阅读说明：本技术 手性荧光共聚焦成像系统及成像方法 (Chiral fluorescence confocal imaging system and imaging method ) 是由 袁景和 岑柏桦 方晓红 于 2021-06-18 设计创作，主要内容包括：本发明涉及光学技术领域,具体公开了一种手性荧光共聚焦成像系统及成像方法,包括：激光器、激发光路、显微镜、样品台、探测光路、光电探测器以及控制装置,其中,所述激光器发射的激光经激发光路调制产生偏振光,所述偏振光通过显微镜照射到样品台的待测样品上,并激发待测样品产生荧光信号,所述荧光信号被所述探测光路调制并传送至所述光电探测器,所述光电探测器将荧光信号转化成电信号并成像,所述控制装置与所述光电探测器以及样品台连接。本发明的手性荧光共聚焦成像系统能够提高传统光学显微镜成像分辨率,实现激光共聚焦成像。(The invention relates to the technical field of optics, and particularly discloses a chiral fluorescence confocal imaging system and an imaging method, wherein the imaging method comprises the following steps: laser instrument, excitation light path, microscope, sample platform, detection light path, photoelectric detector and controlling means, wherein, the laser of laser instrument transmission produces polarized light through exciting the light path modulation, the polarized light shines on the sample that awaits measuring of sample platform through the microscope to the excitation sample that awaits measuring produces fluorescence signal, fluorescence signal quilt detection light path modulation and conveying to photoelectric detector, photoelectric detector converts fluorescence signal into the signal of telecommunication and formation of image, controlling means with photoelectric detector and sample platform are connected. The chiral fluorescence confocal imaging system can improve the imaging resolution of the traditional optical microscope and realize laser confocal imaging.)

1. A chiral fluorescence confocal imaging system, comprising: laser instrument, excitation light path, microscope, sample platform, detection light path, photoelectric detector and controlling means, wherein, the laser of laser instrument transmission produces polarized light through exciting the light path modulation, the polarized light shines on the sample that awaits measuring of sample platform through the microscope to the excitation sample that awaits measuring produces fluorescence signal, fluorescence signal quilt detection light path modulation and conveying to photoelectric detector, photoelectric detector converts fluorescence signal into the signal of telecommunication and formation of image, controlling means with photoelectric detector and sample platform are connected.

2. The chiral fluorescence confocal imaging system according to claim 1, wherein the excitation light path is provided with a first linear polarizer, a first reflector, a second reflector, a first quarter-wave plate and a dichroic filter, and the laser light emitted by the laser device sequentially passes through the linear polarizer, the first reflector, the second reflector, the first quarter-wave plate and the dichroic filter and then is converged on a microscope.

3. The chiral fluorescence confocal imaging system according to claim 2, wherein a second fluorescence filter, a third reflector, a fourth reflector, a fifth reflector, a second quarter-wave plate, a second linear polarizer, a third fluorescence filter and a fluorescence collecting lens are disposed in the detection light path, and the fluorescence signal is separated by the dichroic filter and then sequentially passes through the second fluorescence filter, the third reflector, the fourth reflector, the fifth reflector, the second quarter-wave plate, the second linear polarizer, the third fluorescence filter and the fluorescence collecting lens to enter the photodetector.

4. The chiral fluorescence confocal imaging system according to claim 2, wherein a collimating lens is further disposed in the excitation light path, and the laser emitted by the laser device is collimated by the collimating lens and then passes through the first linear polarizer.

5. The chiral confocal fluorescence imaging system of claim 1, wherein the control device controls the synchronous motion of the sample stage and the photodetector.

6. The chiral fluorescence confocal imaging system of claim 1, wherein the laser emits a laser wavelength at a desired excitation wavelength of the chiral fluorescent molecule.

7. The chiral confocal fluorescent imaging system of claim 1, wherein the photodetector is selected from avalanche photodetectors.

8. The chiral fluorescence confocal imaging system according to any one of claims 1 to 7, wherein the sample to be tested is a chiral fluorescent molecule.

9. The method of imaging by the chiral fluorescence confocal imaging system of any one of claims 1 to 8, comprising the steps of:

controlling the laser to output laser with required wavelength;

the laser generates polarized light through the modulation of an excitation light path;

the polarized light irradiates a sample to be detected of the sample stage through a microscope and excites the sample to be detected to generate a fluorescence signal;

the fluorescence signal is modulated by the detection light path and is transmitted to the photoelectric detector, and the photoelectric detector converts the fluorescence signal into an electric signal and images.

Technical Field

The invention relates to the technical field of optics, and particularly discloses a chiral fluorescence confocal imaging system and an imaging method.

Background

Laser scanning confocal microscopy (Laser scanning confocal microscope) is a new technology which is developed and widely applied in the middle of the 80 th 20 th century, is an advanced cell molecular biology analytical instrument which permeates modern high-tech means such as Laser, electronic camera shooting and computer image processing and is generated by combining with a traditional optical microscope, is increasingly widely applied in the fields of biology, medicine and the like, and has become a necessary tool for biomedical experimental research.

Conventional fluorescence microscopes use fluorescent materials to mark specific structures in cells, not only to enhance the contrast of the image with the background, but also to greatly improve the resolution due to the use of ultraviolet light of short wavelength as the light source of many fluorescence microscopes (δ 0.61 λ/NA, where δ is the resolution of the microscope; λ is the wavelength of the illuminating light; NA is the numerical aperture of the objective lens). However, when the observed fluorescence specimen is slightly thicker, a drawback that is difficult to overcome by the conventional fluorescence microscope appears: the fluorescent structure outside the focal plane is blurred and weakened. The reason is that most biological specimens are hierarchically differentiated overlapping structures (such as a cochlear basilar membrane, which is a spatial structure consisting of outer hair cells, various supporting cells, nerve fibers and the like), and the change of a focal plane under a common optical microscope can show different morphologies. If the fluorescent marker structures are distributed at different levels and overlap, the optical resolution of the fluorescence microscope is greatly reduced because not only is the amount of light collected from the focal plane by the reflection fluorescence microscope (epifluorescence microscope) but also scattered fluorescence from above or below the focal plane is received by the objective lens.

On the basis of a traditional optical microscope, a laser scanning confocal microscope uses laser as a light source, adopts a conjugate focusing principle and a device, and utilizes a computer to carry out digital image processing observation, analysis and output on an observed object. The method is characterized in that the method can carry out tomography and imaging on a sample, and carry out nondestructive observation and analysis on the three-dimensional space structure of cells. Meanwhile, by using immunofluorescence labeling and ionic fluorescence labeling probes, the technology can observe fixed cells and tissue slices, can also dynamically observe and detect the structures, molecules, ions and life activities of living cells in real time, can observe physiological signals such as Ca2+, pH value, membrane potential and the like and changes of cell morphology at a subcellular level, becomes a new powerful research tool in the fields of morphology, molecular cell biology, neuroscience, pharmacology, genetics and the like, and greatly enriches the understanding of people on cell life phenomena.

Chirality refers to the phenomenon that the entity of a chemical molecule and its mirror image cannot overlap, and it is a phenomenon commonly existing in nature, for example, amino acids in structural protein are all L-type amino acids, naturally occurring saccharides are D-type, and the stable configuration of nucleic acid in human body is dextrorotation, etc. A pair of enantiomers have similar physical and chemical properties, but they have different optical or pharmacological activities in chiral environments. The chiral luminescent material can respectively emit left-handed and right-handed Circularly Polarized Light (CPL), and has potential application value in circular polarization electroluminescent devices (CP-OLED) and 3D display. Displays based on chiral luminescent materials, which emit circularly polarized light, can be passed directly through the filter without energy loss. At present, the chiral fluorescent material has very important significance in the fields of chiral identification and resolution, medicinal chemistry, material science, food detection, life science and the like. However, in the field of microscopy imaging, there is a lack of imaging instruments for studying chiral fluorescence. Circular dichroism means that a chiral material can selectively absorb or emit left-handed circularly polarized light and right-handed circularly polarized light, which is also a property unique to chiral materials. Therefore, the chiral fluorescence confocal microscope has important innovative significance by utilizing chiral fluorescence to image, and simultaneously expands the application prospect of the chiral fluorescence material.

Disclosure of Invention

The present invention is directed to a chiral fluorescence confocal imaging system and an imaging method, which are used to solve at least one of the above technical problems.

In order to achieve the above object, the present invention provides a chiral fluorescence confocal imaging system, comprising: laser instrument, excitation light path, microscope, sample platform, detection light path, photoelectric detector and controlling means, wherein, the laser of laser instrument transmission produces polarized light through exciting the light path modulation, the polarized light shines on the sample that awaits measuring of sample platform through the microscope to the excitation sample that awaits measuring produces fluorescence signal, fluorescence signal quilt detection light path modulation and conveying to photoelectric detector, photoelectric detector converts fluorescence signal into the signal of telecommunication and formation of image, controlling means with photoelectric detector and sample platform are connected.

In addition, the invention provides an imaging method of the chiral fluorescence confocal imaging system, which comprises the following steps:

controlling the laser to output laser with required wavelength;

the laser generates polarized light through the modulation of an excitation light path;

the polarized light irradiates a sample to be detected of the sample stage through a microscope and excites the sample to be detected to generate a fluorescence signal;

the fluorescence signal is modulated by the detection light path and is transmitted to the photoelectric detector, and the photoelectric detector converts the fluorescence signal into an electric signal and images.

In addition, the chiral fluorescence confocal imaging system of the invention can also have the following additional technical characteristics.

According to an embodiment of the invention, a first linear polarizer, a first reflecting mirror, a second reflecting mirror, a first quarter-wave plate and a dichroic filter are arranged in the excitation light path, and laser light emitted by the laser sequentially passes through the linear polarizer, the first reflecting mirror, the second reflecting mirror, the quarter-wave plate and the dichroic filter and is converged in a microscope.

According to an embodiment of the present invention, a second fluorescence filter, a third reflector, a fourth reflector, a fifth reflector, a second quarter-wave plate, a second linear polarizer, a third fluorescence filter, and a fluorescence collecting lens are disposed in the detection light path, and the fluorescence signal is separated by the dichroic filter, and then sequentially passes through the second fluorescence filter, the third reflector, the fourth reflector, the fifth reflector, the second quarter-wave plate, the second linear polarizer, the third fluorescence filter, and the fluorescence collecting lens, and then enters the photodetector.

According to an embodiment of the present invention, a collimating lens is further disposed in the excitation light path, and the laser emitted by the laser device is collimated by the collimating lens and then passes through the first linear polarizer.

According to one embodiment of the invention, the control device controls the synchronous movement of the sample stage and the photodetector.

According to one embodiment of the invention, the laser emits a laser wavelength at the desired excitation wavelength for the chiral fluorescent molecules.

According to one embodiment of the invention, the photodetector is selected from avalanche photodetectors.

Compared with the prior art, the invention has the following beneficial effects:

1. the chiral fluorescence confocal imaging system can improve the imaging resolution of the traditional optical microscope and realize laser confocal imaging;

2. the chiral fluorescence confocal imaging system adopts one path of exciting light, the exciting light is modulated by the quarter-wave plate and is irradiated to an imaging area by the dichroic plate, and the operation is simple and feasible.

3. The chiral fluorescence confocal imaging system adopts chiral fluorescent molecules as a sample to be detected, and can be used for researching the interaction between the chiral fluorescent molecules and polarized light and exploring the strength and chiral characteristics of fluorescent signals of the fluorescent molecules under the action of the polarized light through the confocal imaging system and the imaging method;

4. the chiral fluorescence confocal imaging system and the imaging method provided by the invention are simple and feasible, the imaging optical path system is simpler, and the economic cost is saved;

5. the chiral fluorescence confocal imaging system and the imaging method provided by the invention are beneficial to further understanding the optical characteristics of chiral fluorescence molecules and developing the application potential of chiral fluorescence materials in the aspect of optical research.

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 some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.

Fig. 1 is a schematic structural diagram of a chiral fluorescence confocal imaging system according to an embodiment of the present invention.

1, a laser; 2 a first linear polarizer; 3 a first mirror; 4 a second mirror; 5 a first quarter wave plate; 6 dichroic filters; 7, a microscope; 8, a sample stage; 9 a first fluorescence filter; 10 a third mirror; 11 a fourth mirror; 12 a fifth mirror; 13 a second quarter wave plate; 14 a second linear polarizer; 15 a second fluorescence filter; 16 a fluorescence collection lens; 17 photo detector.

The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.

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.

It should be noted that all the directional indicators (such as up, down, left, right, front, and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the movement situation, etc. in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indicator is changed accordingly.

In addition, the descriptions related to "first", "second", etc. in the present invention are only for descriptive purposes 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 at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In the present invention, unless otherwise expressly stated or limited, the terms "connected," "secured," and the like are to be construed broadly, and for example, "secured" may be a fixed connection, a removable connection, or an integral part; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

In addition, the technical solutions in the embodiments of the present invention may be combined with each other, but it must be based on the realization of those skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination of technical solutions should not be considered to exist, and is not within the protection scope of the present invention.

The embodiment of the invention provides a chiral fluorescence confocal imaging system, which firstly modulates laser to be modulated into circularly polarized light to irradiate an imaging area, and then modulates fluorescence emitted by a sample to be detected into linearly polarized light, thereby realizing chiral confocal imaging.

As shown in fig. 1, the chiral fluorescence confocal imaging system includes a laser 1, an excitation light path, a microscope 7, a detection light path, a photodetector 17, and a control device, wherein laser emitted by the laser 1 is modulated by the excitation light path to generate polarized light, the polarized light is irradiated onto a sample to be detected of a sample stage 8 through the microscope 7, and the sample to be detected is excited to generate a fluorescence signal, the fluorescence signal is modulated by the detection light path, the detection light path transmits the fluorescence signal to the photodetector 17, the photodetector 17 converts the fluorescence signal into an electrical signal and images, and the control device is connected to the photodetector 17 and the sample stage 8. It should be noted that the sample to be detected in this embodiment may be a chiral fluorescent molecule.

The laser device comprises an excitation light path, wherein a collimating lens, a first linear polarizer 2, a first reflector 3, a second reflector 4, a first quarter-wave plate 5 and a dichroic filter 6 are arranged in the excitation light path, and laser emitted by the laser device 1 sequentially passes through the collimating lens, the first linear polarizer 2, the first reflector 3, the second reflector 4, the first quarter-wave plate 5 and the dichroic filter 6 and then is converged in a microscope 7, and then irradiates a sample to be measured on a sample stage 8.

It should be noted that the laser output from the laser 1 is focused on the microscope after passing through the first reflecting mirror 3 and the second reflecting mirror 4. Specifically, the laser light enters the microscope after passing through two reflecting mirrors to facilitate alignment of the light paths.

Further, be equipped with second fluorescence filter 9, third speculum 10, fourth speculum 11, fifth speculum 12, second quarter wave plate 13, second linear polarizer 14, third fluorescence filter 15 and fluorescence collection lens 16 in the detection light path, the fluorescence signal passes through after dichroic filter 9's the separation, again pass through second fluorescence filter 9, third speculum 10, fourth speculum 11, fifth speculum 12, second quarter wave plate 13, second linear polarizer 14, third fluorescence filter 15 and fluorescence collection lens 16 in proper order and then get into photoelectric detector 17.

It should be noted that the fluorescence signal of the sample to be measured passes through the fourth mirror 11 and the fifth mirror 12 before being incident on the photoelectric detector 17, so as to facilitate the alignment of the optical paths. The fluorescence signal of the sample to be measured can be filtered by the third fluorescence filter 1 before entering the photodetector 17. The fluorescence signal of the sample to be measured passes through the fluorescence collecting lens 16 to collect fluorescence before being incident on the photodetector 17.

In this embodiment, the fluorescence signal of the sample to be measured passes through the second quarter-wave plate 13 and the second linear polarizer 14 before being incident on the photodetector 17, so as to modulate the circularly polarized light into linearly polarized light and calibrate the linearly polarized light.

In addition, the control device is respectively connected with the photoelectric detector 17 and the displacement controller on the sample stage 8, and is used for controlling the work of the photoelectric detector 17, collecting optical signals and the movement of the sample stage 8, so as to obtain the chiral fluorescence microscopic image.

Specifically, the imaging method of the chiral fluorescence confocal imaging system in the embodiment may include the following steps:

laser output by the laser passes through the collimating lens, the polarizing disc and the two reflectors, then sequentially passes through the quarter-wave plate and the dichroic filter, then is converged to the microscope to irradiate the sample stage, and is used for exciting a sample to be detected, fluorescent signals of the obtained sample to be detected are converged by the same microscope, then pass through the reflectors and the dichroic filter, then pass through the fluorescent filter, the quarter-wave plate, the polarizing disc, the fluorescent filter and the collecting lens, and then enter the photoelectric detector, photoelectric signals output by the photoelectric detector are input to the optical signal collector, and further chiral fluorescent microscopic images are obtained.

The embodiments of the present disclosure have been described above. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. The scope of the disclosure is defined by the appended claims and equivalents thereof. Various alternatives and modifications can be devised by those skilled in the art without departing from the scope of the present disclosure, and such alternatives and modifications are intended to be within the scope of the present disclosure.