CN113768472B - Three-dimensional image acquisition device with fluorescent marker and method - Google Patents
Three-dimensional image acquisition device with fluorescent marker and method Download PDFInfo
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- CN113768472B CN113768472B CN202111325520.6A CN202111325520A CN113768472B CN 113768472 B CN113768472 B CN 113768472B CN 202111325520 A CN202111325520 A CN 202111325520A CN 113768472 B CN113768472 B CN 113768472B
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0033—Features or image-related aspects of imaging apparatus classified in A61B5/00, e.g. for MRI, optical tomography or impedance tomography apparatus; arrangements of imaging apparatus in a room
Abstract
The invention discloses a three-dimensional image acquisition device with a fluorescent marker and a method thereof. The device comprises an inverted light sheet three-dimensional fluorescence imaging light path, an inverted three-dimensional reflection bright field imaging light path and an upper sample carrier; the three-dimensional fluorescence imaging light path and the three-dimensional reflection bright field imaging light path share a detection objective lens and a conjugate correction lens assembly. The method applies the three-dimensional image acquisition device with the fluorescent label to scan and image the sample. The invention realizes the three-dimensional fluorescence imaging light path of the light sheet and the three-dimensional reflection bright field imaging light path to the same sample imaging plane of the sample through sharing the detection objective lens and the conjugate correction lens component, and simultaneously obtains the fluorescence mark information and the bright field imaging information, thereby realizing the two-dimensional image registration of hardware and realizing the more accurate and high-speed three-dimensional image imaging with the fluorescence mark.
Description
Technical Field
The invention belongs to the field of biomedical microscopy, and particularly relates to a three-dimensional image acquisition device with a fluorescent marker and a method.
Background
The three-dimensional image with the fluorescent marker is obtained by the combined application of a three-dimensional imaging device and a three-dimensional image reconstruction method, and the three-dimensional profile image with the three-dimensional fluorescent marker specific information is obtained for the sample with the fluorescent marker.
For a fluorescence-labeled sample, because fluorescence excitation requires laser with a specific wavelength, the laser with the wavelength can damage the sample after long-term irradiation, particularly for a living body sample, and therefore, the imaging cannot be carried out for a long time and the sample cannot be observed for a long time, so that quantitative bright-field imaging of three-dimensional non-specific labeling is developed, the imaging method obtains information of the refractive index distribution and the light absorption distribution of the sample, and the sample can be observed for a long time without damage to the sample, but the method has no specificity. In combination with fluorescence imaging, specific information of the fluorescent label of the sample can be obtained. Therefore, the combination of fluorescence three-dimensional imaging and bright-field three-dimensional imaging can obtain different three-dimensional information of the same sample.
The existing technical means for combining fluorescence three-dimensional imaging and bright-field three-dimensional imaging mainly comprise the following steps: the confocal fluorescence three-dimensional imaging is combined with optical diffraction chromatography (ODT) three-dimensional imaging, and the imaging speed is very low. The wide-field fluorescence z-axis scanning deconvolution is used for obtaining a three-dimensional image and combining a bright-field image obtained by ODT, the signal-to-noise ratio of the wide-field fluorescence is lower than that of the fluorescence of an optical sheet, the obtained three-dimensional fluorescence information is insufficient in effect, and the imaging speed is limited. The structured light illumination (SIM) technology is used for acquiring a fluorescent image and combining an ODT bright field image, the imaging speed is low, a complex mask plate needs to be designed in a light path, and the complexity of a hardware light path is increased. The method is characterized in that a wide-field fluorescence and ODT combined mode is used for acquiring a three-dimensional image, a sample has nonlinear motion in the imaging process, a complex software alignment method is needed for later image alignment and processing, and the sample is relatively complex to control. The bright field three-dimensional imaging technology combining fluorescence super-resolution optical fluctuation imaging (SOFI) and white light partial coherent imaging has a relatively slow imaging speed.
The transmission type quantitative phase image can hardly obtain two-dimensional information consistent with the reflection type fluorescence image, and a fluorescence three-dimensional image and a bright field quantitative phase image need to be respectively reconstructed, then software simulation matching is carried out, and the fluorescence three-dimensional image and the bright field positioning phase image are matched to obtain a three-dimensional image with a fluorescence mark. Hardware registration of a fluorescent three-dimensional image and a bright-field three-dimensional image cannot be realized in principle.
The existing fluorescent mark three-dimensional image acquisition method has high three-dimensional image quality requirement due to the fact that hardware registration cannot be achieved, software registration is needed after respective three-dimensional reconstruction, the imaging speed of high-speed fluorescent imaging and bright field imaging is limited, the imaging speed is low, the method is not applicable to living body samples, and the fluorescent mark three-dimensional image acquisition speed is further reduced.
Generally, the existing fluorescent marker three-dimensional image acquisition method cannot realize the registration of fluorescent information and bright field information from hardware, so that the hardware imaging speed and the software processing speed are both limited, and the imaging speed is slow.
Disclosure of Invention
The invention provides a three-dimensional image acquisition device with a fluorescent mark and a method thereof, aiming at realizing the same sample imaging plane of an inverted light sheet three-dimensional fluorescent imaging light path and an inverted three-dimensional reflection bright field imaging light path for a sample and simultaneously acquiring fluorescent mark information and bright field imaging information through a multiplexing detection objective lens and a conjugate correction lens component so as to realize the two-dimensional image registration of hardware, thereby solving the technical problems of high two-dimensional image precision requirement, low imaging speed, high three-dimensional imaging cost and low speed caused by only registering three-dimensional images when the three-dimensional images with the fluorescent mark are acquired in the prior art.
To achieve the above object, according to one aspect of the present invention, there is provided a three-dimensional image capturing device with fluorescence mark, comprising an inverted light sheet three-dimensional fluorescence imaging optical path, an inverted three-dimensional reflection bright field imaging optical path, and an overhead sample carrier; the three-dimensional fluorescence imaging light path of the light sheet and the reflected bright field imaging light path share a detection objective lens and a conjugate correction lens assembly;
in the inverted light sheet three-dimensional fluorescence imaging light path, a fluorescence illumination light sheet passes through an illumination objective lens and obliquely illuminates a sample at a light sheet inclination angle alpha; the fluorescence excitation light is collected by a detection objective lens, and fluorescence imaging is carried out on a sample imaging plane in the direction orthogonal to the fluorescence illumination light sheet through a conjugate correction lens assembly;
the three-dimensional bright field imaging optical path is reflective differential interference phase difference quantitative bright field imaging, and illumination light of the three-dimensional bright field imaging optical path sequentially passes through the conjugate correction lens assembly and the detection objective lens and is projected on a sample in a direction orthogonal to the fluorescent illumination light sheet; sample reflected light sequentially passes through the detection objective lens and the conjugate correction lens assembly in the direction orthogonal to the fluorescent lighting light sheet, and bright field imaging is carried out on the sample imaging plane;
the conjugate correction lens assembly comprises a front objective lens and a rear objective lens which are confocal and obliquely arranged; the main optical axes of the front objective and the rear objective form an intersection angle beta, and the intersection angle beta is complementary with the inclination angle alpha of the light sheet.
Preferably, the three-dimensional image acquisition device with the fluorescent marker has the front objective lens coaxial with the detection objective lens and the back pupil surface conjugate.
Preferably, the conjugate correction lens assembly of the three-dimensional image capturing device with the fluorescent mark comprises a lens group arranged between the front objective lens and the detection objective lens, and is used for matching the magnification of the front objective lens and the magnification of the detection objective lens.
Preferably, the three-dimensional image acquisition device with the fluorescent marker multiplexes the illumination objective lens and the detection objective lens with the same objective lens, the objective lens is perpendicular to the sample loading plane, and the fluorescent illumination light is incident through the rear pupil near edge of the objective lens.
Preferably, the three-dimensional image acquisition device with the fluorescent mark adopts two objective lenses as an illumination objective lens and a detection objective lens, the detection objective lens is perpendicular to the sample carrying plane, and the optical axes of the illumination objective lens and the detection objective lens form a light sheet inclination angle alpha; preferably also a multi-detection objective for low-power fluorescence imaging.
Preferably, the three-dimensional image acquisition device with the fluorescent mark has the same magnification of the three-dimensional fluorescence imaging optical path of the light sheet and the reflection bright field imaging optical path.
Preferably, the three-dimensional image acquisition device with the fluorescent marker has a sample carrier with a movement mechanism.
Preferably, the three-dimensional image capturing device with the fluorescence mark is provided with a galvanometer on a conjugate plane of a back pupil plane of the detection objective lens and between the detection objective lens and the conjugate correction lens assembly, so that the fluorescence illumination light, the detection light, the bright field illumination light and the detection light are reflected by the galvanometer, and the excitation light path and the detection light path move together to perform image scanning through vibration of the galvanometer.
According to another aspect of the present invention, there is provided a three-dimensional image acquisition method with a fluorescent marker, comprising the steps of:
the three-dimensional image acquisition device with the fluorescent label provided by the invention is applied to scan and image a sample: for each sample imaging plane, acquiring a fluorescence image of the sample imaging plane through a light sheet three-dimensional fluorescence imaging optical path, acquiring a bright field image of the sample imaging plane through a three-dimensional reflection bright field imaging optical path, and registering the fluorescence image and the bright field image to obtain a two-dimensional image of each sample imaging plane with a fluorescence mark; and performing three-dimensional reconstruction on the two-dimensional images with the fluorescent labels of all the sample imaging planes obtained by scanning to obtain three-dimensional images with the fluorescent labels of the samples.
Preferably, the three-dimensional image acquisition method with fluorescent marker, the registering the fluorescent image and the bright field image comprises position registration and/or pixel registration;
the position registration, preferably, a standard with a fluorescence profile consistent with a sample profile is adopted to register the fluorescence image and the bright field image, specifically:
for a certain sample imaging plane of a standard sample, acquiring a fluorescence image of the standard sample through a light sheet three-dimensional fluorescence imaging optical path, acquiring a bright field image of the standard sample through a three-dimensional reflection bright field imaging optical path, and performing contour registration on the fluorescence image and the bright field image of the certain sample imaging plane to obtain the visual field coordinate offset of the light sheet three-dimensional fluorescence imaging optical path and the three-dimensional reflection bright field imaging optical path; and according to the visual field coordinate offset, carrying out coordinate translation registration on the fluorescence image and the bright field image or adjusting the visual field of a camera to carry out visual field registration. The standard substance can be selected from fluorescent beads.
The pixel registration, comprising the steps of: and carrying out interpolation according to the magnification of the three-dimensional fluorescence imaging light path and the reflection bright field imaging light path and the size of the camera pixel, so that the magnification of the fluorescence image is consistent with that of the bright field image. When the amplification factors of the light sheet three-dimensional fluorescence imaging light path and the three-dimensional reflection bright field imaging light path are consistent with the size of the camera pixel, pixel registration is not needed.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:
the invention realizes the three-dimensional fluorescence imaging light path of the light sheet and the three-dimensional reflection bright field imaging light path to the same sample imaging plane of the sample through sharing the detection objective lens and the conjugate correction lens component, and simultaneously obtains the fluorescence mark information and the three-dimensional bright field imaging information, thereby realizing the two-dimensional image registration of hardware and realizing the more accurate and high-speed three-dimensional image imaging with the fluorescence mark. Meanwhile, the light path multiplexing is realized through the inverted light sheet three-dimensional fluorescence imaging light path and the inverted three-dimensional reflection bright field imaging light path, so that the imaging light path and the sample carrying are arranged.
According to the three-dimensional image acquisition method with the fluorescent marker, the two-dimensional image realizes hardware registration of the fluorescent marker information and the bright field image information, three-dimensional reconstruction and software registration of the fluorescent image and the bright field image are not needed, the three-dimensional image with the fluorescent marker can be acquired only by one-time three-dimensional reconstruction, and the method is high in speed and good in real-time performance. Meanwhile, the two-dimensional fluorescent mark information and the bright field image information of the same sample imaging plane are acquired, so that the hardware view registration can be conveniently realized, the image registration of the fluorescent mark information and the bright field image information can be realized only by simple coordinate transformation, the calculated amount is small, and the speed is high.
Drawings
FIG. 1 is a schematic diagram of the combination of vertical detection of single-objective lens fluorescence with reflective differential interference contrast quantitative bright field imaging (epi-DIC) provided in example 1;
FIG. 2 is a schematic diagram of the combination of dual-objective vertical detection of fluorescence with inverted reflection differential interference contrast quantitative bright field imaging (epi-DIC) provided in example 3.
The same reference numbers will be used throughout the drawings to refer to the same or like elements or structures, wherein:
the module 1 is a low power detection and vision finding module, 13 is an objective lens, 12 is a sleeve lens, and 11 is a camera; the module 2 is a fluorescence lighting module, 21 is a fluorescence lighting laser, 22 is a lens group for shaping a fluorescence lighting light path, and 23 is a fluorescence lighting objective lens; 8 is a sample carrier, 3 is a single objective lens module, 31 is a detection objective lens, 32 is a first lens, 33 is a second lens, 34 is a galvanometer, 35 is a third lens, 36 is a fourth lens, 37 is a front objective lens, 38 is a rear objective lens, and 39 is a first dichroic mirror; the module 2 is a fluorescence lighting module, 21 is a fluorescence lighting laser, and 22 is a lens group for shaping a fluorescence lighting light path; the module 4 is a fluorescence detection module, 42 is a sleeve lens, and 41 is a camera; 9 is a second dichroic mirror, and 10 is a first spectroscope; 6 is a bright field lighting module, 62 is a light source and a simple lens group, 63 is a second spectroscope, and 61 is a prism; the module 5 is an automatic focusing module; and 7, a bright field detection module, 71, a sleeve lens and 72, a camera.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention provides a three-dimensional image acquisition device with a fluorescent mark, which comprises an inverted light sheet three-dimensional fluorescent imaging light path, an inverted three-dimensional reflection bright field imaging light path and an overhead sample carrier, wherein the inverted light sheet three-dimensional fluorescent imaging light path is arranged on the sample carrier; the three-dimensional fluorescence imaging optical path and the three-dimensional reflection bright field imaging optical path of the light sheet share a detection objective lens 31 and conjugate correction lens assemblies 35-38;
the three-dimensional fluorescence imaging adopts a light sheet fluorescence technical means to improve the imaging speed, and the inverted light sheet is widely applied to sample imaging and has mature technology; in order to be compatible with hardware of a light sheet imaging system, an inverted bright field imaging light path is adopted to realize compatibility of an imaging light path and sample carrying setting, so that fluorescence imaging is carried out on a sample by adopting light sheet illumination and quantitative phase imaging is carried out by adopting bright field illumination. Furthermore, by properly combining the light paths of the fluorescence imaging and the bright field imaging, the fluorescence imaging and the bright field imaging can be coplanar, namely, the fluorescence imaging of the light sheet and the quantitative phase imaging of the reflection-type bright field can simultaneously acquire the image information of the same plane, so that the hardware registration of the two-dimensional images of the fluorescence and the bright field is realized, and the three-dimensional image reconstruction fusion has higher precision and speed. Meanwhile, hardware registration of the fluorescence and bright field two-dimensional images is realized, resolution is not required to be improved for software registration, and fluorescence imaging and bright field imaging can be both applicable to light sheet imaging and reflective differential interference phase difference quantitative bright field with higher imaging speed.
In the inverted light sheet three-dimensional fluorescence imaging light path, a fluorescence illumination light sheet passes through an illumination objective lens and obliquely illuminates a sample at a light sheet inclination angle alpha; the fluorescence excitation light is collected by a detection objective lens, passes through a conjugate correction lens assembly 35-38, and performs fluorescence imaging on a sample imaging plane in the direction orthogonal to the fluorescence illumination light sheet;
the optical sheet fluorescence imaging optical path, the illumination objective lens and the detection objective lens can use the same objective lens or two objective lenses; when the illumination objective lens and the detection objective lens multiplex the same objective lens, the objective lens is vertical to the sample carrying plane, the fluorescence illumination light enters through the position close to the edge of the back pupil of the objective lens, the number of optical elements is small, and the structure is compact. When the illumination objective lens and the detection objective lens adopt two objective lenses, the detection objective lens is vertical to the sample carrying plane, the optical axis of the illumination objective lens and the optical axis of the detection objective lens form a light sheet inclination angle alpha, and the detection visual field is larger; when the scheme is adopted, a multi-detection objective lens for low-power fluorescence imaging can be additionally arranged, so that the low-power detection function is increased.
The three-dimensional bright field imaging optical path is reflective differential interference phase difference quantitative bright field imaging (epi-DIC), and illumination light of the three-dimensional bright field imaging optical path sequentially passes through conjugate correction lens assemblies 35-38 and the detection objective lens 31 and is projected on a sample in a direction orthogonal to fluorescence illumination light; the sample reflected light sequentially passes through the detection objective lens 31 and the conjugate correction lens assemblies 35-38 to carry out bright field imaging on the sample imaging plane.
The single objective lens sheet uses the same objective lens to form a sheet and detect fluorescence, the sheet is obliquely incident on a sample at the edge close to the objective lens, the inclination angle is generally close to the light receiving angle of the objective lens, the same objective lens is used for detecting the fluorescence, the sheet is excited to be an inclined plane, when two objective lenses are adopted, the illumination sheet is also an inclined plane due to the steric hindrance caused by a sample carrier, and the plane of the information on the inclined plane after being imaged by the objective lens is also an inclined plane; therefore, in order to be combined with a bright field imaging light path, the imaging plane of bright field imaging needs to be adjusted.
The conjugate correction lens assembly comprises a front objective lens 37 and a rear objective lens 38 which are arranged in a confocal inclined mode; the primary optical axes of the front objective lens 37 and the rear objective lens 38 form an intersection angle β, and the intersection angle β is complementary to the light sheet inclination angle α; the front objective 37 is coaxial with the detection objective 31 and is conjugate in the back pupil plane. Preferably comprising a lens group arranged between the front objective 37 and the detection objective 31 for matching the magnification of the front objective 37 and the detection objective 31.
In order to achieve clearer low aberration fluorescence imaging, two additional objectives are used to achieve correction of the tilt plane, namely a front objective 37 and a rear objective 38. The probe and front objectives 37 and the intermediate lens group are coaxially arranged and function to perfectly conjugate the tilted sample imaging plane to the focal plane position of the front objective 37, while the conjugate image of the focal plane position is still tilted. The rear objective 38 will make an angle β with the optical axis of the front objective 37, which is complementary to the light sheet tilt angle α. The tilted image plane coincides with the front focal plane of the rear objective 38 so that the sample imaging plane information will be collected by the rear objective 38 for imaging on the camera plane. The front objective 37 and the rear objective 38 and the intermediate lens group constitute the conjugate correction lens assembly of the detection objective. In the bright field imaging optical path, in order to ensure that the object plane information acquired by the bright field is consistent with the object plane information acquired by the fluorescence of the light sheet, the bright field imaging optical path comprises the conjugate correction lens assembly, so that the bright field illumination and detection optical path is consistent with the fluorescence detection optical path, and two kinds of imaging information of fluorescence and bright field are obtained on the same plane of the same sample, namely the sample imaging plane, so that real-time two-dimensional plane imaging hardware registration is realized.
In a preferred scheme, the amplification factors of the optical sheet three-dimensional fluorescence imaging optical path and the three-dimensional reflection bright field imaging optical path are consistent, so that the visual field registration of optical sheet three-dimensional fluorescence imaging and three-dimensional reflection bright field imaging is realized.
The invention can realize image scanning through sample carrier movement or light path movement; in the sampler motion scenario: the sample carrier is provided with a movement mechanism, and preferably performs three-dimensional movement in space; in the light path movement scheme: a vibrating mirror is arranged on a conjugate surface of a rear pupil surface of the detection objective lens and between the detection objective lens and the conjugate correction lens assembly, so that fluorescent illumination light, detection light, bright field illumination light and detection light are reflected by the vibrating mirror, and the vibration of the vibrating mirror realizes the movement of an optical path to scan images. When the illumination objective lens and the detection objective lens multiplex the same objective lens, the optical path movement is preferably adopted to realize image scanning, so that the vibration caused by the physical movement of a sample is avoided; when the illumination objective lens and the detection objective lens adopt two objective lenses, the movement of the sample carrier is preferably adopted to realize image scanning, and the synchronous control of the multi-galvanometer is avoided.
The three-dimensional image is obtained by scanning the illuminating light through the galvanometer or controlling the sample to move through the electric displacement platform, and the fluorescence light path and the bright field light path detect the same plane no matter the illuminating light moves or the sample moves. The existing technology combining three-dimensional fluorescence and three-dimensional bright field cannot obtain the one-to-one corresponding image relationship between fluorescence and bright field on a two-dimensional plane, and can only complete fusion and alignment after three-dimensional image reconstruction, so that the alignment precision is limited and the imaging speed is slow. The combination of single-objective lens fluorescence and reflective differential interference phase-contrast quantitative phase imaging completes detection of two imaging modes of the same physical object plane from a hardware light path, realizes accurate image registration, has higher fluorescence imaging speed of the lens, and has simpler process because the post-image processing firstly carries out two-dimensional image registration and then only needs to carry out three-dimensional image reconstruction. The imaging efficiency is improved. According to the optimal scheme, the field of view registration is realized through hardware, the fluorescent two-dimensional image and the bright-field two-dimensional image can be registered only by coordinate alignment, and the three-dimensional imaging speed is more real-time and accurate.
The invention provides a three-dimensional image acquisition method with a fluorescent marker, which comprises the following steps:
the three-dimensional image acquisition device with the fluorescent label provided by the invention is applied to scan and image a sample: for each sample imaging plane, acquiring a fluorescence image of the sample imaging plane through a light sheet three-dimensional fluorescence imaging optical path, acquiring a bright field image of the sample imaging plane through a three-dimensional reflection bright field imaging optical path, and registering the fluorescence image and the bright field image to obtain a two-dimensional image of each sample imaging plane with a fluorescence mark; and performing three-dimensional reconstruction on the two-dimensional images with the fluorescent labels of all the sample imaging planes obtained by scanning to obtain three-dimensional images with the fluorescent labels of the samples.
Said registering said fluorescence image and said bright field image comprises a positional registration and/or a pixel registration;
the position registration, preferably, a standard with a fluorescence profile consistent with a sample profile is adopted to register the fluorescence image and the bright field image, specifically:
for a certain sample imaging plane of a standard sample, acquiring a fluorescence image of the standard sample through a light sheet three-dimensional fluorescence imaging optical path, acquiring a bright field image of the standard sample through a three-dimensional reflection bright field imaging optical path, and performing contour registration on the fluorescence image and the bright field image of the certain sample imaging plane to obtain the visual field coordinate offset of the light sheet three-dimensional fluorescence imaging optical path and the reflection bright field imaging optical path; and according to the visual field coordinate offset, carrying out coordinate translation registration on the fluorescence image and the bright field image or adjusting the visual field of a camera to carry out visual field registration. The standard substance can be selected from fluorescent beads.
The pixel registration, comprising the steps of: and carrying out interpolation according to the magnification ratios of the light sheet three-dimensional fluorescence imaging light path and the three-dimensional reflection bright field imaging light path and the size of the camera element, so that the magnification ratios of the fluorescence image and the bright field image are consistent. When the amplification factors of the light sheet three-dimensional fluorescence imaging light path and the three-dimensional reflection bright field imaging light path are consistent with the size of the camera pixel, pixel registration is not needed.
The following are examples:
example 1
The light path motion scanning realizes the vertical detection of single-objective lens light sheet fluorescence and the reflective differential interference phase difference quantitative bright field imaging:
the three-dimensional image acquisition device with the fluorescence label, which combines the vertical detection of single-objective lens fluorescence and the reflective micro-interference phase-contrast quantitative phase imaging, and realizes the three-dimensional imaging by adopting the optical path motion scanning, is taken as an example for explanation. As shown in fig. 1, the device comprises an inverted light sheet three-dimensional fluorescence imaging optical path, an inverted three-dimensional reflection bright field imaging optical path and an overhead sample carrier; the three-dimensional fluorescence imaging optical path and the three-dimensional reflection bright field imaging optical path of the light sheet share the detection objective lens 31 and the conjugate correction lens assembly, and the conjugate correction lens assembly comprises a third lens 35, a fourth lens 36, a front objective lens 37 and a rear objective lens 38;
the light sheet three-dimensional fluorescence imaging light path comprises a fluorescence excitation light path and a fluorescence detection light path:
the fluorescence excitation light sequentially passes through a laser 21 and a lens group 22 of an illumination light path for shaping Gaussian spots in the fluorescence illumination module 2, is reflected by a first dichroic mirror 39 and a vibrating mirror 34, reaches the detection objective lens 31 through a lens group consisting of a first lens 32 and a second lens 33, and obliquely irradiates on a sample through the detection objective lens 31; the second lens 33 and the first lens 32 are relay lens groups with focal lengths of 75mm and 150mm, respectively, and the detection objective lens 31 is a water lens with a magnification of 20 times and a numerical aperture of 1.0. The angle of the vibrating mirror 34 is controlled to make the fluorescent illumination light beam irradiate on the position of the deviated edge of the back pupil of the objective lens 31 after passing through the lens group consisting of the first lens 32 and the second lens 33, and then a light sheet is formed by the objective lens 31, wherein the light sheet parameters are determined by the shaping lens group 22, the position of the back pupil of the objective lens 31 where the light beam enters and the parameters of the objective lens 31. The inclination angle of the optical sheet with respect to the optical axis of the objective lens 31 is approximately 45 ° with respect to the optical axis of the objective lens 31.
Fluorescence generated by the fluorescence detection optical path sequentially passes through the element objective lens 31 in the module 3, the lens group consisting of the first lens 32 and the second lens 33, is reflected by the vibrating mirror 34, penetrates through the first dichroic mirror 39, and the lens group consisting of the third lens 35 and the fourth lens 36 of the conjugate correction lens assembly, wherein the third lens 35 and the fourth lens 36 are lens groups with a focal length of 60mm and a focal length of 100 mm. The front objective 37 and the rear objective 38 of the correction lens assembly are conjugate. The front objective lens 37 and the rear objective lens 38 are an air mirror with a magnification of 20 times and a numerical aperture of 0.75 and an air mirror with a magnification of 10 times and a numerical aperture of 0.45, respectively. And then reflected to the module 4 via the second dichroic mirror 9, and imaged onto the fluorescence camera 41 via the sleeve lens 42 of the module 4. The wavelength of laser is less than that of fluorescence and less than that of bright field, so the dichroscopes select long-pass short-reflection type (transmitting long-wavelength light and reflecting short-wavelength light). The lens group consisting of the first lens 32 and the second lens 33 is a group of relay lenses with equal focal length, the lens group consisting of the third lens 35 and the fourth lens 36 is a sleeve lens which is adapted according to the parameters of the detection objective lens 31 and the front objective lens 37 of the conjugate correction lens assembly, and the requirement of perfect imaging is that the magnification from the object plane to the back focal plane of the front objective lens 37 of the conjugate correction lens assembly meets the refractive index ratio between the object space and the space where the front objective lens 37 of the conjugate correction lens assembly is located.
The laser 21 may be a single wavelength laser or a multi-wavelength laser, which may be used for multi-channel imaging.
The optical sheet fluorescence imaging optical path comprises two objective lenses of a conjugate correction system 37 and 38 for correcting the angle of the detection surface and a lens group consisting of a first lens 32, a second lens 33, a third lens 35 and a fourth lens 36. The angle β by which the optical axis of the rear objective 38 of the conjugate correction lens assembly is tilted with respect to the optical axis of the front objective 37 is the complementary angle, i.e. 45 °, to the tilt angle α of the light sheet with respect to the optical axis of the detection objective 31.
The reflection type differential interference phase difference (epi-DIC) quantitative bright field optical path comprises a module 6, a module 7, a first spectroscope 10, a second dichroic mirror 9 and a module 3. The illumination light sequentially passes through a bright field light source and a lens group 62 in the module 6, is reflected to a prism 61 through a second beam splitter 63, sequentially passes through a first beam splitter 10, a second dichroic mirror 9, is incident to a rear objective lens 38 of a conjugate correction lens assembly in the module 3, sequentially passes through a front objective lens 37 of the conjugate correction lens assembly, a lens group consisting of a third lens 35 and a fourth lens 36 of the conjugate correction lens assembly, and is reflected to a lens group consisting of a first lens 32 and a second lens 33 through a galvanometer 34, and is irradiated on a sample through a detection objective lens 31. The reflected light passes through the module 3 in sequence, passes through the same path as the incident light from the objective lens 31 to the prism 61, is in reverse order, and then passes through the second beam splitter 63 to the sleeve lens 71 to be imaged on the bright field camera 72. Illumination light is emitted from module 6 at 62 and reflected by a second beam splitter 63 onto prism 61. 62 comprises a bright field light source, a shaping lens group and a polarizer.
The bright field camera and the fluorescence camera will detect signals of the same two-dimensional plane of the same sample. By controlling the reflection direction of the galvanometer 34, the sample carrier 8 is kept stationary. Two-dimensional signals of different planes of the sample can be acquired in a time sequence manner, so that a three-dimensional signal is reconstructed.
Example 2
The sample carrier motion scanning realizes the vertical detection of single-objective lens light-piece fluorescence and the reflective differential interference phase difference quantitative bright field imaging:
the acquisition of signals of the same two-dimensional plane of the same sample is the same as that in embodiment 1, and when a three-dimensional signal is acquired, the galvanometer 34 is kept still, and the sample is scanned by controlling the movement of the sample carrier 8, so that two-dimensional signals of different planes of the sample are acquired in time sequence, and three-dimensional reconstruction is completed.
Example 3
Sample carrier motion scanning double-objective lens vertical detection light piece fluorescence and inverted reflection type differential interference phase difference quantitative bright field imaging:
the combination of vertical detection of double-objective lens fluorescence and reflective differential interference phase-contrast quantitative phase imaging is an example, as shown in fig. 2, the difference from the embodiment 1 is that a fluorescence illumination module is separated, and one more fluorescence illumination objective lens 23 is added, so that a larger field of view can be realized. The positions of the fluorescent lighting modules 2 are different, and 23 is a fluorescent lighting objective lens, so that the first dichroic mirror 39 is reduced. In this mode, when the sample carrier 8 is moved to perform scanning three-dimensional imaging, the galvanometer 34 may be kept still or replaced with a mirror, and the rest of the mode is the same as that of embodiment 1. If three-dimensional scanning imaging of optical path movement is to be realized, a galvanometer needs to be additionally arranged in the module 2 to be synchronously controlled with the galvanometer 34, so that registration of three-dimensional fluorescence and a three-dimensional bright field is realized.
In the above embodiments, the magnification of the fluorescence detection optical path is the same as that of the bright field detection optical path, that is, the sleeve lenses 42 and 71 are of the same type, and the cameras are of the same type and have the same pixel size. The same object plane is imaged on the same camera plane through the same magnification, and the visual field registration is easier to realize.
Example 4
The invention provides a three-dimensional image acquisition method with a fluorescent marker, which comprises the following steps:
the three-dimensional image acquisition apparatus having a fluorescent marker of any one of embodiments 1 to 3 is applied;
scanning and imaging: and scanning and imaging the samples, and acquiring two-dimensional fluorescence labeling images and two-dimensional bright field phase information by using a CMOS camera for each sample imaging plane, namely a detection plane.
Position registration: adopting a fluorescent ball standard, obtaining a fluorescent image of the fluorescent ball standard through a light sheet three-dimensional fluorescent imaging light path, obtaining a bright field image of the fluorescent ball standard through a reflective bright field imaging light path, and carrying out contour registration on the fluorescent image and the bright field image of an imaging plane of the certain sample to obtain the visual field coordinate offset of the light sheet three-dimensional fluorescent imaging light path and the reflective bright field imaging light path; adjusting the camera view according to the view coordinate offset to perform view registration, or performing simple coordinate rigid transformation according to the view coordinate offset to realize registration; due to the design of the common light path of the fluorescence light path and the bright field light path, the two cameras acquire the information of the same plane of the sample. In the actual imaging process, the registration condition of the images can be detected by using a fluorescent ball, and when the deviation is large, the registration effect is achieved by mounting a camera in one of the optical paths on a displacement table for fine adjustment.
Pixel registration: and performing interpolation according to the magnification of the two optical paths and the size of the camera element to enable the two images to be matched. If the parameters of the embodiments 1 to 3 are selected to make the amplification factors of the three-dimensional fluorescence imaging light path and the reflection bright field imaging light path of the light sheet and the size of the camera pixel consistent, the pixel registration is not necessary.
And scanning to obtain images with fluorescent labels on all detection surfaces of the sample, and performing three-dimensional reconstruction to obtain three-dimensional images with fluorescent labels. The scanning mode includes scanning the illumination light and scanning the sample. The sample is scanned using a control sample carrier using a galvanometer 34 for illumination light scanning.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (12)
1. A three-dimensional image acquisition device with a fluorescent mark is characterized by comprising an inverted light sheet three-dimensional fluorescent imaging light path, an inverted three-dimensional reflection bright field imaging light path and an overhead sample carrier; the three-dimensional fluorescence imaging light path and the three-dimensional reflection bright field imaging light path share a detection objective lens and a conjugate correction lens assembly;
in the inverted light sheet three-dimensional fluorescence imaging light path, a fluorescence illumination light sheet passes through an illumination objective lens and obliquely illuminates a sample at a light sheet inclination angle alpha; the fluorescence excitation light is collected by a detection objective lens, and fluorescence imaging is carried out on a sample imaging plane in the direction orthogonal to the fluorescence illumination light sheet through a conjugate correction lens assembly;
the three-dimensional reflection bright field imaging optical path is reflection type differential interference phase difference quantitative bright field imaging, and illumination light of the three-dimensional reflection bright field imaging optical path is projected on a sample in a direction orthogonal to a fluorescence illumination light sheet through a conjugate correction lens assembly and the detection objective lens in sequence; sample reflected light sequentially passes through the detection objective lens and the conjugate correction lens assembly in the direction orthogonal to the fluorescent lighting light sheet, and bright field imaging is carried out on the sample imaging plane;
the conjugate correction lens assembly comprises a front objective lens and a rear objective lens which are confocal and obliquely arranged; the main optical axes of the front objective and the rear objective form an intersection angle beta, and the intersection angle beta is complementary with the inclination angle alpha of the light sheet.
2. The three-dimensional image capturing device with fluorescent markers of claim 1, wherein the front objective is coaxial with the detection objective and the back pupil plane is conjugate.
3. The three-dimensional image capturing device with fluorescent markers according to claim 1 or 2, wherein the conjugate correction lens assembly includes a lens group disposed between the front objective lens and the detection objective lens for matching the magnifications of the front objective lens and the detection objective lens.
4. The three-dimensional image capturing device with fluorescent markers of claim 1, wherein the illumination objective and the detection objective are multiplexed with the same objective, which is perpendicular to the sample-carrying plane, through which the fluorescent illumination light is incident at the near edge of the back pupil.
5. The apparatus according to claim 1, wherein the illumination objective and the detection objective are two objectives, the detection objective is perpendicular to the sample-carrying plane, and the optical axis of the illumination objective and the optical axis of the detection objective form a light sheet inclination angle α.
6. The three-dimensional image capture device with fluorescent markers of claim 1, further comprising a multi-probe objective for low power fluorescence imaging.
7. The three-dimensional image capture device with a fluorescent marker of claim 1, where the light sheet three-dimensional fluorescence imaging optical path is coincident with the magnification of the three-dimensional reflected bright field imaging optical path.
8. The three-dimensional image capturing apparatus with fluorescent marker according to claim 1, wherein the sample carrier has a moving mechanism.
9. The apparatus according to claim 1, wherein a galvanometer mirror is disposed on the conjugate surface of the back pupil surface of the detecting objective lens and between the detecting objective lens and the conjugate correcting lens assembly, so that the fluorescence illumination light, the detection light, the bright field illumination light, and the detection light are reflected by the galvanometer mirror, and the image scanning is performed by the joint movement of the excitation light path and the detection light path through the vibration of the galvanometer mirror.
10. A method for acquiring a three-dimensional image with a fluorescent marker, comprising the steps of:
scanning and imaging a sample by using the three-dimensional image acquisition device with the fluorescent marker as claimed in any one of claims 1 to 9: for each sample imaging plane, acquiring a fluorescence image of the sample imaging plane through a light sheet three-dimensional fluorescence imaging optical path, acquiring a bright field image of the sample imaging plane through a three-dimensional reflection bright field imaging optical path, and registering the fluorescence image and the bright field image to obtain a two-dimensional image of each sample imaging plane with a fluorescence mark; and performing three-dimensional reconstruction on the two-dimensional images with the fluorescent labels of all the sample imaging planes obtained by scanning to obtain three-dimensional images with the fluorescent labels of the samples.
11. The method of acquiring a three-dimensional image with a fluorescent marker according to claim 10, wherein the registering the fluorescent image and the bright field image comprises position registration and/or pixel registration.
12. The method of claim 11, wherein the position registration is performed by using a standard whose fluorescence profile is consistent with the sample profile to register the fluorescence image and the bright field image, and specifically comprises:
for a certain sample imaging plane of a standard sample, acquiring a fluorescence image of the standard sample through a light sheet three-dimensional fluorescence imaging optical path, acquiring a bright field image of the standard sample through a three-dimensional reflection bright field imaging optical path, and performing contour registration on the fluorescence image and the bright field image of the certain sample imaging plane to obtain the visual field coordinate offset of the light sheet three-dimensional fluorescence imaging optical path and the three-dimensional reflection bright field imaging optical path; according to the view coordinate offset, carrying out coordinate translation registration on the fluorescence image and the bright field image or adjusting the view of a camera to carry out view registration; the standard substance can be selected from fluorescent beads;
the pixel registration, comprising the steps of: and carrying out interpolation according to the magnification of the three-dimensional fluorescence imaging light path of the optical sheet and the reflection bright field imaging light path and the size of the camera element, so that the magnification of the fluorescence image is consistent with that of the bright field image.
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