CN109813687A - Up-conversion fluorescence imaging system and up-conversion fluorescence imaging method - Google Patents

Abstract

The invention discloses a kind of up-conversion fluorescence imaging systems, the wave-length coverage of the incident light of the light source transmitting of the system is 1000nm to 2500nm, the wave-length coverage is in the area near-infrared II and the area near-infrared III, and the penetration depth of the infrared light of the wave band in the sample is deep；By the incident light excitation in the area near-infrared II or the area near-infrared III, the upper conversion fluorescent nano particle being marked in the sample in advance can issue the emergent light near infrared region, and the penetration depth of the emergent light in the sample is equally relatively deep；And since the wavelength of the incident light of light source sending includes the area near-infrared II and the area near-infrared III, which is very unlikely to cause the autofluorescence phenomenon of sample, so as to effectively increase fluorescence imaging depth and sensitivity；The invention also discloses a kind of imaging methods of up-conversion fluorescence, equally have above-mentioned beneficial effect.

Description

Up-conversion fluorescence imaging system and up-conversion fluorescence imaging method

technical field

The invention relates to the field of fluorescence imaging, in particular to an up-conversion fluorescence imaging system and an up-conversion fluorescence imaging method.

Background technique

With the continuous progress of science and technology in recent years, fluorescence imaging technology is more and more widely used in people's daily life.

Fluorescence imaging technology has been widely used in the field of biomedicine due to its advantages of non-ionizing low-energy radiation, non-invasiveness, high sensitivity, and high sensitivity. At the same time, fluorescence can visualize and image biomolecules, cells, tissues, organs, etc., and is usually used for early diagnosis of tumors, detection of biomarkers in vitro, metabolic pathways and distribution of drugs in vivo, etc.

Among a variety of fluorescent materials, the luminescence mechanism of Upconversion Nanoparticls (UCNPs) is to emit a high-energy photon by absorbing two or more low-energy photons, that is, up-conversion fluorescent nanoparticle. Particles can emit light with shorter wavelengths by absorbing light with longer wavelengths. Compared with other fluorescent materials, upconversion fluorescent nanoparticles have the characteristics of large Stokes shift, high imaging signal-to-noise ratio, narrow emission spectrum, and long fluorescence lifetime.

In the prior art, 808 nm or 980 nm laser excitation is usually used, and fluorescence signals at 540 nm, 660 nm or 800 nm are collected to realize up-conversion fluorescence imaging. However, in the prior art, the fluorescence imaging depth of upconversion fluorescence imaging using 808 nm or 980 nm laser excitation is usually low.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an up-conversion fluorescence imaging system, which can effectively increase the depth of fluorescence imaging; another purpose of the present invention is to provide an up-conversion fluorescence imaging method, which can effectively increase the depth of fluorescence imaging.

In order to solve the above technical problems, the present invention provides an up-conversion fluorescence imaging system, the up-conversion fluorescence imaging system includes a light source, a sample stage, an optical signal receiver and a processor;

The wavelength range of the incident light emitted by the light source is: 1000nm to 2500nm, inclusive;

The sample stage is used for placing a sample marked with up-conversion fluorescent nanoparticles, and the sample is used for absorbing the incident light and emitting outgoing light;

The optical signal receiver is connected to the processor, and the optical signal receiver is configured to receive the outgoing light and convert the outgoing light into image data to be sent to the processor;

The processor is configured to generate a fluorescence image based on the image data.

Optionally, the up-conversion fluorescent nanoparticles are AReF ₄ (Re ³⁺ , n%)@mAReF ₄ or Re ₂ O ₃ (Re ³⁺ , n%); wherein, A includes Li, Na, K; Re Including Sc, Y, La, Gd, Lu; Re ³⁺ includes Pr ³⁺ , Nd ³⁺ , Sm ³⁺ , Dy ³⁺ , Ho ³⁺ , Er ³⁺ , Tm ³⁺ ; the value range of n is: 0 to 100, including the right endpoint; the value range of m is: 0 to 30, including the endpoint.

Optionally, the light source is a laser light source, and a laser beam adjusting lens group is arranged between the laser light source and the sample stage.

Optionally, the laser beam adjustment lens group includes a collimator lens, a beam expander lens and a shaping lens sequentially distributed along the optical axis.

Optionally, a band-pass filter is arranged between the sample stage and the optical signal receiver, and the band-pass filter is used to select monochromatic outgoing light with a preset wavelength.

Optionally, a fluorescent beam adjusting lens group is arranged between the bandpass filter and the optical signal receiver.

Optionally, the fluorescent beam adjustment lens group includes a collimating lens, a beam expander lens and a shaping lens sequentially distributed along the optical axis.

Optionally, the optical signal receiver is a CCD detector.

The present invention also provides an imaging method for up-conversion fluorescence, the method comprising:

The light source emits incident light to illuminate the sample set on the sample stage, wherein the wavelength range of the incident light is: 1000nm to 2500nm, inclusive, and the sample is a sample labeled with up-converted fluorescent nanoparticles;

the sample absorbs the incident light and emits outgoing light;

an optical signal receiver receives the outgoing light and converts the outgoing light into image data for sending to the processor;

The processor generates a fluorescence image from the image data.

Optionally, the up-conversion fluorescent nanoparticles are AReF ₄ (Re ³⁺ , n%)@mAReF ₄ or Re ₂ O ₃ (Re ³⁺ , n%); wherein, A includes Li, Na, K; Re Including Sc, Y, La, Gd, Lu; Re ³⁺ includes Pr ³⁺ , Nd ³⁺ , Sm ³⁺ , Dy ³⁺ , Ho ³⁺ , Er ³⁺ , Tm ³⁺ ; the value range of n is: 0 to 100, including the right endpoint; the value range of m is: 0 to 30, including the endpoint.

In the up-conversion fluorescence imaging system provided by the present invention, the wavelength range of the incident light emitted by the light source of the system is 1000nm to 2500nm, the wavelength range is in the near-infrared II region and the near-infrared III region, and the infrared light in this band is in the sample The penetration depth in the sample is relatively deep; by excitation by incident light in the near-infrared II region or near-infrared III region, the upconverting fluorescent nanoparticles pre-labeled in the sample can emit outgoing light in the near-infrared region, and the outgoing light is in the near-infrared region. The penetration depth in the sample is also deep; and since the wavelength of the incident light emitted by the light source includes the near-infrared II region and the near-infrared III region, the incident light pole is not easy to cause the autofluorescence phenomenon of the sample, which can effectively increase the fluorescence imaging. depth and sensitivity.

The present invention also provides an imaging method for up-conversion fluorescence, which also has the above beneficial effects, and will not be repeated here.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the following will briefly introduce the accompanying drawings used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only For some embodiments of the present invention, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

1 is a schematic structural diagram of an up-conversion fluorescence imaging system according to an embodiment of the present invention;

2 is a schematic structural diagram of another up-conversion fluorescence imaging system provided by an embodiment of the present invention;

FIG. 3 is a flowchart of an imaging method for up-conversion fluorescence provided by an embodiment of the present invention.

In the figure: 10. Light source, 20. Laser beam adjustment lens group, 21. Collimating lens, 22. Beam expander lens, 23. Shaping lens, 30. Sample stage, 31. Sample, 32. Living sample, 40. Bandpass Filter, 50. Fluorescence beam adjustment lens group, 60. Fluorescence signal acquisition objective lens, 70. Optical signal receiver, 80. Processor, 90. Reflector, 91. Obscura, 92. Automatic lifting control part.

Detailed ways

The core of the present invention is to provide an up-conversion fluorescence imaging system. In the prior art, 808 nm or 980 nm laser excitation is usually used, and fluorescence signals at 540 nm, 660 nm or 800 nm are collected to realize up-conversion fluorescence imaging. Since the wavelength range of the incident light emitted by the light source is in the near-infrared I region in the prior art, its wavelength is relatively short, and the penetration depth in the sample is relatively shallow; The outgoing light emitted by the fluorescent nanoparticles is usually in the visible light range, the wavelength of the outgoing light is also relatively short, and the penetration depth in the sample is relatively shallow; at the same time, since the wavelength of the incident light emitted by the light source in the prior art is in the near-infrared I region , which may cause autofluorescence of the sample, resulting in a shallow depth of fluorescence imaging.

In an up-conversion fluorescence imaging system provided by the present invention, the wavelength range of the incident light emitted by the light source of the system is 1000 nm to 2500 nm, the wavelength range is in the near-infrared II region and the near-infrared III region, and the infrared light in this band is in the The penetration depth in the sample is relatively deep; through excitation by incident light in the near-infrared II region or near-infrared III region, the up-converted fluorescent nanoparticles pre-labeled in the sample can emit outgoing light in the near-infrared region, and the outgoing light is in the near-infrared region. The penetration depth in the sample is also deep; and since the wavelength of the incident light emitted by the light source includes the near-infrared II region and the near-infrared III region, the incident light pole is not easy to cause the autofluorescence phenomenon of the sample, so that the fluorescence can be effectively increased. Imaging depth and sensitivity.

In order to make those skilled in the art better understand the solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Please refer to FIG. 1 , which is a schematic structural diagram of an up-conversion fluorescence imaging system according to an embodiment of the present invention.

Referring to FIG. 1 , in an embodiment of the present invention, the up-conversion fluorescence imaging system includes a light source 10 , a sample stage 30 , an optical signal receiver 70 and a processor 80 .

The wavelength range of the incident light emitted by the light source 10 is: 1000 nm to 2500 nm, inclusive, that is, the wavelength of the incident light emitted by the light source 10 includes 1000 nm to 2500 nm, including both 1000 nm and 2500 nm. The above-mentioned sample stage 30 is used for placing the sample 31 marked with the up-converted fluorescent nanoparticles, and the sample 31 is used for absorbing the incident light and emitting the outgoing light.

In the embodiment of the present invention, up-conversion fluorescent nanoparticles are added to the sample 31 placed on the sample stage 30, and the up-conversion fluorescent nanoparticles are AReF ₄ (Re ³⁺ , n%)@mAReF ₄ or Re ₂ O ₃ (Re ³⁺ , n%); wherein, A includes Li, Na, K; Re includes Sc, Y, La, Gd, Lu; Re ³⁺ includes Pr ³⁺ , Nd ³⁺ , Sm ³⁺ , Dy ^{3 +} , Ho ³⁺ , Er ³⁺ , Tm ³⁺ ; the value range of n is: 0 to 100, including the right endpoint value; the value range of m is: 0 to 30, including the endpoint value. In the above-mentioned AReF ₄ (Re ³⁺ , n%)@mAReF ₄ , @ represents a core-shell structure, that is, the up-conversion fluorescent nanoparticles have a coating structure. The above m represents the number of shell layers of AReF _4. If the value of m is 0, there is only one layer of AReF ₄ from the inside to the outside for each up-conversion fluorescent nanoparticle; if the value of m is 5, then for each up-conversion fluorescent nanoparticle The particles are coated with a total of 5 layers of AReF ₄ from the inside to the outside, that is, a total of 6 layers of AReF ₄ from the inside to the outside. In the embodiment of the present invention, the number of shell layers of the AReF ₄ (Re ³⁺ , n%)@mAReF ₄ may be any number from 0 to 30, including 0 and 30.

In the above AReF ₄ (Re ³⁺ , n%)@mAReF ₄ , A represents an alkali metal, including three elements of Li, Na and K; the above AReF ₄ (Re ³⁺ , n%)@mAReF ₄ and Re ₂ O ₃ In (Re ³⁺ , n%), Re is five rare earth elements suitable for the upconversion fluorescent nanoparticle matrix, which are Sc, Y, La, Gd, and Lu, respectively. Other rare earth elements are not suitable for the matrix of upconverting fluorescent nanoparticles. The above-mentioned Re ³⁺ is the rare earth ion added in the matrix of the up-conversion fluorescent nanoparticle, and the rare earth ion added in the embodiment of the present invention usually exists in the matrix in the form of +3 valence. The above Re ³⁺ includes Pr ³⁺ , Nd ³⁺ , Sm ³⁺ , Dy ³⁺ , Ho ³⁺ , Er ³⁺ , Tm ³⁺ ; the above AReF ₄ and Re ₂ O ³ are _two kinds of up-conversion fluorescent nanoparticles, respectively substrate. For doping different kinds of rare earth ions, the upconversion fluorescent nanoparticles will have different absorption peaks and correspondingly different emission peaks; at the same time, the matrix of the upconversion fluorescent nanoparticles will also affect their absorption peaks and emission peaks. For example, if the doped Re ³⁺ in AReF ₄ (Re ³⁺ , n%)@mAReF ₄ is Tm ³⁺ , the absorption peak of the entire upconverting fluorescent nanoparticle is 1630 nm, and the corresponding emission of the upconverting fluorescent nanoparticle The peaks include 465nm, 684nm, 785nm, 1190nm and 1800nm; if the Re ^{3+ doped} in Re ₂ O ₃ (Re ³⁺ , n%) is Tm ³⁺ , the absorption peaks of the entire upconversion fluorescent nanoparticles include 800 nm and 1200 nm , the corresponding upconverting fluorescent nanoparticles have an emission peak at 1800 nm. Because in the embodiment of the present invention, the up-conversion fluorescent nanoparticles can be doped with multiple kinds of Re ³⁺ , but usually only one kind of Re ³⁺ is ^doped for each kind of up-conversion fluorescent nanoparticles. Of course, a variety of upconversion fluorescent nanoparticles doped with different Re ³⁺ can be used simultaneously in the use process, and upconversion fluorescent nanoparticles of a variety of substrates can also be used simultaneously. The absorption peaks corresponding to the above various Re ³⁺ are distributed in the near-infrared II region and the near-infrared III region from 1000 nm to 2500 nm. Of course, for a certain kind of Re ³⁺ added, there are usually multiple emission peaks. For example, if the doped Re ³⁺ in AReF ₄ (Re ³⁺ , n%)@mAReF ₄ is Dy ³⁺ , the absorption peak of the whole upconverting fluorescent nanoparticle is 1680 nm, and the corresponding emission of the upconverting fluorescent nanoparticle Peaks include 483nm, 582nm, 910nm, 1080nm and 1290nm, and the above emission peaks include a wide range from visible light to near-infrared light, but due to the characteristics of up-converting fluorescent nanoparticles, for a certain Re ³⁺ , the wavelengths of the absorption peaks are all larger than the wavelengths of the emission peaks. Since the ^doped Re has multiple emission peaks, multicolor fluorescence imaging can be achieved, which will be described in detail in the subsequent paragraphs.

Of course, for any kind of absorption peak and emission peak of Re ³⁺ , there are corresponding peak widths and half-peak widths, so the specific numerical values used in the above examples are only those corresponding to the peaks of absorption peaks or emission peaks. The wavelength, the wavelength of the incident light that the upconverting fluorescent nanoparticles can absorb and the wavelength of the outgoing light that the upconverting fluorescent nanoparticle can absorb is a range in practical situations. In the embodiment of the present invention, the doped rare earth ions are not limited to the above seven rare earth ions, and the valence state of the doped rare earth ions is not limited to +3, as long as the absorption peak of the doped rare earth ions is It can be between 1000 nm and 2500 nm. In the embodiment of the present invention, the valence state and ion species of the doped rare earth ions are not specifically limited.

In the above expressions, n represents the concentration of doped rare earth ions Re ³⁺ . In the embodiment of the present invention, the doping concentration of rare earth ions may range from 0 to 100, including the right end value, that is, the doping concentration of rare earth ions includes 100%. For upconversion fluorescent nanoparticles doped with different rare earth ions, the doping concentration of rare earth ions only affects the intensity of the emission light emitted by the upconversion fluorescent nanoparticles, and does not affect the position of the absorption peak.

In the embodiment of the present invention, since the absorption peak of the up-conversion fluorescent nanoparticles marked in the sample 31 ranges from 1000 nm to 2500 nm, the corresponding wavelength range of the incident light emitted by the above-mentioned light source 10 is: 1000 nm to 2500 nm, inclusive value. Usually, since the laser has the characteristics of good monochromaticity, directivity, and high brightness, the light source 10 used in the example of the present invention is usually a laser light source. Due to the strong monochromaticity of the incident light emitted by the laser light source and the narrow wave peak, the wavelength of the incident light emitted by the laser light source needs to correspond to the absorption peak of the above-mentioned up-conversion fluorescent nanoparticles in the specific use process. The absorption peak of nanoparticles is located at 2000 nm, so a corresponding laser light source that can emit incident light with a wavelength of 2000 nm is required. At present, depending on the type of rare earth ions doped in the up-conversion fluorescent nanoparticles, a semiconductor laser with a wavelength of 1050 nm, 1150 nm, 1250 nm, 1500 nm, 1600 nm, 1700 nm or 1950 nm can be selected as the light source 10 for emitting incident light. Of course, in an example of the present invention, the wavelength of the incident light emitted by the light source 10 is not specifically limited, as long as it is between 1000 nm and 2500 nm.

Since the irradiation area of the incident light emitted by the laser light source is usually small, a laser beam adjusting lens group 20 is usually provided to adjust the shape of the incident light, so that the incident light can be uniformly irradiated on the sample 31 . Generally, the incident light emitted by the laser light source is usually transmitted to the laser beam adjustment lens group 20 through an optical fiber. The laser beam adjustment lens group 20 includes a collimating lens 21, a beam expander lens 22 and a shaping lens 23. These three The lenses are sequentially distributed along the optical axis. That is, the incident light emitted by the light source 10 first passes through the collimating lens 21 to collimate the incident light, then passes through the beam expander lens 22 to expand the incident light, and finally passes through the shaping lens 23 to adjust the shape of the incident light. Of course, the structure of the laser beam adjusting lens group 20 is not limited to the above three lenses, and can also include other lenses, and it is not limited to use the above three lenses, and the distribution of the above three lenses along the optical axis is also Other distribution methods are possible. The specific structure of the laser beam adjusting lens group 20 is not specifically limited in the embodiments of the present invention.

In the embodiment of the present invention, the light source 10 emits incident light to illuminate the sample 31 disposed on the sample stage 30, and the sample 31 is the sample 31 marked with up-converted fluorescent nanoparticles; the sample 31 can absorb the incident light, and emits outgoing light, which is fluorescence. Specifically, the up-conversion fluorescent nanoparticles added in the sample 31 absorb the incident light and emit fluorescence. In the embodiment of the present invention, due to the limitation of the overall structure, a reflector 90 is usually provided to emit the incident light emitted by the light source 10 to the sample 31 . Meanwhile, the specific structure of the sample stage 30 is not specifically limited. In addition to placing the sample 31, the sample stage 30 can also have an automatic lifting function to adjust the height of the sample stage 30; it can also have a constant temperature function to ensure the activity of the biological sample when the sample 31 is a biological sample. . Of course, the sample stage 30 may also have other functions, which will not be listed one by one here.

In this embodiment of the present invention, the up-conversion fluorescence imaging system further includes an optical signal receiver 70 and a processor 80 , and the optical signal receiver 70 is connected to the processor 80 . The optical signal receiver 70 is configured to receive the outgoing light and convert the outgoing light into image data to be sent to the processor 80 .

After the sample 31 emits outgoing light, the optical signal receiver 70 will receive the outgoing light and convert the outgoing light into image data to be sent to the processor 80 . That is, the function of the optical signal receiver 70 is to convert the received optical signal into an electrical signal, so as to facilitate the processor 80 to process the fluorescent signal emitted by the sample 31 . Because in the embodiment of the present invention, the outgoing light emitted by the sample 31 to which the up-conversion fluorescent nanoparticles is added usually includes monochromatic outgoing light with multiple wavelengths, but due to the different wavelengths of the monochromatic outgoing light, the corresponding outgoing light in the sample 31 Medium penetration depths also vary. Normally, the above-mentioned optical signal receiver 70 only receives one type of monochromatic outgoing light at a time, and converts the received monochromatic outgoing light into corresponding image data and sends it to the processor 80 . Correspondingly, a band-pass filter 40 needs to be arranged between the sample stage 30 and the optical signal receiver 70 , and the band-pass filter 40 is used to select monochromatic outgoing light of a preset wavelength.

The bandpass filter 40 is a kind of filter, which can separate monochromatic light of a certain wavelength band. The wavelength range of the monochromatic outgoing light allowed to pass through the bandpass filter 40 needs to correspond to the peak position and half-peak width of the emission peak of the above-mentioned up-conversion fluorescent nanoparticles. In the embodiment of the present invention, a variety of bandpass filters 40 may be provided to sequentially select monochromatic emission light of different wavelengths, and the corresponding optical signal receiver 70 may also sequentially receive monochromatic emission light of different wavelengths. , and corresponding image data can be generated for each monochromatic emission light, and finally the plurality of image data are sent to the processor 80 for processing. Since the penetration depths of monochromatic outgoing light of different wavelengths in the sample 31 are different, a plurality of bandpass filters 40 are used to allow the optical signal receiver 70 to sequentially acquire monochromatic outgoing light of different wavelengths, which is convenient for the later description. Processor 80 generates polychromatic fluorescence images with different imaging depths.

Of course, the optical signal receiver 70 can simultaneously receive a variety of monochromatic outgoing lights and convert them into image data, but this is not conducive to the post-processor 80 to process the image data, and will also affect the final generated fluorescence image. Effect.

Usually, a fluorescent beam adjusting lens group 50 is arranged between the bandpass filter 40 and the optical signal receiver 70 to adjust the shape of the outgoing light, so that the optical signal receiver 70 can finally obtain a clear image, so as to realize the Collection of outgoing light. Similar to the laser beam adjusting lens group 20 , the fluorescent beam adjusting lens group 50 also generally includes a collimating lens, a beam expanding lens and a shaping lens which are sequentially distributed along the optical axis. For details, please refer to the above-mentioned specific introduction about the laser beam adjusting lens group 20 , which will not be repeated here. The specific structure of the fluorescent beam adjusting lens group 50 is also not specifically limited in the embodiment of the present invention, as long as the shape of the outgoing light can be adjusted.

Between the above-mentioned fluorescent light beam lens group and the optical signal receiver 70, a fluorescent signal collecting objective lens 60 is usually also disposed for collecting the above-mentioned outgoing light. In the embodiment of the present invention, the working wavelength range of the fluorescence signal collecting objective lens 60 is generally between 200 nm and 2500 nm. According to the needs of practical applications, a plurality of fluorescence signal acquisition objective lenses 60 with different magnifications and numerical apertures may be specifically set. The specific magnification and numerical aperture values of the fluorescence signal acquisition objective lenses 60 are not specifically limited in the embodiments of the present invention.

In the embodiment of the present invention, the optical signal receiver 70 is generally a CCD (Charge-coupled Device) detector. Since the emission peaks of the above-mentioned up-conversion fluorescent nanoparticles are usually distributed in the ultraviolet, visible and near-infrared wavelength bands, the CCD detector usually needs to include at least two fluorescence detectors, one for detecting The other is a focal plane array detector used to detect fluorescence signals in the near-infrared band. Through the above two focal plane array detectors, it can be realized that the CCD detector can receive the fluorescence signal from the ultraviolet light band, to the visible light band, and then to the infrared light band, and convert the fluorescence signal into image data. The fluorescent signal corresponds to the above-mentioned outgoing light in the embodiment of the present invention.

In this embodiment of the present invention, the optical signal receiver 70 may receive the above-mentioned outgoing light, and convert the outgoing light into image data to send to the processor 80; the processor 80 generates a fluorescence image according to the image data.

The above-mentioned processor 80 is mainly used for calculating, analyzing, processing and reconstructing the received image data, so as to finally generate a fluorescence image according to the image data. In the embodiment of the present invention, the above-mentioned sample stage 30 may be further provided with a stepper motor, the processor 80 may be connected to the stepper motor, and the processor 80 may automatically control the operation of the stepper motor to realize the operation of the sample stage 30 Lift up and down to achieve high-precision imaging. At the same time, the processor 80 can also generally implement focusing, switching of the band-pass filter 40, selection of a sample imaging position, and the like in the above-mentioned imaging process. The specific functions that can be implemented by the processor 80 are not specifically limited in this embodiment of the present invention, as long as a fluorescence image can be generated according to the image data.

In the embodiment of the present invention, a visible light source may be further added to the up-conversion fluorescence imaging system to realize bright field imaging. That is, the sample 31 is irradiated by emitting visible light from a visible light source, so as to observe the appearance information such as the color, shape and the like of the sample 31 . Typically, sodium lamps are used as visible light sources. Of course, other devices may also be selected as the visible light source, which is not specifically limited in the embodiment of the present invention.

In the up-conversion fluorescence imaging system provided by the present invention, the wavelength range of the incident light emitted by the light source 10 of the system is 1000 nm to 2500 nm, the wavelength range is in the near-infrared II region and the near-infrared III region, and the infrared light in this band is in the The penetration depth in the sample 31 is relatively deep; through excitation by the incident light in the near-infrared II region or the near-infrared III region, the up-conversion fluorescent nanoparticles marked in advance in the sample 31 can emit outgoing light in the near-infrared region, which is The penetration depth of the outgoing light in the sample 31 is also deep; and since the wavelength of the incident light emitted by the light source 10 includes the near-infrared II region and the near-infrared III region, the incident optode is not easy to cause the autofluorescence phenomenon of the sample 31, thereby It can effectively increase the depth of fluorescence imaging.

In the present invention, when the sample 31 is a living body sample, the living body sample usually needs to be put into a dark box, and the incident light from the light source 10 needs to illuminate the living body sample more uniformly. For details, please refer to the following invention embodiments.

Please refer to FIG. 2 , which is a schematic structural diagram of another up-conversion fluorescence imaging system according to an embodiment of the present invention.

Referring to FIG. 2 , the up-conversion fluorescence imaging system provided by the embodiment of the present invention is substantially the same as the up-conversion fluorescence imaging system provided by the above-mentioned embodiment of the present invention, and the difference lies in most of the components of the entire up-conversion fluorescence imaging system in the embodiment of the present invention Both are in a dark box 91 , and the light source 10 emits multiple incident lights to illuminate the living body sample 32 .

In the embodiment of the present invention, the dark box 91 is provided with a plurality of laser beam adjustment lens groups 20 , and the plurality of the laser beam adjustment lens groups 20 are generally uniformly distributed in the inner wall of the dark box 91 . The incident light emitted by the light source 10 will be transmitted to the plurality of laser beam adjusting lens groups 20 through a plurality of optical paths respectively. Usually, the incident light is transmitted to the plurality of the laser beam adjusting lens groups 20 through multiple optical fibers. After adjusting the shape of the incident light, the laser beam adjusting lens group 20 can directly irradiate the living sample 32 or irradiate the living sample 32 through the mirror 90 . In the embodiment of the present invention, the area where the living sample 32 is located is the imaging area. In the embodiment of the present invention, the incident light is to be uniformly irradiated in the imaging area. Usually, it is necessary to ensure that the light from different points in the imaging area is irradiated uniformly. Strong deviation is less than 5.0%.

The sample stage 30 on which the living sample 32 is placed usually needs to have a constant temperature function, that is, the sample stage 30 is provided with heating and constant temperature components to ensure that the vital signs of the living sample 32 are normal during the imaging process. The lower surface of the sample stage 30 is usually connected with an automatic lift control part 92, the processor 80 is usually connected with the automatic lift control part 92, the processor 80 can adjust the height of the sample stage 30 through the automatic lift control part 92, This is used to achieve focusing and to ensure the clarity of the final fluorescent image.

In the embodiment of the present invention, the up-conversion fluorescent nanoparticles labeled in the living sample 32 are usually AReF ₄ (Tm ³⁺ , n%)@mAReF ₄ , that is, the rare earth ions doped in the up-conversion fluorescent nanoparticles are Tm ³⁺ . Since the absorption peak of AReF ₄ (Tm ³⁺ , n%)@mAReF ₄ is located at 1630 nm, the emission peaks include 465 nm, 684 nm, 785 nm, 1190 nm and 1800 nm, and the bands of absorption and emission peaks include near-infrared II region and near-infrared III region , the light waves of these two wavelength bands have good penetration depths in the living sample 32, and the corresponding fluorescence imaging using AReF ₄ (Tm ³⁺ , n%)@mAReF ₄ can greatly increase the final fluorescence image. Imaging depth. Of course, the up-conversion fluorescent nanoparticles can also be doped with other kinds of rare earth ions, and the absorption peak and the emission peak can also be in the near-infrared region at the same time. The types of rare earth ions doped in the up-conversion fluorescent nanoparticles are not specifically limited in the embodiments of the present invention.

If AReF ₄ (Tm ³⁺ , n%)@mAReF ₄ is used as the up-conversion fluorescent nanoparticles, the corresponding light source 10 is preferably a laser light source that can emit a wavelength of 1630 nm, and the bandpass filter 40 used subsequently is preferably The wavelength of light that is allowed to pass is 1190 nm.

After the living sample 32 emits the outgoing light, the up-conversion fluorescence imaging system provided by the embodiment of the present invention usually selects the pre-set monochromatic outgoing light through the corresponding bandpass filter 40, and passes the fluorescent light beam. The adjustment lens group 50 adjusts the shape of the monochromatic outgoing light, and finally transmits the monochromatic outgoing light to the optical signal receiver 70 through the fluorescence signal collecting objective lens 60 . After the light signal receiver 70 converts the monochromatic outgoing light into image data, the image data is sent to the processor 80, and finally the processor 80 generates a fluorescence image according to the image data.

The remaining contents of the up-conversion fluorescence imaging system described in the embodiments of the present invention have been described in detail in the above-mentioned embodiments of the present invention.

The up-conversion fluorescence imaging system provided by the present invention can uniformly irradiate the incident light emitted by the light source 10 on the living sample 32 through a plurality of laser beam adjustment lens groups 20 to improve the quality of the final generated fluorescence image. And the sample stage 30 has heating and constant temperature components, which can ensure that the vital signs of the living sample 32 are normal during the imaging process.

The up-conversion fluorescence imaging system provided by the present invention can be further applied to a fluorescence surgical navigation system. For details, please refer to the following embodiments of the invention.

The up-conversion fluorescence imaging system provided by the embodiment of the present invention is substantially the same as the up-conversion fluorescence imaging system provided by the above-mentioned embodiment of the present invention, and the difference is: in the embodiment of the present invention, the incident light emitted by the light source 10 passes through the laser beam. After the lens group 20 is adjusted, incident light with an irradiation area of not less than 200 cm ² can be formed to irradiate the lesion area of the patient.

Before surgery, the patient will inject AReF ₄ (Er ³⁺ , n%)@mAReF ₄ with the target molecule modified on the surface, that is, the rare earth ions doped in the up-conversion fluorescent nanoparticles are Er ³⁺ , and the up-conversion fluorescent nanoparticles The surface of the particles is modified with target molecules, and the up-conversion fluorescent nanoparticles can selectively and massively aggregate in the pathological tissue of the patient. Since the absorption peak of AReF ₄ (Er ³⁺ , n%)@mAReF ₄ is located at 1500 nm, and the emission peaks are located at 540 nm, 660 nm, 800 nm, 980 nm and 1730 nm, respectively, since AReF ₄ (Er ³⁺ , n%)@mAReF ₄ has more Therefore, multi-color fluorescence imaging can be achieved. Correspondingly, multi-color fluorescence imaging using AReF ₄ (Er ^{3 +} , n%)@mAReF ₄ can obtain the information of the entire pathological tissue hierarchically. Of course, the up-conversion fluorescent nanoparticles can also be doped with other kinds of rare earth ions, and can also have multiple absorption peaks at the same time to realize multicolor fluorescence imaging. The types of rare earth ions doped in the up-conversion fluorescent nanoparticles are not specifically limited in the embodiments of the present invention.

If AReF ₄ (Er ³⁺ , n%)@mAReF ₄ is used as the up-converting fluorescent nanoparticles, the corresponding light source 10 is preferably a laser light source that can emit a wavelength of 1500 nm, and a plurality of bandpass filters 40 for subsequent use Preferably, the wavelengths of light waves allowed to pass are 540 nm, 660 nm, 800 nm, 980 nm and 1730 nm, respectively.

After the optical signal receiver 70 acquires a plurality of monochromatic outgoing lights through a plurality of bandpass filters 40 , a plurality of image data may be generated, and the image data may be sent to the processor 80 . In the embodiment of the present invention, a three-dimensional reconstruction algorithm is stored in the processor 80, and the three-dimensional boundary and volume of the pathological tissue can be reconstructed based on the monochromatic outgoing light of different wavelengths acquired by the optical signal receiver 70, so as to provide accurate surgical procedures. information, guide the precise and controllable implementation of surgical operations, and significantly improve the quality of surgical treatment.

The remaining contents of the up-conversion fluorescence imaging system described in the embodiments of the present invention have been described in detail in the above two embodiments of the present invention.

The up-conversion fluorescence imaging system provided by the invention can reconstruct the three-dimensional boundary and volume of the pathological tissue of a patient on the basis of generating a multi-color fluorescence image, so as to provide accurate information for surgical operations and guide precise and controllable surgical operations. Implementation, significantly improve the quality of surgical treatment.

The following describes an upconversion fluorescence imaging method provided by an embodiment of the present invention, and the imaging method described below and the upconversion fluorescence imaging system described above can be referred to each other correspondingly.

FIG. 3 is a flowchart of an imaging method for up-conversion fluorescence provided by an embodiment of the present invention. Referring to FIG. 3 , the imaging method may include:

S101: The light source emits incident light to illuminate the sample set on the sample stage.

In an embodiment of the present invention, the wavelength range of the incident light is: 1000 nm to 2500 nm, inclusive, and the sample is a sample labeled with up-converted fluorescent nanoparticles.

S102: The sample absorbs incident light and emits outgoing light.

S103: The optical signal receiver receives the outgoing light, converts the outgoing light into image data, and sends it to the processor.

S104: The processor generates a fluorescence image according to the image data.

In the embodiment of the present invention, the up-conversion fluorescent nanoparticles are specifically AReF ₄ (Re ³⁺ , n%)@mAReF ₄ or Re ₂ O ₃ (Re ³⁺ , n%); wherein, A includes Li, Na , K; Re includes Sc, Y, La, Gd, Lu; Re ³⁺ includes Pr ³⁺ , Nd ³⁺ , Sm ³⁺ , Dy ³⁺ , Ho ³⁺ , Er ³⁺ , Tm ³⁺ ; the n The value range of m is: 0 to 100, including the right endpoint value; the value range of m is: 0 to 30, including the endpoint value.

The up-conversion fluorescence imaging method of this embodiment is used to use the aforementioned up-conversion fluorescence imaging system. Therefore, the specific implementation of the up-conversion fluorescence imaging method can be found in the previous section of the embodiment of the up-conversion fluorescence imaging system. Therefore, its For the specific implementation manner, reference may be made to the descriptions of the corresponding partial embodiments, which will not be repeated here.

The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same or similar parts between the various embodiments may be referred to each other. As for the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant part can be referred to the description of the method.

Professionals may further realize that the units and algorithm steps of each example described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, computer software, or a combination of the two, in order to clearly illustrate the possibilities of hardware and software. Interchangeability, the above description has generally described the components and steps of each example in terms of functionality. Whether these functions are performed in hardware or software depends on the specific application and design constraints of the technical solution. Skilled artisans may implement the described functionality using different methods for each particular application, but such implementations should not be considered beyond the scope of the present invention.

The steps of a method or algorithm described in conjunction with the embodiments disclosed herein may be directly implemented in hardware, a software module executed by a processor, or a combination of the two. A software module can be placed in random access memory (RAM), internal memory, read only memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, registers, hard disk, removable disk, CD-ROM, or any other in the technical field. in any other known form of storage medium.

The up-conversion fluorescence imaging system and the up-conversion fluorescence imaging method provided by the present invention have been described in detail above. The principles and implementations of the present invention are described herein by using specific examples, and the descriptions of the above embodiments are only used to help understand the method and the core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (10)

1. a kind of up-conversion fluorescence imaging system, which is characterized in that the up-conversion fluorescence imaging system includes light source, sample Platform, optical signal receiver and processor；

The wave-length coverage of the incident light of the light source transmitting are as follows: 1000nm to 2500nm, including endpoint value；

The sample stage is used to be placed through the sample of upper conversion fluorescent nano particle label, the sample for absorb it is described enter It penetrates light and launches and penetrate light；

The optical signal receiver connects the processor, and the optical signal receiver is for receiving the emergent light, and by institute It states emergent light and is converted into image data, to be sent to the processor；

The processor is used to generate fluorescent image according to described image data.

2. system according to claim 1, which is characterized in that the upper conversion fluorescent nano particle is AReF₄(Re³⁺, N%)@mAReF₄Or Re₂O₃(Re³⁺, n%)；Wherein, A includes Li, Na, K；Re includes Sc, Y, La, Gd, Lu；Re³⁺Including Pr³⁺、 Nd³⁺、Sm³⁺、Dy³⁺、Ho³⁺、Er³⁺、Tm³⁺；The value range of the n are as follows: 0 to 100, including right end point value；The value model of the m It encloses are as follows: 0 to 30, including endpoint value.

3. system according to claim 2, which is characterized in that the light source is laser light source, the laser light source and institute It states and is provided with laser beam adjusting lens group between sample stage.

4. system according to claim 3, which is characterized in that the laser beam adjust lens group include along optical axis successively Collimation lens, extender lens and the shaping lens of distribution.

5. system according to claim 2, which is characterized in that be arranged between the sample stage and the optical signal receiver There is bandpass filter, the bandpass filter is for choosing the monochromatic emergent light for presetting wavelength.

6. system according to claim 5, which is characterized in that between the bandpass filter and the optical signal receiver It is provided with fluorescent light beam and adjusts lens group.

7. system according to claim 6, which is characterized in that the fluorescent light beam adjust lens group include along optical axis successively Collimation lens, extender lens and the shaping lens of distribution.

8. system according to claim 2, which is characterized in that the optical signal receiver is CCD detector.

9. a kind of imaging method of up-conversion fluorescence, which is characterized in that the described method includes:

The sample of sample stage is arranged in light source transmitting incident light irradiation, wherein the wave-length coverage of the incident light are as follows: 1000nm is extremely 2500nm, including endpoint value, the sample are the sample marked by upper conversion fluorescent nano particle；

The sample absorbs the incident light, and launches and penetrate light；

Optical signal receiver receives the emergent light, and the emergent light is converted into image data, to be sent to processor；

The processor generates fluorescent image according to described image data.

10. according to the method described in claim 9, it is characterized in that, the upper conversion fluorescent nano particle is AReF₄(Re³⁺, N%)@mAReF₄Or Re₂O₃(Re³⁺, n%)；Wherein, A includes Li, Na, K；Re includes Sc, Y, La, Gd, Lu；Re³⁺Including Pr³⁺、 Nd³⁺、Sm³⁺、Dy³⁺、Ho³⁺、Er³⁺、Tm³⁺；The value range of the n are as follows: 0 to 100, including right end point value；The value model of the m It encloses are as follows: 0 to 30, including endpoint value.