CN110888229A - Light field micro-microscope system - Google Patents
Light field micro-microscope system Download PDFInfo
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- CN110888229A CN110888229A CN201911229932.2A CN201911229932A CN110888229A CN 110888229 A CN110888229 A CN 110888229A CN 201911229932 A CN201911229932 A CN 201911229932A CN 110888229 A CN110888229 A CN 110888229A
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- excitation light
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- 230000005284 excitation Effects 0.000 claims abstract description 61
- 238000003384 imaging method Methods 0.000 claims abstract description 51
- 230000003287 optical effect Effects 0.000 claims abstract description 16
- 238000000386 microscopy Methods 0.000 claims description 15
- 210000004556 brain Anatomy 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 4
- 210000002569 neuron Anatomy 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 238000002610 neuroimaging Methods 0.000 description 1
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/02—Objectives
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
- G02B21/08—Condensers
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/361—Optical details, e.g. image relay to the camera or image sensor
Abstract
The utility model provides a miniature microscopic system in light field, includes excitation light source, arouses light path, emission light path, objective and imaging sensor, wherein, the light that excitation light source sent passes through arouse light path projects the observation object, and is right the observation object carries out fluorescence excitation, and the fluorescence that is aroused passes through the emission light path by imaging sensor gathers, be provided with a beam splitting focusing device in the emission light path before imaging sensor for gather the multidimensional information of observation object, wherein, beam splitting focusing device set up in the miniature microscopic system in light field the place ahead of the focus plane that objective working distance corresponds makes the observation object is in behind each optical device of the inside miniature microscopic system in light field become the virtual image on the imaging sensor. The invention not only reduces the size and weight of the light field micro-microscope system, but also ensures that the resolution ratio has consistency in the imaging depth range.
Description
Technical Field
The invention relates to the field of optical microscopic imaging and computer vision, in particular to a light field micro-microscope system.
Background
Revealing the mystery of the brain has been one of the biggest challenges facing humans, and therefore brain imaging has been a research hotspot in the academic and industrial circles at home and abroad. In order to understand the relationship between specific regions in the brain and their functions, researchers have proposed various head-mounted micro-imaging systems in recent years in order to observe the neuron activity in the brain of an animal under natural behavior conditions in real time. However, existing micro-microscopy imaging systems are not capable of acquiring three-dimensional time-varying information of neuronal activity within a single exposure. In order to realize the recording of the three-dimensional neuron activity, researchers fuse the light field acquisition with a micro-imaging system, and provide the light field micro-imaging system.
However, the current light field micro-microscope system still has the defects. First, the optical architecture used by the existing light field micro-microscopy system results in a long overall system size and low weight, which may result in the natural behavior of the animal being affected. Secondly, the spatial and angular sampling of the existing light field micro-microscope system in the imaging depth range is not properly distributed, so that the resolution ratio does not have consistency in the imaging depth range.
Disclosure of Invention
It is a primary object of the present invention to provide a light field micro-microscopy system that reduces the size and weight of the system and provides uniformity of system resolution over the depth of imaging.
In order to achieve the purpose, the invention adopts the following technical scheme:
the utility model provides a miniature microscopic system in light field, includes excitation light source, arouses light path, emission light path, objective and imaging sensor, wherein, the light that excitation light source sent passes through arouse light path projects the observation object, and is right the observation object carries out fluorescence excitation, and the fluorescence that is aroused passes through the emission light path by imaging sensor gathers, be provided with a beam splitting focusing device in the emission light path before imaging sensor for gather the multidimensional information of observation object, wherein, beam splitting focusing device set up in the miniature microscopic system in light field the place ahead of the focus plane that objective working distance corresponds makes the observation object is in behind each optical device of the inside miniature microscopic system in light field become the virtual image on the imaging sensor.
Further:
the emission light path comprises a relay lens, and the beam splitting focusing device is arranged between the imaging sensor and the relay lens.
The optical parameters and the setting position of the beam splitting focusing device satisfy the following relational expression:
d1-d2>0, (1)
wherein M is the magnification factor formed by the objective lens and the relay lens in the light field micro-microscope system during focusing, NA is the numerical aperture of the objective lens, fmlaAnd dmlaRespectively the focal length and diameter of each focusing lens unit on the beam splitting and focusing device, d1The distance between the position of the focal plane corresponding to the working distance of the objective lens and the relay lens, d2Is the distance between the position where the beam splitting focusing device is placed and the relay lens, b is the distance between the beam splitting focusing device and the imaging sensor, MmlaFor the magnification, magnification M and magnification M of the objective lens and the relay lens at the position where the beam splitting focusing device is placedmlaOnly at d1－d2Equal when being equal to 0.
The product of the magnification factor formed by the objective lens and the relay lens and the imaging field of view of the light field micro microscope system does not exceed the effective area of the target surface of the imaging sensor.
The excitation light source is a plurality of light sources with different excitation wavelengths.
The excitation light source comprises a first excitation light source and a second excitation light source, the light field micro-microscope system further comprises a first dichroic mirror, the first excitation light source is reflected to the observation object through the first dichroic mirror, and the second excitation light source is transmitted to the observation object through the first dichroic mirror.
The light field micro-microscope system also comprises a second dichroic mirror, the first excitation light source is reflected to the observation object by the excitation light reflected by the first dichroic mirror through the second dichroic mirror, the second excitation light source is reflected to the observation object by the excitation light transmitted by the first dichroic mirror through the second dichroic mirror, and the fluorescence excited by the excitation light of the first excitation light source and the excitation light of the second excitation light source is transmitted through the second dichroic mirror and collected by the imaging sensor through the emission light path.
The invention has the following beneficial effects:
the invention provides a light field micro-microscope system, wherein a light splitting focusing device is arranged in front of a focusing surface corresponding to the working distance of an objective lens in the light field micro-microscope system, so that an observation object forms a virtual image on an imaging sensor after passing through each optical device in the light field micro-microscope system, the arrangement mode of the light splitting focusing device not only can shorten the overall size of the system, but also can ensure that the system has proper space sampling and angle sampling within the imaging depth range, and the resolution ratio has consistency within the imaging depth range, thereby compared with the prior art, the invention provides a micro-microscope system which has small size and light weight and can realize the acquisition of three-dimensional information of the observation object by single exposure,
the light field micro-microscope system provided by the invention ensures that the three-dimensional neuron activity recording in the animal brain is realized under the condition of natural behavior, simultaneously reduces the size and the weight of the system, and ensures that the resolution ratio has consistency in the imaging depth range.
Drawings
FIG. 1 is an overall architecture diagram of a light field micro microscopy system according to an embodiment of the present invention;
FIG. 2 is an optical architecture diagram of the emission optical path of the light field micro-microscope system according to the embodiment of the present invention.
Detailed Description
The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element. In addition, the connection may be for either a fixed function or a circuit/signal communication function.
It is to be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in an orientation or positional relationship indicated in the drawings for convenience in describing the embodiments of the present invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be in any way limiting of the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the embodiments of the present invention, "a plurality" means two or more unless specifically limited otherwise.
Referring to fig. 1 and fig. 2, an embodiment of the invention provides a light field micro-microscope system, which includes an excitation light source, an excitation light path, an emission light path, an objective lens 6 and an imaging sensor 11, wherein, the exciting light 4 emitted by the exciting light sources 1, 3 is projected to an observation object 5 through the exciting light path, fluorescence excitation is performed on the observation object 5, the excited fluorescence 9 is collected by the imaging sensor 11 through the emission optical path, a light splitting focusing device 10 is arranged in the emission light path in front of the imaging sensor 11 and is used for collecting the multi-dimensional information of the observation object 5, wherein, the beam splitting focusing device 10 is arranged in front of a focusing surface corresponding to the working distance of the objective 6 in the light field micro-microscope system, so that the observation object 5 forms a virtual image on the imaging sensor 11 after passing through each optical device inside the light field micro-microscope system.
Referring to fig. 1, in some embodiments, the emission optical path includes a relay lens 8, and the beam splitting and focusing device 10 is disposed between the imaging sensor 11 and the relay lens 8.
In some embodiments, the excitation light source is a plurality of light sources having different excitation wavelengths.
Referring to fig. 1, in a preferred embodiment, the excitation light source includes a first excitation light source 1 and a second excitation light source 3, the light field micro microscopy system further includes a first dichroic mirror 2, the first excitation light source 1 is reflected onto the observation object 5 through the first dichroic mirror 2, and the second excitation light source 3 is transmitted onto the observation object 5 through the first dichroic mirror 2.
As shown in fig. 1, in a further preferred embodiment, the light field micro-microscopy system further includes a second dichroic mirror 7, the excitation light reflected by the first dichroic mirror 2 of the first excitation light source 1 is reflected onto the observation object 5 through the second dichroic mirror 7, the excitation light transmitted by the first dichroic mirror 2 of the second excitation light source 3 is reflected onto the observation object 5 through the second dichroic mirror 7, and the fluorescence excited by the excitation light of the first excitation light source 1 and the second excitation light source 3 is transmitted through the second dichroic mirror 7 and collected by the imaging sensor 11 through the emission light path.
The general architecture of the light field micro-microscope system of the embodiment of the invention is shown in fig. 1. The novel light field micro-microscope system comprises an excitation light path and an emission light path, wherein an excitation light source in the excitation light path is used for fluorescence excitation of an observation object 5, and a light splitting focusing device 10 is placed in front of an imaging sensor 11 in the emission light path and used for collecting multi-dimensional information of the observation object 5. The light splitting focusing device 10 is placed in front of a focusing surface corresponding to the working distance of the objective lens 6 in the light field micro-microscope system, so that the observation object 5 forms a virtual image on the imaging sensor 11 after passing through each optical device in the light field micro-microscope system, as shown in fig. 2.
In the existing light field micro-microscope system, a beam splitting focusing device 10 is placed at a focusing plane corresponding to the working distance of an objective lens 6. The placement mode not only enables the whole system to be long in size and light in weight, but also enables angular sampling of the system at the working distance to be redundant, and the resolution is low, so that the resolution of the system is not consistent in the imaging depth range.
In order to avoid the defects, the light field micro-microscope system provided by the embodiment of the invention moves the beam splitting focusing device towards the relay lens, the whole size of the system is shortened by changing the optical architecture of the system, and the moving distance enables the system to have proper spatial sampling and angle sampling in the imaging depth range, so that the resolution ratio is ensured to have consistency in the imaging depth range. The optical parameters and the placement position of the selected beam splitting and focusing device need to satisfy the following relational expression
d1-d2>0, (1)
Wherein M is the magnification factor formed by the objective lens and the relay lens in the light field micro-microscope system during focusing, NA is the numerical aperture of the objective lens, fmlaAnd dmlaRespectively for each beam splitter and focusing deviceFocal length and diameter of individual focusing lens units, d1The distance between the position of the focal plane corresponding to the working distance of the objective lens and the relay lens, d2Is the distance between the position where the beam splitting focusing device is placed and the relay lens, b is the distance between the beam splitting focusing device and the imaging sensor, MmlaThe magnification of the objective lens and the relay lens at the position where the beam splitting focusing device is placed. Magnification factor M and magnification factor MmlaOnly at d1－d2Equal when being equal to 0.
The system selects the objective lens with larger numerical aperture NA as much as possible when selecting the objective lens so as to ensure that the system has higher imaging performance. The product of the magnification factor formed by the selected objective lens and the relay lens and the imaging view field of the novel light field micro-microscope system does not exceed the effective area of the target surface of the imaging sensor.
The novel light field micro-microscope system provided by the invention can realize the acquisition of multi-dimensional information of an observation object in single exposure, and can reconstruct the three-dimensional information of the observation object by combining a reconstruction algorithm. In addition, the novel light field micro-microscope system has the advantages of small size and light weight, and can be used for body imaging of freely moving animal brains and observing time-varying activities of neurons in the animal brains.
The background of the present invention may contain background information related to the problem or environment of the present invention and does not necessarily describe the prior art. Accordingly, the inclusion in the background section is not an admission of prior art by the applicant.
The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention. In the description herein, references to the description of the term "one embodiment," "some embodiments," "preferred embodiments," "an example," "a specific example," or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction. Although embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the scope of the claims.
Claims (7)
1. The light field micro-microscope system is characterized by comprising an excitation light source, an excitation light path, an emission light path, an objective lens and an imaging sensor, wherein light emitted by the excitation light source is projected to an observation object through the excitation light path, fluorescence excitation is carried out on the observation object, the excited fluorescence is collected by the imaging sensor through the emission light path, a light splitting and focusing device is arranged in the emission light path in front of the imaging sensor and used for collecting multi-dimensional information of the observation object, and the light splitting and focusing device is arranged in front of a focusing surface corresponding to the working distance of the objective lens in the light field micro-microscope system, so that the observation object forms a virtual image on the imaging sensor after passing through each optical device in the light field micro-microscope system.
2. The light field micro microscopy system as defined in claim 1 wherein the emission light path includes a relay lens, the beam splitting focusing device being disposed between the imaging sensor and the relay lens.
3. The light field micro-microscopy system as claimed in claim 2, wherein the optical parameters and the setting position of the beam splitting focusing device satisfy the following relation:
d1-d2>0,(1)
wherein M is the magnification factor formed by the objective lens and the relay lens in the light field micro-microscope system during focusing, NA is the numerical aperture of the objective lens, fmlaAnd dmlaRespectively the focal length and diameter of each focusing lens unit on the beam splitting and focusing device, d1The distance between the position of the focal plane corresponding to the working distance of the objective lens and the relay lens, d2Is the distance between the position where the beam splitting focusing device is placed and the relay lens, b is the distance between the beam splitting focusing device and the imaging sensor, MmlaFor the magnification, magnification M and magnification M of the objective lens and the relay lens at the position where the beam splitting focusing device is placedmlaOnly at d1－d2Equal when being equal to 0.
4. The light field micro-microscopy system as defined in any one of claims 2 to 3 wherein the product of the magnification of the objective lens and the relay lens and the imaging field of view of the light field micro-microscopy system does not exceed the effective area of the imaging sensor target surface.
5. The light field micro microscopy system as defined in any one of claims 1 to 4, wherein the excitation light source is a plurality of light sources having different excitation wavelengths.
6. The light field micro-microscopy system as claimed in any one of claims 1 to 5, wherein the excitation light source comprises a first excitation light source and a second excitation light source, the light field micro-microscopy system further comprising a first dichroic mirror, the first excitation light source being reflected onto the observation object through the first dichroic mirror, the second excitation light source being transmitted onto the observation object through the first dichroic mirror.
7. The light field micro microscopy system as defined in claim 6, wherein the light field micro microscopy system further comprises a second dichroic mirror, wherein the first excitation light source is reflected by the excitation light reflected by the first dichroic mirror onto the observation object through the second dichroic mirror, wherein the second excitation light source is reflected by the excitation light transmitted by the first dichroic mirror onto the observation object through the second dichroic mirror, and wherein fluorescence excited by the excitation light of the first excitation light source and the second excitation light source is transmitted through the second dichroic mirror and collected by the imaging sensor through the emission light path.
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