CN112666135A - Three-dimensional microscopic imaging device and method - Google Patents
Three-dimensional microscopic imaging device and method Download PDFInfo
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- CN112666135A CN112666135A CN202011355858.1A CN202011355858A CN112666135A CN 112666135 A CN112666135 A CN 112666135A CN 202011355858 A CN202011355858 A CN 202011355858A CN 112666135 A CN112666135 A CN 112666135A
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Abstract
The invention provides a three-dimensional microscopic imaging device and a method, comprising the following steps: a laser light source device; the light field shaping device is arranged on a light path of the laser beam and is used for shaping the laser beam and obtaining a plurality of illumination plane waves with three-dimensional distribution one by one, wherein all the illumination plane waves are divided into a plurality of groups with the same spatial frequency and different initial phase angles; the device comprises a first microscope objective arranged on an optical path of an illumination plane wave, a sample stage arranged on a light-emitting optical path of the first microscope objective and used for placing a sample object, and a second microscope objective arranged on the light-emitting optical path of the sample stage; the light detection device is arranged on a light-emitting path of the second microscope objective and is used for collecting the light intensity of fluorescence generated by the sample object and converting the light intensity of the fluorescence into an electric signal; and the imaging device is connected with the light detection device and acquires a three-dimensional shape distribution image of the sample object according to the electric signal. The purpose of three-dimensional microscopic imaging is realized.
Description
Technical Field
The invention relates to the technical field of microscopic imaging, in particular to a three-dimensional imaging microscopic imaging device and a three-dimensional imaging microscopic imaging method.
Background
The conventional imaging technology mainly uses an array type photodetector (e.g., a CCD (charge coupled device), a CMOS (Complementary Metal-Oxide-Semiconductor), etc.) to detect the intensity distribution of an object. The photosensitive detector widely used at present is mainly suitable for array detectors of visible light wave bands and non-visible light wave bands, such as wave bands of infrared, terahertz, X-ray and the like, and is very expensive and even undeveloped, however, in the fields of biomedicine and the like, the non-visible light is required to be used for illumination imaging, and the application of the array type photosensitive detector is greatly limited. Therefore, single-pixel imaging techniques with broad spectral response have received great attention and have been a focus of research.
The single-pixel imaging technology is a new computational imaging technology, and because the single-pixel imaging uses a photodiode or a photomultiplier as a detector, the single-pixel imaging technology has a wide applicable spectral range, can be used for imaging of wave bands such as infrared, terahertz and X-ray, and can also be applied to imaging under the condition of weak light. Existing single-pixel imaging techniques are primarily used for imaging two-dimensional scenes, and three-dimensional imaging remains a significant challenge.
Disclosure of Invention
In view of this, the present invention provides a three-dimensional microscopic imaging apparatus and method, which effectively solve the technical problems in the prior art and achieve the purpose of three-dimensional microscopic imaging.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a three-dimensional microscopic imaging apparatus comprising:
a laser light source device for outputting a laser beam;
the light field shaping device is arranged on a light path of the laser beam and is used for shaping the laser beam and obtaining a plurality of illumination plane waves with three-dimensional distribution one by one, wherein all the illumination plane waves are divided into a plurality of groups with the same spatial frequency and different initial phase angles;
the device comprises a first microscope objective arranged on the light path of the illumination plane wave, a sample stage arranged on the light emergent path of the first microscope objective and used for placing a sample object, and a second microscope objective arranged on the light emergent path of the sample stage;
the optical detection device is arranged on a light-emitting path of the second microscope objective and is used for collecting the light intensity of fluorescence generated by the sample object and converting the light intensity of the fluorescence into an electric signal;
and the imaging device is connected with the optical detection device and is used for acquiring a three-dimensional shape distribution image of the sample object according to the electric signal.
Optionally, the laser light source device includes: a laser device;
alternatively, the laser light source device includes: the optical field shaping device is arranged on a light emitting light path of the Nth beam combiner, N is an integer larger than 1, i is an integer larger than or equal to 1 and smaller than or equal to N, and j is an integer larger than or equal to 1 and smaller than or equal to N.
Optionally, the laser wavelengths of the first to nth lasers are not identical.
Optionally, the three-dimensional microscopic imaging apparatus further includes:
and the light field shaping device is arranged on the light outlet path of the light beam expanding device.
Optionally, the optical field shaping device includes:
the digital micro-mirror array is arranged on the light path of the laser beam;
the first lens is arranged on a light-emitting path of the digital micromirror array;
the first small-hole filter is arranged on a light-emitting path of the first lens;
and the second lens is arranged on the light-emitting optical path of the first pinhole filter, wherein the focal plane of the first microscope objective is conjugated with the focal plane of the second lens.
Optionally, the optical field shaping device further includes:
the digital micro-mirror array is arranged on the light emergent path of the first reflector;
and the third lens is arranged on the light-emitting optical path of the second lens, the second reflector is arranged on the light-emitting optical path of the third lens, and the first microscope objective is arranged on the light-emitting optical path of the second reflector.
Optionally, the light detection device includes: the first dichroic mirror to the Mth dichroic mirror, the first filter to the M +1 th filter, the first collecting lens to the M +1 th collecting lens, and the first single-pixel detector to the M +1 th single-pixel detector, wherein M is an integer greater than or equal to 1;
the first dichroic mirror is arranged on a light-emitting light path of the second microscope objective, the h +1 dichroic mirror is arranged on a light-transmitting light path of the h dichroic mirror, the M +1 filter is arranged on a light-transmitting light path of the M dichroic mirror, the h filter is arranged on a light-reflecting light path of the h dichroic mirror, and h is an integer which is more than or equal to 1 and less than M;
the kth collecting lens is arranged on a light emitting path of the kth filter, the kth single-pixel detector is arranged on the light emitting path of the kth collecting lens, and k is an integer which is larger than or equal to 1 and smaller than or equal to M.
Correspondingly, the invention also provides a three-dimensional microscopic imaging method, which comprises the following steps:
providing a laser beam;
shaping the laser beams and obtaining a plurality of illumination plane waves with three-dimensional distribution one by one, wherein all the illumination plane waves are divided into a plurality of groups with the same spatial frequency and different initial phase angles;
the illumination plane wave passes through a first microscope objective and then irradiates a sample object, and fluorescence generated by the sample object is emitted by a second microscope objective;
collecting the light intensity of the fluorescence generated by the sample object and converting the light intensity of the fluorescence into an electric signal;
and acquiring a three-dimensional shape distribution image of the sample object according to the electric signal.
Optionally, the collecting the light intensity of the fluorescence generated by the sample object and converting the light intensity of the fluorescence into an electrical signal includes:
and collecting the light intensity at the zero-frequency position of the Fourier spectrum of the fluorescence generated by the sample object, and converting the light intensity at the zero-frequency position of the Fourier spectrum of the fluorescence into an electric signal.
Optionally, acquiring a three-dimensional morphological distribution image of the sample object according to the electrical signal includes:
and calculating a frequency spectrum coefficient corresponding to the space frequency according to the electric signals corresponding to the illumination plane waves with the same space frequency and different initial phases by adopting a four-step phase shift method, and acquiring a three-dimensional shape distribution image of the sample object according to the frequency spectrum coefficient and by utilizing three-dimensional inverse Fourier transform.
Compared with the prior art, the technical scheme provided by the invention at least has the following advantages:
the invention provides a three-dimensional microscopic imaging device and a method, comprising the following steps: a laser light source device for outputting a laser beam; the light field shaping device is arranged on a light path of the laser beam and is used for shaping the laser beam and obtaining a plurality of illumination plane waves with three-dimensional distribution one by one, wherein all the illumination plane waves are divided into a plurality of groups with the same spatial frequency and different initial phase angles; the device comprises a first microscope objective arranged on the light path of the illumination plane wave, a sample stage arranged on the light emergent path of the first microscope objective and used for placing a sample object, and a second microscope objective arranged on the light emergent path of the sample stage; the optical detection device is arranged on a light-emitting path of the second microscope objective and is used for collecting the light intensity of fluorescence generated by the sample object and converting the light intensity of the fluorescence into an electric signal; and the imaging device is connected with the optical detection device and is used for acquiring a three-dimensional shape distribution image of the sample object according to the electric signal. According to the technical scheme provided by the invention, the illumination plane wave with three-dimensional distribution obtained by the light field shaping device irradiates the sample object, and then the three-dimensional shape distribution image of the sample object is obtained according to the light intensity of the fluorescence generated by the sample object, so that the purpose of three-dimensional microscopic imaging is realized.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
Fig. 1 is a schematic structural diagram of a three-dimensional microscopic imaging apparatus provided by an embodiment of the invention;
fig. 2 is a schematic structural diagram of a laser light source device according to an embodiment of the present invention;
FIG. 3 is a schematic structural diagram of another three-dimensional microscopic imaging apparatus provided by an embodiment of the present invention;
fig. 4 is a schematic structural diagram of a light detection device according to an embodiment of the present invention;
FIG. 5 is a flow chart of a three-dimensional microscopic imaging method according to an embodiment of the present invention;
fig. 6 is a schematic diagram of a simulation result according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As described in the background art, the single-pixel imaging technology is a new computational imaging technology, and because the single-pixel imaging uses a photodiode or a photomultiplier as a detector, the applicable spectral range is wide, and the single-pixel imaging technology can be used for imaging in bands such as infrared, terahertz and X-ray, and can also be applied to imaging under the condition of weak light. Existing single-pixel imaging techniques are primarily used for imaging two-dimensional scenes, and three-dimensional imaging remains a significant challenge.
Based on this, the embodiment of the invention provides a three-dimensional microscopic imaging device and a three-dimensional microscopic imaging method, which effectively solve the technical problems in the prior art and achieve the purpose of three-dimensional microscopic imaging.
To achieve the above object, the technical solutions provided by the embodiments of the present invention are described in detail below, specifically with reference to fig. 1 to 6.
Referring to fig. 1, a schematic structural diagram of a three-dimensional microscopic imaging apparatus according to an embodiment of the present invention is shown, where the three-dimensional microscopic imaging apparatus includes:
a laser light source device 100, the laser light source device 100 for outputting a laser beam.
The light field shaping device 200 is arranged on the light path of the laser beam, and the light field shaping device 200 is used for shaping the laser beam and obtaining a plurality of illumination plane waves with three-dimensional distribution one by one, wherein all the illumination plane waves are divided into a plurality of groups with the same spatial frequency and different initial phase angles; when the laser beam is shaped into a series of illuminating plane waves with three-dimensional distribution by the light field shaping device, only one illuminating plane wave with specific spatial frequency and initial phase angle is generated each time, and all illuminating plane waves can be divided into multiple groups with the same spatial frequency and different initial phase angles; alternatively, all the illumination plane waves in each group of illumination plane waves may be illumination plane waves generated adjacently in time sequence, or may be illumination plane waves not adjacent in time sequence, and the present invention is not particularly limited. .
The microscope comprises a first microscope objective 310 arranged on the light path of the illumination plane wave, a sample stage 320 arranged on the light-emitting light path of the first microscope objective 310 and used for placing a sample object, and a second microscope objective 330 arranged on the light-emitting light path of the sample stage 320.
And the optical detection device 400 is arranged on the light-emitting path of the second microscope objective 330, and the optical detection device 400 is used for collecting the light intensity of the fluorescence generated by the sample object and converting the light intensity of the fluorescence into an electric signal.
And an imaging device 500 connected to the light detecting device 400, the imaging device 500 being configured to obtain a three-dimensional morphological distribution image of the sample object according to the electrical signal.
According to the technical scheme provided by the embodiment of the invention, the single illuminating plane wave with three-dimensional distribution, which is generated successively by the light field shaping device, irradiates the sample object, and then the three-dimensional shape distribution image of the sample object is obtained according to the light intensity of the fluorescence generated by the sample object, so that the purpose of three-dimensional microscopic imaging is realized.
In an embodiment of the present invention, the laser light source device provided by the present invention is used for outputting a laser beam, and further, the laser beam is shaped relatively and then excites a sample object to generate fluorescence. The laser light source device provided by the embodiment of the invention can comprise a laser, wherein a light-emitting light path of the laser is provided with a light field shaping device; alternatively, the laser light source device provided by the embodiment of the invention may further include a plurality of lasers, and the application range of the three-dimensional microscopic imaging apparatus may be increased by controlling the plurality of lasers to be simultaneously or non-simultaneously turned on. That is, the laser light source device provided by the embodiment of the present invention includes: a laser device; alternatively, the laser light source device includes: the optical field shaping device is arranged on a light emitting light path of the Nth beam combiner, N is an integer larger than 1, i is an integer larger than or equal to 1 and smaller than or equal to N, and j is an integer larger than or equal to 1 and smaller than or equal to N.
Specifically, as shown in fig. 2, a schematic structural diagram of a laser light source device 100 according to an embodiment of the present invention is provided, where the laser light source device 100 according to an embodiment of the present invention is described by taking a first laser 111 to a third laser 113 as an example, where the laser light source device 100 further includes a first beam combiner 121 to a third beam combiner 123, an optical path of the first laser 111 is provided with the first beam combiner 121, an optical path of the second laser 112 is provided with the second beam combiner 122, and an optical path of the third laser 113 is provided with the third beam combiner 123. And the light-emitting optical path of the first beam combiner 121 is provided with a second beam combiner 122, and the light-emitting optical path of the second beam combiner 122 is provided with a third beam combiner 123.
It can be understood that the embodiment of the invention meets the application of the three-dimensional microscopic imaging device in different scenes by lighting the first laser to the third laser simultaneously or not. Optionally, the laser wavelengths of the first laser and the nth laser provided by the embodiment of the invention are not completely the same, so that the application range of the three-dimensional microscopic imaging device can be further improved. The laser wavelength of one laser provided in the embodiment of the present invention may be 405nm, the laser wavelength of the second laser may be 488nm, and the laser wavelength of the third laser may be 532nm, which is not limited in the present invention.
As shown in fig. 3, a schematic structural diagram of another three-dimensional microscopic imaging apparatus provided in an embodiment of the present invention is shown, where the three-dimensional microscopic imaging apparatus provided in the embodiment of the present invention further includes:
the light beam expanding device 600 is arranged on the light path of the laser beam, and the light field shaping device 200 is arranged on the light outlet light path of the light beam expanding device 600, so that the laser beam is expanded through the light beam expanding device 600, and the laser irradiation effect of the three-dimensional microscopic imaging device is improved. As shown in fig. 3, a light beam expander 600 provided in an embodiment of the present invention may include a lens 610 (e.g., a focal length of the lens 610 is 200mm) and a lens 620 (e.g., a focal length of the lens 620 is 1000mm, and a distance between the lens 610 and the lens 620 may be 1200mm, through which a laser beam may be expanded to 5 times, which is not limited in the present invention), the lens 610 is disposed on a light path of the laser beam, and the lens 620 is disposed on a light-emitting light path of the lens 610, and the laser beam is expanded through the light beam expander composed of the lens 610 and the lens 620.
As shown in fig. 3, the light field shaping device 200 provided by the embodiment of the present invention includes:
and a digital micromirror array 210 disposed on the optical path of the laser beam.
A first lens 220 disposed on the light path of the dmd array 210.
And a first pinhole filter 230 disposed on the light-emitting path of the first lens 220.
And a second lens 240 disposed on an optical path of the first pinhole filter 230, wherein a focal plane of the first microscope objective 310 is conjugate to a focal plane of the second lens 240.
It will be appreciated that embodiments of the present invention provide for the laser beam to be incident upon the digital micromirror array, which shapes the laser beam to successively load thereon holograms of illuminating plane waves having a predetermined spatial frequency and a predetermined initial phase angle. Wherein the digital micromirror array may be at a 12 ° angle to the laser beam. Then, the digital micro lens array outputs the shaped light beam to a 4-f system composed of a first lens (for example, the focal length of the first lens can be 300mm) and a second lens (for example, the focal length of the second lens can be 100mm), and a first small-hole filter between the first lens and the second lens selects light of +1 level or-1 level of the Fourier spectrum of the light beam, so that a series of illumination plane waves with three-dimensional distribution is formed after passing through the focal plane of the second lens (for which the spatial frequency and the initial phase angle of the illumination plane waves are determined by a hologram on the digital micro lens array). The focal plane of the first micro-objective lens and the focal plane of the second lens are conjugated, so that the two positions have the same distribution of illuminating plane waves.
In an embodiment of the present invention, the optical field shaping device provided by the present invention may further include some optical devices to change the direction of the propagating light beam, so as to optimize parameters such as the volume of the optical field shaping device, and the present invention is not particularly limited thereto. As shown in fig. 3, the light field shaping device 200 provided by the embodiment of the present invention further includes:
the first reflecting mirror 250 is arranged on the light path of the laser beam, and the digital micro-mirror array 210 is arranged on the light outgoing path of the first reflecting mirror 250;
and a third lens 260 disposed on the light-emitting path of the second lens 240, and a second reflecting mirror 270 disposed on the light-emitting path of the third lens 260, wherein the first micro-objective 310 is disposed on the light-emitting path of the second reflecting mirror 270.
It should be noted that, the embodiment of the present invention does not specifically limit the type and number of the optical devices included in the optical shaping device for optimizing the parameters thereof, and the optical shaping device needs to be specifically designed according to the actual application.
In an embodiment of the present invention, the light detection device provided by the present invention is used for collecting the light intensity of the fluorescence generated by the sample object, and converting the light intensity into an electrical signal for outputting. The optical detection device provided by the embodiment of the invention may include: the first dichroic mirror to the Mth dichroic mirror, the first filter to the M +1 th filter, the first collecting lens to the M +1 th collecting lens, and the first single-pixel detector to the M +1 th single-pixel detector, wherein M is an integer greater than or equal to 1;
first dichroic mirror set up in on the light-emitting path of second micro objective, the h +1 th dichroic mirror sets up on the printing opacity light path of h dichroic mirror, the M +1 th filter set up in on the printing opacity light path of M dichroic mirror, the h filter set up in on the reflection of light path of h dichroic mirror, h is more than or equal to 1 and is less than M's integer. The kth collecting lens is arranged on a light emitting path of the kth filter, the kth single-pixel detector is arranged on the light emitting path of the kth collecting lens, and k is an integer which is larger than or equal to 1 and smaller than or equal to M.
Specifically, as shown in fig. 4, a schematic structural diagram of a light detection device according to an embodiment of the present invention is provided, in which the light detection device 400 includes a first dichroic mirror 411 to a second dichroic mirror 412 for illustration, and the light detection device 400 further includes a first filter 421 to a third filter 423, a first collecting lens 431 to a third collecting lens 433, and a first single-pixel detector 441 to a third single-pixel detector 443. Wherein first dichroic mirror 411 set up in on the light-emitting path of second micro objective 330, second dichroic mirror 412 sets up on the printing opacity light path of first dichroic mirror 411, third filter 423 set up in on the printing opacity light path of second dichroic mirror 412, first filter 421 set up in on the reflection of light path of first dichroic mirror 411, second filter 422 sets up on the reflection of light path of second dichroic mirror 412. The first collecting lens 431 is arranged on the light emitting path of the first filter 421, the second collecting lens 432 is arranged on the light emitting path of the second filter 422, and the third collecting lens 433 is arranged on the light emitting path of the third filter 423; the first single-pixel detector 441 is disposed on the light-emitting path of the first collecting lens 431, the second single-pixel detector 442 is disposed on the light-emitting path of the second collecting lens 432, and the third single-pixel detector 443 is disposed on the light-emitting path of the third collecting lens 433.
It can be understood that, after entering the first dichroic mirror, the illumination plane wave provided by the embodiment of the present invention is divided into two paths of light transmission and light reflection by the first dichroic mirror, and the fluorescence generated by the sample object is reflected onto the first filter, and the stray light is filtered by the first filter, and then output to the first collecting lens (for example, the focal length of the first collecting lens may be 50mm), so as to perform fourier transform, and then transmit to the first single-pixel detector; the first single-pixel detector can be a single-pixel detector comprising a second small-hole filter, collects the light intensity of the zero-frequency position of the Fourier spectrum of the fluorescence and converts the light intensity into an analog electric signal; the analog electrical signal may be subsequently analog-to-digital converted by the data acquisition card to convert the analog electrical signal into a digital signal, which is not limited in the present invention. In addition, when the optical detector provided in the embodiment of the present invention includes a branch optical path composed of other dichroic mirrors, an optical filter, a collecting lens, and a single-pixel detector, other light except for fluorescence may be collected by the other branch optical path, and further, other light may be processed to obtain more types of sample objects, for example, when the illumination light wave includes a reference beam, the reference beam may be collected by the branch optical path, and the present invention is not particularly limited thereto.
Therefore, after the light intensity related information of the fluorescence detected by the light detection device is transmitted to the imaging device, the imaging device can process the light intensity related information according to a corresponding recovery algorithm to obtain a three-dimensional shape distribution image of the sample object. Optionally, the imaging device provided in the embodiment of the present invention may be a computer, and the present invention is not particularly limited thereto.
It can be understood that the technical solution provided by the embodiment of the present invention can switch the space illumination light field at a high speed by using the digital micromirror array, thereby improving the efficiency. Meanwhile, the illumination mode of the laser light source device can be flexibly selected, and multi-wavelength fluorescence microscopic imaging is realized. In addition, the single-pixel detector is adopted to collect the light beams, so that the device has weak light detection capability and anti-scattering capability, and three-dimensional microscopic imaging in a complex environment is realized.
Corresponding to the three-dimensional microscopic imaging apparatus provided in the above embodiment of the present invention, an embodiment of the present invention further provides a three-dimensional microscopic imaging method, and as shown in fig. 5, the method is a flowchart of the three-dimensional microscopic imaging method provided in the embodiment of the present invention, and the method includes:
and S1, providing a laser beam.
S2, shaping the laser beams and obtaining a plurality of illumination plane waves with three-dimensional distribution one by one, wherein all the illumination plane waves are divided into a plurality of groups with the same spatial frequency and different initial phase angles.
And S3, irradiating the illumination plane wave to the sample object after passing through the first microscope objective, and enabling the fluorescence generated by the sample object to be emitted by the second microscope objective.
S4, collecting the light intensity of the fluorescence generated by the sample object, and converting the light intensity of the fluorescence into an electric signal.
And S5, acquiring a three-dimensional shape distribution image of the sample object according to the electric signal.
In an embodiment of the present invention, the collecting the light intensity of the fluorescence generated by the sample object and converting the light intensity of the fluorescence into an electrical signal includes:
and collecting the light intensity at the zero-frequency position of the Fourier spectrum of the fluorescence generated by the sample object, and converting the light intensity at the zero-frequency position of the Fourier spectrum of the fluorescence into an electric signal.
The method for acquiring the three-dimensional shape distribution image of the sample object according to the electric signal, provided by the embodiment of the invention, comprises the following steps:
and calculating a frequency spectrum coefficient corresponding to the space frequency according to the electric signals corresponding to the illumination plane waves with the same space frequency and different initial phases by adopting a four-step phase shift method, and acquiring a three-dimensional shape distribution image of the sample object according to the frequency spectrum coefficient and by utilizing three-dimensional inverse Fourier transform.
It can be understood that, in the three-dimensional microscopic imaging method provided by the embodiment of the present invention, after the laser beam is obtained, the light field shaping device shapes the laser beam to generate a plurality of illumination plane waves with different complex amplitude distributions, and the three-dimensional light field with specific spatial distribution and different optical axis orientations is formed after spatial transmission. The illumination plane wave is utilized to irradiate a sample object after passing through the first microscope objective, the sample object then emits fluorescence, and the fluorescence is emitted by the second microscope objective. And then collecting the light intensity at the zero-frequency position of the Fourier spectrum of the fluorescence generated by the sample object, and converting the light intensity at the zero-frequency position of the Fourier spectrum of the fluorescence into an electric signal, wherein one of the illumination plane waves corresponds to one of the electric signals. And finally, calculating corresponding spectral coefficients by using corresponding electric signals of the illuminating plane waves with the same spatial frequency so as to obtain three-dimensional spectral coefficient distribution of fluorescence, and finally obtaining a three-dimensional form distribution image of the sample object based on three-dimensional inverse Fourier transform.
In order to generate the illumination plane wave, the embodiment of the invention adopts the light field shaping device to shape the laser beam, wherein the light field shaping device can only generate a two-dimensional distributed light field, so that the required three-dimensional light field distribution needs to be converted into two-dimensional distribution. Firstly, a three-dimensional light field is converted into superposition of two three-dimensional plane waves, and the vectors of the two three-dimensional plane waves are respectively
And
the plane wave of (2). For two three-dimensional plane waves, the optical system is constrained by the numerical aperture and the optical wavelength of the objective lens due to three-dimensionalThe space light field is a plane wave light field, and k of two three-dimensional plane waves is taken based on scalar diffraction theoryx，kyTwo items. As the generated light beam propagates, a specific three-dimensional light field is obtained. In order to achieve fast light field switching, the embodiments of the present invention use the digital micromirror array as a component of the light field shaping device to modulate the structured light field that generates the illumination plane wave. A digital micromirror array is a mems-based device consisting of millions of micromirrors. Each micromirror can be independently controlled by the address electrode voltage to change the state of the micromirror. The micromirror states include three types, on, off, and static, which are expressed using the reflection angles of the micromirror of 12 °, -12 °, and 0 °, respectively.
The light field shaping device provided by the embodiment of the invention generates a plurality of groups with different spatial frequencies (k)x,ky,kz) In order to obtain the complex coefficients corresponding to the spatial frequencies, the embodiment of the present invention uses a four-step phase shift method for processing. Each spatial frequency corresponds to four light fields with different initial phases (that is, each set of illumination plane waves provided by the embodiment of the present invention includes four illumination plane waves, the spatial frequencies of the four illumination plane waves are the same, the four illumination plane waves have different initial phase angles, respectively), and the difference between two adjacent initial phases is pi/2, which is 0, pi/2, pi, 3 pi/2, respectively. Using the single-pixel detector response values of these four light fields, the spatial frequency (k) can be calculatedx,ky,kz) Corresponding fourier spectral coefficients of the four light fields:
wherein (k)x,ky,kz) The spatial frequencies of the illuminating plane waves in the x, y, z directions, g (k), respectivelyx,ky,kz) Represents and (k)x,ky,kz) A corresponding one of the fourier spectral coefficients;
Because the light field shaping device can only load two-dimensional holograms, the three-dimensional k vector needs to be converted into two-dimensional distribution, and the three-dimensional light field distribution is not changed, so that two beams of wave vectors are respectively set as
And
the plane wave of (2). The following equations are used in parallel:
the calculated k1 and k2 only need to take x and y terms, which is equivalent to changing the direction of the optical axis. Each group k1, k2 has a unique k corresponding to it. Thus, along with the transmission of the light beam in the space, the required three-dimensional space distribution light field can be obtained.
After the light field shaping device generates a plurality of illumination plane waves, the sample object is illuminated through the first microscope objective, and the microscope objective enables the size of an illumination light field to be matched with the sample object. The desired light field with a specific spatial distribution is generated in the sample space and the sample object is illuminated, and after the fluorescence excited by the specific wavelength is excited, the resulting fluorescence is collected by the light detection device.
The fluorescence intensity is Fourier transformed by a Fourier lens (a first collecting lens), a single-pixel detector with a second small-hole filter is placed on the Fourier plane of the lens, and the zero-frequency intensity of the fluorescence spectrum is collected. The plurality of zero-frequency light intensities are detected by the single-pixel detector and then sequentially generate analog signals, and the analog signals are subjected to analog-to-digital conversion through the data acquisition card to output digital signals. Finally, processing by an imaging device, wherein after signal acquisition is finished, firstly, calculating a frequency spectrum coefficient corresponding to each spatial frequency by using a four-step phase shift method, and then, directly obtaining the fluorescence distribution of the sample object by using three-dimensional inverse Fourier transform; that is, for any sample object, it can be considered as a superposition of a series of plane waves; the complex-valued coefficients corresponding to each plane wave are fourier coefficients representing the corresponding spectral positions, i.e.:
O(x,y,z)＝∑g(kx,ky,kz)P(x,y,z,kx,ky,kz)
wherein, P (x, y, z, k)x,ky,kz)＝exp(kxx+kyy+kzz) represents a spatial frequency of (k)x,ky,kz) (ii) a bundle of three-dimensional spatial plane waves, (x, y, z) representing three-dimensional spatial cartesian coordinates, and Σ representing spatial frequency as a variable, for (k)x,ky,kz) The three-dimensional coordinates are summed. According to the inverse Fourier transform, O (x, y, z) ═ F-1{g(kx,ky,kz) In which F-1{ } denotes an inverse fourier transform. For any sample object, when the three-dimensional fourier coefficients are known, then the inverse fourier transform can be used to obtain an image of the object; when the signal is collected, the frequency spectrum is symmetrical by positive and negative half axes of a z axis, so that the fluorescence imaging of the object can be obtained by collecting only half of the frequency spectrum. As shown in fig. 6, a simulation result diagram provided in the embodiment of the present invention shows that the imaging effect is high.
The embodiment of the invention provides a three-dimensional microscopic imaging device and a method, comprising the following steps: a laser light source device for outputting a laser beam; the light field shaping device is arranged on a light path of the laser beam and is used for shaping the laser beam and obtaining a plurality of illumination plane waves with three-dimensional distribution one by one, wherein all the illumination plane waves are divided into a plurality of groups with the same spatial frequency and different initial phase angles; the device comprises a first microscope objective arranged on the light path of the illumination plane wave, a sample stage arranged on the light emergent path of the first microscope objective and used for placing a sample object, and a second microscope objective arranged on the light emergent path of the sample stage; the optical detection device is arranged on a light-emitting path of the second microscope objective and is used for collecting the light intensity of fluorescence generated by the sample object and converting the light intensity of the fluorescence into an electric signal; and the imaging device is connected with the optical detection device and is used for acquiring a three-dimensional shape distribution image of the sample object according to the electric signal. According to the technical scheme provided by the embodiment of the invention, a series of irradiated sample objects with three-dimensional distribution are successively obtained through the light field shaping device, and then the three-dimensional shape distribution image of the sample object is obtained according to the light intensity of fluorescence generated by the sample object, so that the purpose of three-dimensional microscopic imaging is realized.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A three-dimensional microscopic imaging apparatus, comprising:
a laser light source device for outputting a laser beam;
the light field shaping device is arranged on a light path of the laser beam and is used for shaping the laser beam and obtaining a plurality of illumination plane waves with three-dimensional distribution one by one, wherein all the illumination plane waves are divided into a plurality of groups with the same spatial frequency and different initial phase angles;
the device comprises a first microscope objective arranged on the light path of the illumination plane wave, a sample stage arranged on the light emergent path of the first microscope objective and used for placing a sample object, and a second microscope objective arranged on the light emergent path of the sample stage;
the optical detection device is arranged on a light-emitting path of the second microscope objective and is used for collecting the light intensity of fluorescence generated by the sample object and converting the light intensity of the fluorescence into an electric signal;
and the imaging device is connected with the optical detection device and is used for acquiring a three-dimensional shape distribution image of the sample object according to the electric signal.
2. The three-dimensional microscopic imaging apparatus according to claim 1, wherein said laser light source device comprises: a laser device;
alternatively, the laser light source device includes: the optical field shaping device is arranged on a light emitting light path of the Nth beam combiner, N is an integer larger than 1, i is an integer larger than or equal to 1 and smaller than or equal to N, and j is an integer larger than or equal to 1 and smaller than or equal to N.
3. The three-dimensional microscopic imaging apparatus according to claim 2, wherein the laser wavelengths of said first to nth lasers are not identical.
4. The three-dimensional microscopic imaging apparatus according to claim 1, characterized in that said three-dimensional microscopic imaging apparatus further comprises:
and the light field shaping device is arranged on the light outlet path of the light beam expanding device.
5. The three-dimensional microscopic imaging apparatus according to claim 1, wherein said light field shaping device comprises:
the digital micro-mirror array is arranged on the light path of the laser beam;
the first lens is arranged on a light-emitting path of the digital micromirror array;
the first small-hole filter is arranged on a light-emitting path of the first lens;
and the second lens is arranged on the light-emitting optical path of the first pinhole filter, wherein the focal plane of the first microscope objective is conjugated with the focal plane of the second lens.
6. The three-dimensional microscopic imaging apparatus according to claim 5, wherein said light field shaping device further comprises:
the digital micro-mirror array is arranged on the light emergent path of the first reflector;
and the third lens is arranged on the light-emitting optical path of the second lens, the second reflector is arranged on the light-emitting optical path of the third lens, and the first microscope objective is arranged on the light-emitting optical path of the second reflector.
7. The three-dimensional microscopic imaging apparatus according to claim 1, wherein said light detecting device comprises: the first dichroic mirror to the Mth dichroic mirror, the first filter to the M +1 th filter, the first collecting lens to the M +1 th collecting lens, and the first single-pixel detector to the M +1 th single-pixel detector, wherein M is an integer greater than or equal to 1;
the first dichroic mirror is arranged on a light-emitting light path of the second microscope objective, the h +1 th dichroic mirror is arranged on a light-transmitting light path of the h dichroic mirror, the M +1 th filter is arranged on a light-transmitting light path of the M +1 th dichroic mirror, the h filter is arranged on a light-reflecting light path of the h dichroic mirror, and h is an integer which is greater than or equal to 1 and less than M;
the kth collecting lens is arranged on a light emitting path of the kth filter, the kth single-pixel detector is arranged on the light emitting path of the kth collecting lens, and k is an integer which is larger than or equal to 1 and smaller than or equal to M.
8. A method of three-dimensional microscopic imaging, comprising:
providing a laser beam;
shaping the laser beams and obtaining a plurality of illumination plane waves with three-dimensional distribution one by one, wherein all the illumination plane waves are divided into a plurality of groups with the same spatial frequency and different initial phase angles;
the illumination plane wave passes through a first microscope objective and then irradiates a sample object, and fluorescence generated by the sample object is emitted by a second microscope objective;
collecting the light intensity of the fluorescence generated by the sample object and converting the light intensity of the fluorescence into an electric signal;
and acquiring a three-dimensional shape distribution image of the sample object according to the electric signal.
9. The three-dimensional microscopic imaging method according to claim 8, wherein collecting the light intensity of fluorescence generated from the sample object and converting the light intensity of the fluorescence into an electrical signal comprises:
and collecting the light intensity at the zero-frequency position of the Fourier spectrum of the fluorescence generated by the sample object, and converting the light intensity at the zero-frequency position of the Fourier spectrum of the fluorescence into an electric signal.
10. The three-dimensional microscopic imaging method according to claim 9, wherein acquiring a three-dimensional morphological distribution image of the sample object based on the electrical signal comprises:
and calculating a frequency spectrum coefficient corresponding to the space frequency according to the electric signals corresponding to the illumination plane waves with the same space frequency and different initial phases by adopting a four-step phase shift method, and acquiring a three-dimensional shape distribution image of the sample object according to the frequency spectrum coefficient and by utilizing three-dimensional inverse Fourier transform.
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