CN103385698A - Fluorescence imaging system and application thereof - Google Patents

Abstract

The invention discloses a fluorescence imaging system and its application. The fluorescence imaging system includes: a fluorescence excitation unit, the light beam generated by it is irradiated on the fluorescent substance, and the fluorescent substance is excited to emit fluorescence; a filter group is used to filter the fluorescence excited by the fluorescent substance, so as to filter the non- The light in the fluorescence band is filtered out; the imaging objective lens receives the fluorescence filtered by the filter group; the detector, whose effective wavelength range of detection is 800-1700nm, obtains the imaging of the fluorescent substance through the imaging objective lens. The invention uses near-infrared laser beams to irradiate small animals to excite quantum dots in the small animals to emit fluorescence, and can quickly, efficiently and non-destructively observe deep tissues, organs and cells in living small animals in real time. The researches in such aspects provide efficient technical support, and can observe the morphology characteristics of other materials in the near-infrared band.

Description

Fluorescence imaging system and its application

technical field

The invention relates to the field of optical technology, in particular to a fluorescence imaging system with an operating wavelength range in the near-infrared band and an application thereof.

Background technique

At present, bioluminescence and fluorescence imaging are mainly used for in vivo imaging of small living animals. Bioluminescence is to use luciferase gene to mark cells or DNA, and in vivo fluorescence imaging technology mainly has three marking methods: fluorescent protein labeling, fluorescent dye labeling and quantum dot labeling. In comparison, quantum dots, as a new type of nano-fluorescent probe, have many advantages such as wide excitation spectrum, narrow fluorescence emission spectrum, adjustable fluorescence spectrum, high quantum yield, high photochemical stability and not easy to decompose.

Due to the different tissue penetration of different wavelengths, the absorption of near-infrared wavelengths by hemoglobin, fat and water is kept at a relatively low level. Therefore, biological tissues have a "near-infrared window" in the near-infrared band, which has high penetration penetration depth. Therefore, for in vivo imaging, choosing a fluorescent labeling method with excitation and emission spectra in the near-infrared region will be beneficial to in vivo optical imaging, especially fluorescence imaging of deep tissues. Therefore, the near-infrared fluorescence quantum dot imaging system has great application prospects, especially for the near-infrared fluorescence imaging of deep tissues in small living animals. In addition to being labeled, it can quickly measure the growth of tumors in various cancer models, It can also observe and evaluate the changes of cancer cells in real time during cancer treatment, and can also quantitatively detect tumors in situ, metastases, and spontaneous tumors in small animals as a whole without trauma; it can be detected by in vivo bioluminescence imaging technology, and can be continuously observed Its infiltration process on the body and the influence of antiviral drugs and antibiotics on its pathological process; it can also be applied to research fields such as immune cells, stem cells, and apoptosis, such as observing markers on other research substances, such as drugs, specific It has unique application advantages in long-term life activity detection and living body tracking. However, there are very few living small animal fluorescence imaging systems that use near-infrared quantum dots at home and abroad.

Contents of the invention

In order to solve the above problems and fill the technical gap in this field, the present invention provides a fluorescence imaging system with a working wavelength range in the near-infrared band and its application.

According to one aspect of the present invention, a fluorescence imaging system is provided, which includes: a fluorescence excitation unit, the light beam generated by it is irradiated on the fluorescent substance, so that the fluorescent substance excites fluorescence; The fluorescence excited by the fluorescent substance is filtered to filter out the light in the non-fluorescence band; the imaging objective lens receives the fluorescence filtered by the filter group; the detector has an effective detection wavelength range of 800-1700nm, The imaging objective lens acquires the imaging of the fluorescent substance.

In addition, the detector is an InGaAs detector.

In addition, the wavelength of the light beam generated by the fluorescence excitation unit is 780-1100 nm.

Further, the wavelength of the light beam generated by the fluorescent excitation unit is 808nm, and the fluorescent substance is Ag ₂ S quantum dots.

In addition, the fluorescence excitation unit includes: a laser, a coupler, an optical fiber, and a beam expansion module. The laser beam emitted by the laser passes through the coupler and the optical fiber respectively to obtain a Gaussian beam, and then the Gaussian beam is transformed by the beam expansion module. The range of the beam spot is enlarged.

In addition, the working power of the laser is greater than 0W and not greater than 15W.

In addition, the beam expander module is a plano-concave lens that transmits more than 90% of near-infrared light.

In addition, the path of the light beam generated by the fluorescence excitation unit coincides with the path of the fluorescence excited by the fluorescent substance entering the detector.

In addition, the imaging system further includes a three-dimensional stage for carrying the fluorescent substance.

In addition, the imaging system further includes an illumination unit, which provides an illumination beam to irradiate the fluorescent substance.

In addition, the imaging system also includes an anesthesia system.

In addition, the imaging system also includes a computer processing system connected to the fluorescence excitation unit, imaging objective lens, and detector respectively, and the computer processing system is used to control and adjust the optical path of the light beam generated by the fluorescence excitation unit, and the imaging objective lens And collecting the imaging signal of the detector, and processing the imaging signal.

According to another aspect of the present invention, an application of the above-mentioned fluorescence imaging system in near-infrared quantum dot imaging is also provided.

Beneficial effect:

The fluorescence imaging system and its application of the present invention irradiate small animals with near-infrared laser beams to excite quantum dots in the small animals to emit fluorescence, and can observe deep tissues, organs and cells in small living animals in real time quickly, efficiently and non-destructively , provides efficient technical support for future research on tumor cells, stem cells, etc., and can observe the morphology characteristics of other materials in the near-infrared band.

Description of drawings

The above-mentioned and other objects, features and advantages of the present invention will become clearer through the following detailed description in conjunction with the accompanying drawings. In the accompanying drawings:

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

Detailed ways

The present invention utilizes the principle that near-infrared light excites quantum dots to emit fluorescence. This is because the hemoglobin widely distributed in the blood has a strong absorption of the optical signal with a wavelength below 600nm, and the optical signal with a wavelength above 1000nm is absorbed by water, so the optical signal with a wavelength below 600nm and above 1000nm is passing through There is obvious attenuation during the process of permeating living cells, which greatly reduces the sensitivity of imaging and is not suitable for living imaging. The 650-850nm wavelength band can well solve the problem of in vivo penetration. Combining the light beam in this wavelength band with Ag ₂ S quantum dots can excellently complete the research of fluorescence imaging.

Embodiments of the invention will now be described in detail, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like parts throughout. The embodiments are described below in order to explain the present invention by referring to the figures. In the drawings, the thicknesses of layers and regions may be exaggerated for clarity. In the following description, unnecessary detailed descriptions of well-known structures and/or functions may be omitted in order to avoid obscuring the inventive concept caused by unnecessary detailed descriptions of well-known structures and/or functions.

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

As shown in FIG. 1 , a fluorescence imaging system according to an embodiment of the present invention may include a detector 110, an imaging objective lens 120, a filter set 130, a three-dimensional stage 180, and illumination units respectively arranged on both sides of the detector 110. 140 and the fluorescence excitation unit 150 may also include an anesthesia system 160 connected to the three-dimensional stage 180, and a computer processing system 170 connected to the detector 110, the imaging objective lens 120 and the fluorescence excitation unit 150 respectively. Wherein, the fluorescence excitation unit 150, the illumination unit 140, and the detector 110 are as close as possible, so that the light beams generated by the fluorescence excitation unit 150 and the light beams generated by the illumination unit 140 are vertically irradiated on the stage 180, so that the irradiation center of each light beam is aligned with the detector. The detection center of 110 is as consistent as possible, so the path of the light beam generated by the fluorescence excitation unit 150 coincides with the path of the fluorescence excited by the fluorescent substance entering the detector 110 . The lighting unit 140 can be, for example, a broad-spectrum halogen lamp, and the white light emitted by it can just be seen by the near-infrared detector 110 as a signal.

Further, the components in the fluorescence excitation unit 150 of this embodiment can all work in the near-infrared band. The fluorescence excitation unit 150 may include a laser 151 , a coupler 152 , an optical fiber 153 and a beam expansion module 154 . In this embodiment, the excitation beam (that is, the laser beam) emitted by the laser 151 is a near-infrared short-wavelength beam with a wavelength of _808nm ; Between 1250nm. The optical fiber 153 is a single-mode optical fiber, and its coupling efficiency is above 50%. The beam expander module 154 is a plano-concave lens that can pass through more than 90% of near-infrared light, and it can make the range of the light spot of the excitation beam irradiated on the surface of the small animal body much larger than the body of the small animal (or the surface area of the measured object .)

The working range of the imaging objective lens 120 is 700-1900nm; the detector 110 is a near-infrared detector, which can be an InGaAs detector, and the effective wavelength range of the InGaAs detector is 800-1700nm.

The filter group 130 of this embodiment can be made up of two optical filters, and its main function is to filter out background light. 808nm near-infrared short-wavelength beam) is filtered out, so that only the fluorescence emitted by the quantum dots enters the detector 110 . However, the number of filters constituting the filter set 130 is not as many as possible. Too many filters will weaken the imaging effect of the fluorescence emitted by the quantum dots after reaching the detector 110. Weakly adjust the composition of the filter set appropriately. In other embodiments, if the signal of the quantum dots is strong, the number and wavelength bands of optical filters can be increased, which is beneficial to improve the imaging effect; otherwise, the number of optical filters should be reduced.

The following describes the working process of the fluorescence imaging system of this embodiment:

First, in this embodiment, mice are used as test samples. The mice need to be processed before the excitation beam is scanned. Specifically, the mouse hair on the back of the body of the mouse was removed with a depilatory agent, or nude mice were used to prevent it from affecting the collection of fluorescence data. Then, a fluorescent substance such as Ag ₂ S quantum dots was injected into the mouse body through the tail vein of the mouse. The range of fluorescence emission spectrum of quantum dots used in this embodiment is between 932nm and 1250nm. Next, the mice are first placed in the anesthesia system 160, and pre-anesthetized in a mixed gas pre-anesthesia box containing 5% isoflurane anesthesia and 95% oxygen. About 30 seconds later, the fully anesthetized mouse was transferred to the stage 180, and the anesthesia mask was worn on the mouse's face, and the fluorescent imaging system of this embodiment was started for live observation.

Start the fluorescence imaging system of this example. The excitation beam emitted by the laser 151 in the fluorescence excitation unit 150 passes through the coupler 152, and then is introduced into the single-mode fiber 153 to obtain a Gaussian beam with a wavelength of 808nm, and then passes through the beam expander module 154 to enlarge the range of the spot of the Gaussian beam obtained , and then illuminate the back of the mouse on the stage 180. At this time, the Ag ₂ S quantum dots pre-implanted in the mouse body will be excited to emit fluorescent photons, these fluorescent photons escape through the surface of the tissue, and filter the background light and other stray light through the filter set 130 off, and then reach the detector 110 through the imaging objective lens 120 to obtain the imaging of the Ag ₂ S quantum dots. Of course, some of the fluorescent light is emitted by the mouse itself, and these short-wave parts of near-infrared light can be captured and entered into the detector 110 . Then the computer processing system 170 controls and collects the luminescent signal of the quantum dot through the detector 110 . At the same time, the white light emitted by the lighting unit 140 is also directly irradiated to the mouse on the stage 180 , and the reflected light from the back of the mouse passes through the filter set 130 , then passes through the imaging objective lens 120 and reaches the detector 110 . The main function of the lighting unit 140 is to provide illumination to the mouse, and the detector 110 sends the obtained imaging signal of the _Ag2S quantum dot to the computer processing system 170, and the computer processing system 170 combines the computer software with the fluorescence signal of the quantum dot Together, they accurately marked the position of the quantum dots in the mice.

In addition, in order to reduce the thermal damage of the near-infrared laser to the mice, the power of the laser 151 and the switch of the laser are controlled by the computer processing system 170, and the laser 151 is only turned on when taking pictures. In addition, the imaging effect is guaranteed to be good. Keep the working power of the laser 151 as low as possible, and the working power of the laser in this embodiment is continuously adjustable from 0-15W.

In addition, the fluorescence imaging system according to the embodiment of the present invention can be applied in near-infrared quantum dot imaging. By irradiating small animals with near-infrared laser beams, the quantum dots in the small animals are excited and made to emit fluorescence, which can quickly It can observe the deep tissues, organs and cells in living small animals in real time, efficiently and without damage, which provides efficient technical support for future research on tumor cells, stem cells, etc., and can observe the shape of other materials in the near-infrared band. features.

Inspired by the above-mentioned ideal implementation according to the present invention, through the above-mentioned description content, relevant workers can completely make various changes and modifications within the scope of not departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the contents in the description, and must be determined according to the scope of the claims.

Claims (13)

1. A fluorescent imaging system, characterized in that, comprising: a fluorescence excitation unit, the light beam generated by it is irradiated on the fluorescent substance, so that the fluorescent substance excites fluorescence; A filter set, which filters the fluorescence excited by the fluorescent substance, so as to filter out the light in the non-fluorescence band; The imaging objective lens receives the fluorescence filtered by the filter set; The detector, whose detection effective wavelength range is 800-1700nm, obtains the imaging of the fluorescent substance through the imaging objective lens. 2. The fluorescence imaging system according to claim 1, wherein the detector is an InGaAs detector. 3. The fluorescence imaging system according to claim 1 or 2, characterized in that the wavelength of the light beam generated by the fluorescence excitation unit is 780-1100 nm. 4 . The fluorescence imaging system according to claim 3 , wherein the wavelength of the light beam generated by the fluorescence excitation unit is 808 nm, and the fluorescent substance is Ag ₂ S quantum dots. 5. The fluorescence imaging system according to claim 1 or 2, wherein the fluorescence excitation unit comprises: a laser, a coupler, an optical fiber and a beam expander module, and the laser beams emitted by the laser pass through the coupler respectively and an optical fiber to obtain a Gaussian beam, and then the spot range of the Gaussian beam is enlarged by the beam expander module. 6. The fluorescence imaging system according to claim 5, wherein the working power of the laser is greater than 0W and not greater than 15W. 7. The fluorescence imaging system according to claim 5, wherein the beam expander module is a plano-concave lens that transmits more than 90% of near-infrared light. 8. The fluorescence imaging system according to claim 1, wherein the path of the light beam generated by the fluorescence excitation unit coincides with the path of the fluorescence excited by the fluorescent substance entering the detector. 9. The fluorescence imaging system according to claim 1, further comprising a three-dimensional stage for carrying the fluorescent substance. 10 . The fluorescence imaging system according to claim 1 , further comprising an illumination unit, which provides an illumination beam to irradiate the fluorescent substance. 11 . 11. The fluorescence imaging system according to claim 1, further comprising an anesthesia system. 12. The fluorescence imaging system according to claim 1, characterized in that, the imaging system further comprises a computer processing system connected to the fluorescence excitation unit, the imaging objective lens, and the detector respectively, and the computer processing system is used to control and adjusting the optical path of the light beam generated by the fluorescence excitation unit, the imaging objective lens and collecting the imaging signal of the detector, and processing the imaging signal. 13. The application of the fluorescent imaging system of claim 1 in near-infrared quantum dot imaging.

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