can accurately carry out three-dimensional imaging and tracking on microscopic moving targets such as target particles and the like, can be used in changeable and flexible application scenes such as an incubator, a field and the like, and can carry out nondestructive, in-situ and dynamic three-dimensional monitoring on microscopic target objects in the fields of biomedicine, material science and the like. In addition, the device is low in cost, convenient to use, and light source and micro objective can change by oneself in order to adapt to the observation of the not unidimensional microcosmic object, are fit for using widely.
Disclosure of Invention
The invention aims to solve the defects that the existing digital holographic microscope needs to occupy larger space, is not easy to move, has high cost and has complex light path adjustment. The digital holographic microscope can be carried about, has light weight, small space restriction and flexible design, can accurately carry out three-dimensional imaging and tracking on microscopic moving targets such as target particles and the like, can be used in changeable and flexible application scenes such as an incubator, a field and the like, and can carry out nondestructive, in-situ and dynamic three-dimensional monitoring on microscopic target objects in the fields of biomedicine, material science and the like. In addition, the device is low in cost, convenient to use, and light source and micro objective can change by oneself in order to adapt to the observation of the not unidimensional microcosmic object, are fit for using widely.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a portable coaxial holographic microscopic device adopting 3D printing technology comprises an LED light source 4, an objective lens 5, a reflector 9, a rotating worm 6, a rotating turbine 7, an image sensor 11, an integrated structure manufactured by the 3D printing technology, an object stage 12 and a reflector loading stage 8, wherein the integrated structure comprises a base 1, a support plate 2 and a light source loading groove 3;
furthermore, a supporting plate 2 is fixedly connected to the upper end of a base 1 in an integrated structure manufactured by a 3D printing technology, the upper end of the base 1 is connected with a microscope objective 5, and a slot of a reflector loading table 8 and a camera loading groove 10 are reserved inside the base so as to be respectively provided with the reflector loading table 8 and an image sensor 11 which are provided with a reflector 9; the upper end of the supporting plate 2 is connected with a light source loading groove 3 for installing and fixing an LED light source 4; the base is provided with a worm hole which is connected with a horizontal worm 6 and a vertical worm wheel 7 which is matched with the horizontal worm, and the vertical worm wheel 7 is connected with the base 1, the object stage 12 and the light source loading groove 3. The worm 6 in the horizontal direction can be rotated to drive the worm wheel 7 in the vertical direction, so that the height of the object stage 12 from the microscope objective 5 can be adjusted.
Further, the wavelength of the LED light source 4 is 250nm < λ <1100 nm.
Further, the magnification of the microscope objective 5 is 1, 2, 5, 10, 20, 40, 50, 60 or 100.
Further, the center of the LED light source 4, the center of the opening of the light source loading slot 3, the sample to be measured 13, the center of the opening of the stage 12, the center of the microscope objective 5, the center of the opening of the reflector loading stage 8, the center of the reflector 9, and the center of the image sensor 11 are arranged in sequence along the light propagation direction.
Further, the image sensor 11 records a hologram, which is reconstructed by a computer 14.
The imaging method of the portable digital holographic microscopic device based on the 3D printing technology is characterized by comprising the following specific steps:
A. turning on the power of the LED light source 4 and the image sensor 11;
B. and adjusting the position of the object stage 7 to determine the focusing position of the sample 13 to be measured. Defocusing, and recording a hologram of the sample to be detected;
C. processing the hologram by the computer 14, calculating and deducting a background image from the hologram to obtain a hologram with the background image deducted;
D. reconstructing the holographic image after deducting the background image to obtain a reconstructed holographic image;
E. and (3) carrying out threshold filtering and searching for a local maximum value of light intensity on the three-dimensional reconstructed intensity distribution information of the particles to obtain the three-dimensional position of each particle.
Further, the background image is obtained by calculating the average light intensity of the multiple frames of holographic images, and the calculation is as follows:
in the formula Ib(x, y) is the gray scale value of the pixel at the (x, y) position in the background image, N is the total frame number of the hologram, t is the time, Io(x, y) is the gray value of the pixel at the (x, y) position in the original hologram at the time t, and the light intensity value I of each pixel point in the hologram after the background is deducteds(x,y)：
Is(x,y)＝Io(x,y)-Ib(x,y)。
Further, the specific process of reconstructing the holographic image after deducting the background image is as follows: reconstructing the holographic light field by using a Rayleigh-Sommerfeld algorithm to obtain light intensity information and phase information corresponding to all particles in a set range, calculating the light intensity information only without storing the phase information due to interference of twin phases, wherein the calculation formula is as follows:
U(r,z)＝FT-1(FT(Is(r,0)·H(q,-z)))，
wherein h (r, -z) is a propagation operator, r is an initial transverse coordinate of the particle, and z is an initial axial coordinate of the particle; i is an imaginary unit; k is the wave number; r is the light propagation distance; i issIs the light intensity of the particle; FT-1Is inverse Fourier transform; FT is Fourier transform; h (q, -z) is the Fourier transform of H (r, -z).
Further, obtaining the three-dimensional position of the particle by a light intensity threshold value and searching for the maximum local light intensity; setting a light intensity threshold lower limit T, and filtering noise lower than the light intensity threshold through the light intensity threshold lower limit T; in a cube with similar observed particle sizes, the three-dimensional position of the maximum light intensity value is searched point by point.
Compared with the prior art, the invention has the following advantages: the invention has flexible design, small volume and light weight, can be used in diversified and flexible application scenes such as in a culture box and in the field, and can carry out nondestructive, in-situ and dynamic three-dimensional monitoring on microscopic target objects in the fields of biomedicine, material science and the like. In addition, the device is low in cost and convenient to use, and the light source and the microscope objective can be replaced by themselves to adapt to observation of different microscopic target objects, so that the device is suitable for popularization and use.
Drawings
FIG. 1 is a schematic diagram of a portable coaxial digital holographic microscope based on 3D printing technology according to the present invention;
FIG. 2 is a diatom hologram recorded by the image sensor of example 1 of the present invention;
FIG. 3 is a background-subtracted hologram according to example 1 of the present invention;
FIG. 4 is a partial magnified view of the background-subtracted hologram of example 1 of the present invention;
FIG. 5 is a schematic diagram of background subtraction according to the present invention;
FIG. 6 is a schematic diagram of a light intensity map obtained by reconstruction calculation after background subtraction according to the present invention;
fig. 7 is a restored image after computed reconstruction of a hologram according to example 1 of the present invention;
fig. 8 is a partially enlarged view of a restored image after computed reconstruction of a hologram according to example 1 of the present invention;
FIG. 9 is a hologram of Escherichia coli recorded by the image sensor in example 2 of the present invention;
FIG. 10 is a background-subtracted hologram according to example 2 of the present invention;
FIG. 11 is a partial magnified view of the background-subtracted hologram of example 2 of the present invention;
fig. 12 is a restored image after computed reconstruction of a hologram according to example 2 of the present invention;
fig. 13 is a partially enlarged view of a restored image after computed reconstruction of a hologram according to example 2 of the present invention;
FIG. 14 is a hologram of a silica pellet recorded by an image sensor according to example 3 of the present invention;
FIG. 15 is a background-subtracted hologram according to example 3 of the present invention;
fig. 16 is a restored image after the hologram calculation reconstruction in example 3 of the present invention;
fig. 17 is a partially enlarged view of a restored image after computed reconstruction of a hologram according to example 3 of the present invention;
FIG. 18 is a hologram of red blood cells recorded by the image sensor according to example 4 of the present invention;
FIG. 19 is a background-subtracted hologram according to example 4 of the present invention;
FIG. 20 is a partial magnified view of the background-subtracted hologram of example 4 of the present invention;
fig. 21 is a restored image after the hologram calculation reconstruction in example 4 of the present invention;
fig. 22 is a partially enlarged view of the restored image after the hologram calculation reconstruction in example 4 of the present invention.
The various components in the figure are as follows:
the device comprises a base 1, a supporting plate 2, a light source loading groove 3, a light source 4, a microscope objective 5, a rotating worm 6, a rotating worm wheel 7, a reflector loading table 8, a reflector 9, an image sensor loading groove 10, an image sensor 11, an objective table 12, a sample to be detected 13 and a computer 14.
Detailed description of the invention
The present invention will be described in further detail with reference to the following examples and drawings, but the scope of application of the present invention is not limited thereto.
Example 1
As shown in the figure I, the portable coaxial digital holographic microscope based on the 3D printing technology adopts an LED light source 4 with adjustable light intensity, and the LED light source 4 is placed and fixed in a light source loading groove 3, wherein the center of the LED light source 4 is coincident with the center of the light source loading groove 3. The horizontal rotating worm 6 is connected with the base, and then the two vertical rotating worms 6 sequentially pass through the light source loading groove 3, the object stage 12 and the base 2 respectively and are finally connected with the horizontal worm 6. The height of the object stage 12 is adjusted by rotating the horizontal rotating worm 6 to drive the two vertical rotating worm wheels 7 to rotate, so as to control the distance between the sample 13 to be measured placed on the object stage 12 and the microscope objective 5. The wavelength of the adjustable LED light source 4 is 528nm, and the method multiple of the microscope objective 5 is 10;
the light that LED light source 4 sent passes through the central hole of light source loading groove 3 back light source light's propagation direction is the vertical direction, and the light of vertical propagation passes the trompil center that places objective table 4 of the sample 13 that awaits measuring and reaches microscope objective 5, and partly light is the object light through the sample 13 scattering that awaits measuring, and another part is as the reference light, and two bundles of light superposes and produces the interference fringe. The light reaches the mirror 9 fitted in the center of the mirror loading table 8 after being amplified by the microscope objective 5, and the reflected light is received by the image sensor 11 fixed in the image sensor loading slot 10 and recorded as a hologram shown in fig. two; subtracting the background image from the holographic image by the computer 14 to obtain a holographic image with the background image subtracted as shown in the third drawing, wherein a partial enlarged view of the holographic image is shown in the fourth drawing;
the sample 13 to be tested is selected as diatom liquid;
the background image is obtained by calculating the average light intensity of the multi-frame holographic image, and the calculation is as follows:
in the formula Ib(x, y) is the gray scale value of the pixel at the (x, y) position in the background image, N is the total frame number of the hologram, t is the time, Io(x, y) is the gray value of the pixel at the (x, y) position in the original hologram at the time t, and the light intensity value I of each pixel point in the hologram after the background is deducteds(x,y)：
Is(x,y)＝Io(x,y)-Ib(x,y)。
In this embodiment, the background light intensity is obtained by an averaging method, that is, the average value of a plurality of holograms continuously shot is calculated and subtracted from each hologram to eliminate the interference caused by the background signal, and a schematic diagram thereof is shown in fig. five.
Reconstructing the holographic image after deducting the background image to obtain a reconstructed holographic image; the specific process is as follows: reconstructing the holographic light field by using a Rayleigh-Sommerfeld algorithm to obtain light intensity information and phase information corresponding to all particles in a set range, calculating the light intensity information only without storing the phase information due to interference of twin phases, wherein the calculation formula is as follows:
U(r,z)＝FT-1(FT(Is(r,0)·H(q,-z)))，
wherein h (r, -z) is a propagation operator, r is an initial transverse coordinate of the particle, and z is an initial axial coordinate of the particle; i is an imaginary unit; k is the wave number; r is the light propagation distance; i issIs the light intensity of the particle; FT-1Is inverse Fourier transform; FT is Fourier transform; h (q, -z) is the Fourier transform of H (r, -z).
Obtaining the three-dimensional position of the particle by the light intensity threshold value and searching the maximum local light intensity; the specific process is as follows: setting a light intensity threshold lower limit T, and filtering noise lower than the light intensity threshold through the light intensity threshold lower limit T; in a cube with similar observed particle sizes, the three-dimensional position of the maximum light intensity value is searched point by point, namely the three-dimensional focal plane position of the target particle. The reconstructed image at the position with the maximum light intensity value is the light intensity image of the corresponding particles in the holographic image at the focal plane. The schematic diagram of the process is shown in fig. six, and fig. seven is a light intensity diagram of the focal plane of the sample to be measured 13 obtained by reconstruction, and a partially enlarged diagram of the light intensity diagram is shown in fig. eight.
Example 2
As shown in the figure I, the portable coaxial digital holographic microscope based on the 3D printing technology adopts an LED light source 4 with adjustable light intensity, and the LED light source 4 is placed and fixed in a light source loading groove 3, wherein the center of the LED light source 4 is coincident with the center of the light source loading groove 3. The horizontal rotating worm 6 is connected with the base, and then the two vertical rotating worms 6 sequentially pass through the light source loading groove 3, the object stage 12 and the base 2 respectively and are finally connected with the horizontal worm 6. The height of the object stage 12 is adjusted by rotating the horizontal rotating worm 6 to drive the two vertical rotating worm wheels 7 to rotate, so as to control the distance between the sample 13 to be measured placed on the object stage 12 and the microscope objective 5. The wavelength of the adjustable LED light source 4 is 528nm, and the method multiple of the microscope objective 5 is 10;
the light that LED light source 4 sent passes through the central hole of light source loading groove 3 back light source light's propagation direction is the vertical direction, and the light of vertical propagation passes the trompil center that places objective table 4 of the sample 13 that awaits measuring and reaches microscope objective 5, and partly light is the object light through the sample 13 scattering that awaits measuring, and another part is as the reference light, and two bundles of light superposes and produces the interference fringe. The light reaches the mirror 9 fitted in the center of the mirror loading table 8 after being amplified by the microscope objective 5, and the reflected light is received by the image sensor 11 fixed in the image sensor loading slot 10 and recorded as a hologram shown in fig. nine; subtracting the background image from the holographic image by the computer 14 to obtain a holographic image with the background image subtracted, as shown in fig. ten, and a partially enlarged view of the holographic image is shown in fig. eleven;
the sample 13 to be detected is selected as escherichia coli liquid;
the background image is obtained by calculating the average light intensity of the multi-frame holographic image, and the calculation is as follows:
in the formula Ib(x, y) is the gray scale value of the pixel at the (x, y) position in the background image, N is the total frame number of the hologram, t is the time, Io(x, y) is the gray value of the pixel at the (x, y) position in the original hologram at the time t, and the light intensity value I of each pixel point in the hologram after the background is deducteds(x,y)：
Is(x,y)＝Io(x,y)-Ib(x,y)。
In this embodiment, the background light intensity is obtained by an averaging method, that is, the average value of a plurality of holograms continuously shot is calculated and subtracted from each hologram to eliminate the interference caused by the background signal, and a schematic diagram thereof is shown in fig. five.
Reconstructing the holographic image after deducting the background image to obtain a reconstructed holographic image; the specific process is as follows: reconstructing the holographic light field by using a Rayleigh-Sommerfeld algorithm to obtain light intensity information and phase information corresponding to all particles in a set range, calculating the light intensity information only without storing the phase information due to interference of twin phases, wherein the calculation formula is as follows:
U(r,z)＝FT-1(FT(Is(r,0)·H(q,-z)))，
wherein h (r, -z) is a propagation operator, r is an initial transverse coordinate of the particle, and z is an initial axial coordinate of the particle; i is an imaginary unit; k is the wave number; r is the light propagation distance; i issIs the light intensity of the particle; FT-1Is inverse Fourier transform; FT is Fourier transform; h (q, -z) is the Fourier transform of H (r, -z).
Obtaining the three-dimensional position of the particle by the light intensity threshold value and searching the maximum local light intensity; the specific process is as follows: setting a light intensity threshold lower limit T, and filtering noise lower than the light intensity threshold through the light intensity threshold lower limit T; in a cube with similar observed particle sizes, the three-dimensional position of the maximum light intensity value is searched point by point, namely the three-dimensional focal plane position of the target particle. The reconstructed image at the position with the maximum light intensity value is the light intensity image of the corresponding particles in the holographic image at the focal plane. The schematic diagram of the process is shown in fig. six, and fig. twelve is a light intensity diagram of the focal plane of the sample to be measured 13 obtained by reconstruction, and a partially enlarged view of the light intensity diagram is shown in fig. thirteen.
Example 3
As shown in the figure I, the portable coaxial digital holographic microscope based on the 3D printing technology adopts an LED light source 4 with adjustable light intensity, and the LED light source 4 is placed and fixed in a light source loading groove 3, wherein the center of the LED light source 4 is coincident with the center of the light source loading groove 3. The horizontal rotating worm 6 is connected with the base, and then the two vertical rotating worms 6 sequentially pass through the light source loading groove 3, the object stage 12 and the base 2 respectively and are finally connected with the horizontal worm 6. The height of the object stage 12 is adjusted by rotating the horizontal rotating worm 6 to drive the two vertical rotating worm wheels 7 to rotate, so as to control the distance between the sample 13 to be measured placed on the object stage 12 and the microscope objective 5. The wavelength of the adjustable LED light source 4 is 528nm, and the method multiple of the microscope objective 5 is 40;
the light that LED light source 4 sent passes through the central hole of light source loading groove 3 back light source light's propagation direction is the vertical direction, and the light of vertical propagation passes the trompil center that places objective table 4 of the sample 13 that awaits measuring and reaches microscope objective 5, and partly light is the object light through the sample 13 scattering that awaits measuring, and another part is as the reference light, and two bundles of light superposes and produces the interference fringe. The light reaches the mirror 9 fitted in the center of the mirror loading table 8 after being amplified by the microscope objective 5, and the reflected light is received by the image sensor 11 fixed in the image sensor loading slot 10 and recorded as a hologram shown in fig. fourteen; subtracting the background image from the holographic image by the computer 14 to obtain a holographic image with the background image subtracted as in the fifteen image, wherein a partially enlarged view of the holographic image is shown as the sixteen image;
the sample 13 to be tested is selected as a silica pellet with a diameter of 12 μm;
the background image is obtained by calculating the average light intensity of the multi-frame holographic image, and the calculation is as follows:
in the formula Ib(x, y) is the gray scale value of the pixel at the (x, y) position in the background image, N is the total frame number of the hologram, t is the time, Io(x, y) is the gray value of the pixel at the (x, y) position in the original hologram at the time t, and the light intensity value I of each pixel point in the hologram after the background is deducteds(x,y)：
Is(x,y)＝Io(x,y)-Ib(x,y)。
In this embodiment, the background light intensity is obtained by an averaging method, that is, the average value of a plurality of holograms continuously shot is calculated and subtracted from each hologram to eliminate the interference caused by the background signal, and a schematic diagram thereof is shown in fig. five.
Reconstructing the holographic image after deducting the background image to obtain a reconstructed holographic image; the specific process is as follows: reconstructing the holographic light field by using a Rayleigh-Sommerfeld algorithm to obtain light intensity information and phase information corresponding to all particles in a set range, calculating the light intensity information only without storing the phase information due to interference of twin phases, wherein the calculation formula is as follows:
U(r,z)＝FT-1(FT(Is(r,0)·H(q,-z)))，
wherein h (r, -z) is a propagation operator, r is an initial transverse coordinate of the particle, and z is an initial axial coordinate of the particle; i is an imaginary unit; k is the wave number; r is the light propagation distance; i issIs the light intensity of the particle; FT-1Is inverse Fourier transform; FT is Fourier transform; h (q, -z) is the Fourier transform of H (r, -z 0.
Obtaining the three-dimensional position of the particle by the light intensity threshold value and searching the maximum local light intensity; the specific process is as follows: setting a light intensity threshold lower limit T, and filtering noise lower than the light intensity threshold through the light intensity threshold lower limit T; in a cube with similar observed particle sizes, the three-dimensional position of the maximum light intensity value is searched point by point, namely the three-dimensional focal plane position of the target particle. The reconstructed image at the position with the maximum light intensity value is the light intensity image of the corresponding particles in the holographic image at the focal plane. The schematic diagram of the process is shown in fig. six, fig. seventeen is a light intensity diagram of the focal plane of the sample to be measured 13 obtained through reconstruction, and a partially enlarged diagram of the light intensity diagram is shown in fig. eighteen.
Example 4
As shown in the figure I, the portable coaxial digital holographic microscope based on the 3D printing technology adopts an LED light source 4 with adjustable light intensity, and the LED light source 4 is placed and fixed in a light source loading groove 3, wherein the center of the LED light source 4 is coincident with the center of the light source loading groove 3. The horizontal rotating worm 6 is connected with the base, and then the two vertical rotating worms 6 sequentially pass through the light source loading groove 3, the object stage 12 and the base 2 respectively and are finally connected with the horizontal worm 6. The height of the object stage 12 is adjusted by rotating the horizontal rotating worm 6 to drive the two vertical rotating worm wheels 7 to rotate, so as to control the distance between the sample 13 to be measured placed on the object stage 12 and the microscope objective 5. The wavelength of the adjustable LED light source 4 is 528nm, and the method multiple of the microscope objective 5 is 40;
the light that LED light source 4 sent passes through the central hole of light source loading groove 3 back light source light's propagation direction is the vertical direction, and the light of vertical propagation passes the trompil center that places objective table 4 of the sample 13 that awaits measuring and reaches microscope objective 5, and partly light is the object light through the sample 13 scattering that awaits measuring, and another part is as the reference light, and two bundles of light superposes and produces the interference fringe. The light reaches the mirror 9 fitted in the center of the mirror loading table 8 after being amplified by the microscope objective 5, and the reflected light is received by the image sensor 11 fixed in the image sensor loading slot 10 and recorded as a hologram shown in nineteenth drawing; subtracting the background image from the holographic image by the computer 14 to obtain a holographic image with the background image subtracted, such as twenty;
the test sample 13 is here selected as red blood cells;
the background image is obtained by calculating the average light intensity of the multi-frame holographic image, and the calculation is as follows:
in the formula Ib(x, y) is the gray scale value of the pixel at the (x, y) position in the background image, N is the total frame number of the hologram, t is the time, Io(x, y) is the gray value of the pixel at the (x, y) position in the original hologram at the time t, and the light intensity value I of each pixel point in the hologram after the background is deducteds(x,y)：
Is(x,y)＝Io(x,y)-Ib(x,y)。
In this embodiment, the background light intensity is obtained by an averaging method, that is, the average value of a plurality of holograms continuously shot is calculated and subtracted from each hologram to eliminate the interference caused by the background signal, and a schematic diagram thereof is shown in fig. five.
Reconstructing the holographic image after deducting the background image to obtain a reconstructed holographic image; the specific process is as follows: reconstructing the holographic light field by using a Rayleigh-Sommerfeld algorithm to obtain light intensity information and phase information corresponding to all particles in a set range, calculating the light intensity information only without storing the phase information due to interference of twin phases, wherein the calculation formula is as follows:
U(r,z)＝FT-1(FT(Is(r,0)·H(q,-z)))，
wherein h (r, -z) is a propagation operator, r is an initial transverse coordinate of the particle, and z is an initial axial coordinate of the particle; i is an imaginary unit; k is the wave number; r is the light propagation distance; i issIs the light intensity of the particle; FT-1Is inverse Fourier transform; FT is Fourier transform; h (q, -z) is the Fourier transform of H (r, -z).
Obtaining the three-dimensional position of the particle by the light intensity threshold value and searching the maximum local light intensity; the specific process is as follows: setting a light intensity threshold lower limit T, and filtering noise lower than the light intensity threshold through the light intensity threshold lower limit T; in a cube with similar observed particle sizes, the three-dimensional position of the maximum light intensity value is searched point by point, namely the three-dimensional focal plane position of the target particle. The reconstructed image at the position with the maximum light intensity value is the light intensity image of the corresponding particles in the holographic image at the focal plane. The schematic diagram of the process is shown in fig. six, fig. twenty-one is a light intensity diagram of the focal plane of the sample to be measured 13 obtained through reconstruction, and a partial enlarged view of the light intensity diagram is shown in fig. twenty-two.
Claims (10)
1. A portable coaxial digital holographic microscope based on a 3D printing technology is manufactured by adopting the 3D printing technology to form an integrated structure, and is characterized in that the integrated structure consists of a base (1) at the lower end, a supporting plate (2) is fixedly connected to the upper end of the base (1), a light source loading groove (3) is fixedly connected to the upper end of the supporting plate (2), an LED light source (4) is arranged and fixed in the light source loading groove, a microscope objective (5) is connected to the upper end of the base (1), an inserted reflector loading table (8) is arranged in the base (1), a reflector (9) is loaded in the center of the reflector loading table (8), an image sensor loading groove (10) is arranged in the base (1), an image sensor (11) is loaded in the image sensor loading groove (10), and a worm hole is reserved at the upper end of the base (1), a horizontal rotating worm (6) is connected in the base (1), two ends of the horizontal rotating worm (6) are connected with a vertical rotating worm wheel (7), the middle section of the vertical rotating worm wheel (7) is connected with an objective table (12), and the upper end of the vertical rotating worm wheel is connected with a light source loading groove (3); the image sensor (11) is connected with a computer (14).
2. The portable coaxial digital holographic microscope based on 3D printing technology according to claim 1, characterized in that the base (1), the support plate (2) and the light source loading slot (3) are of a unitary structure;
micro objective (5) is connected to integrated device's lower extreme base (1) upper end, base (1) internal connection has male speculum load table (8), speculum load table (8) are connected with speculum (9), base (1) inside leaves image sensor load cell (10), image sensor load cell (10) are connected with image sensor (11).
3. The portable coaxial digital holographic microscope based on 3D printing technology according to claim 1, characterized in that the number of the rotating worms is three, and the rotating worms comprise a horizontal rotating worm (6) and two vertical rotating worm wheels (7), one horizontal rotating worm (6) is horizontally connected in the base (1), two vertical rotating worm wheels (7) and horizontal rotating worm (6) are vertically connected, and the two vertical rotating worm wheels (7) are connected with the base (1) and the object stage (12).
4. The portable coaxial digital holographic microscope based on 3D printing technology according to claim 1, characterized in that the LED light source (4) has a wavelength of 250nm < λ <1100 nm;
the magnification of the microscope objective (5) is selected to be 1, 2, 5, 10, 20, 40, 50, 60 or 100.
5. The portable coaxial digital holographic microscope based on 3D printing technology as claimed in claim 1, characterized in that the LED light source (4) center, the light source loading slot (3) center, the stage (12) center, the microscope objective (5) center, the reflector loading stage (8) center, the reflector (9) center are on the same vertical line.
6. The portable coaxial digital holographic microscope based on 3D printing technology as claimed in claim 1, characterized in that the light emitted by the LED light source (4) firstly passes through the light source loading slot (3), propagates through the stage (12) and the microscope objective (5) in turn to reach the reflector (9), and is reflected into the image sensor (11) via the reflector (9) to record the image.
7. The imaging method of the portable digital holographic microscopy device based on the 3D printing technology, which is based on any one of the claims 1-6, is characterized by comprising the following specific steps:
A. turning on power to the LED light source (4) and the image sensor (11);
B. and adjusting the position of the object stage to determine the focusing position. Then, carrying out moderate defocusing, and recording a sample hologram through an image sensor;
C. processing the hologram by a computer (14), and deducting a background image from the hologram to obtain a hologram with the background image deducted;
D. carrying out three-dimensional reconstruction on the holographic image after deducting the background image to obtain a reconstructed holographic image;
E. and (3) carrying out threshold filtering on the three-dimensional reconstructed intensity distribution information of the particles and searching for a local maximum value of light intensity to obtain the three-dimensional position and the focal plane image of each particle.
8. The imaging method of the portable digital holographic microscopy device based on 3D printing technology as claimed in claim 7, wherein in the step C, the background image is obtained by calculating the average light intensity of a plurality of holographic images as follows:
in the formula Ib(x, y) is the gray scale value of the pixel at the (x, y) position in the background image, N is the total frame number of the hologram, t is the time, Io(x, y) is the gray value of the pixel at the (x, y) position in the original hologram at the time t, and the light intensity value I of each pixel point in the hologram after the background is deducteds(x,y)：
Is(x,y)＝Io(x,y)-Ib(x,y)。
9. The imaging method according to claim 7, wherein the step D is a specific process: reconstructing the holographic light field by using a Rayleigh-Sommerfeld algorithm to obtain light intensity information and phase information corresponding to all particles in a set range, calculating the light intensity information only without storing the phase information due to interference of twin phases, wherein the calculation formula is as follows:
U(r,z)＝FT-1(FT(Is(r,0)·H(q,-z)))，
wherein h (r, -z) is a propagation operator, r is an initial transverse coordinate of the particle, and z is an initial axial coordinate of the particle; i is an imaginary unit; k is the wave number; r is the light propagation distance; i issIs the light intensity of the particle; FT-1Is inverse Fourier transform; FT is Fourier transform; h (q, -z) is the Fourier transform of H (r, -z).
10. The imaging method according to claim 7, wherein in step E, the three-dimensional position of the particle and the focal plane intensity map are obtained by the intensity threshold and finding the local maximum intensity; setting a light intensity threshold lower limit T, and filtering noise lower than the light intensity threshold through the light intensity threshold lower limit T; in a cube with similar observed particle sizes, the three-dimensional position of the maximum light intensity value is searched point by point, namely the three-dimensional focal plane position of the target particle. The reconstructed image at the position with the maximum light intensity value is the light intensity image of the particles in the holographic image at the position corresponding to the focal plane.
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