WO2022001520A1 - Method, device and application for second near-infrared region fluorescence imaging using near-infrared fluorescent protein derivative or analog - Google Patents

Abstract

A method, device and application for the second near-infrared region fluorescence imaging using a near-infrared fluorescent protein derivative or an analog. Also provided is an expression of near-infrared fluorescent protein iRFP713 in the second near-infrared region. The near-infrared fluorescent protein has an emission wavelength in a near-infrared region greater than the emission spectrum of traditional GFP-type fluorescent proteins (approximately 400-600 nm) and is thus more suitable for in vivo bioimaging. The application has significant advantages in terms of penetration depth and spatial resolution compared to bioimaging using GFP-type proteins and imaging applications using near infrared proteins at the first near-infrared region (approximately 700-900 nm), and greatly improves the penetration depth and signal-to-noise ratio of in vivo imaging.

Description

A method, device and application for near-infrared second-region fluorescence imaging using near-infrared fluorescent protein derivatives or analogs

This application is required to be submitted to the China Patent Office on June 30, 2020, the application number is 202010620535.4, and the name of the invention is "A method, device and application for near-infrared fluorescent imaging using near-infrared fluorescent protein derivatives or analogs" The priority of the Chinese patent application of , the entire contents of which are incorporated by reference in this application.

technical field

The present invention relates to the technical field of biological fluorescence imaging, in particular to a method, device and application for near-infrared second-region fluorescence imaging using near-infrared fluorescent protein derivatives or analogs.

Background technique

Biofluorescence imaging technology refers to the method of using the fluorescence emitted by biological tissue or structure to conduct imaging, which is used to study biological processes. By using various fluorescent markers, fluorescence imaging can realize imaging studies at the cell level, tissue level and in vivo level. It has the advantages of high resolution, small damage, and fast imaging, and has broad application prospects.

The use of fluorescent probes is a key part of fluorescence imaging technology. Currently common fluorescent probes include small molecule fluorescent dyes, fluorescent nanoparticles and various fluorescent proteins. Among these probes, although chemically modified and synthesized nanoparticles and fluorescent dyes have strong fluorescence brightness, their disadvantages are also very obvious. On the one hand, they are not reproducible, that is, they cannot replicate with the replication of cells, so they are not suitable for studying some biological processes that require long-term observation, such as tumor development and metastasis; on the other hand, nanoparticles and During the transportation of fluorescent dyes in the body, accumulation and precipitation often occur, which are not easy to be excreted from the body, thus affecting the metabolism of the body. Since fluorescent proteins are proteins in nature, they have good biocompatibility and low cytotoxicity; at the same time, fluorescent proteins can achieve specific expression in tissue cells, making fluorescent imaging capable of providing specific location information. Fluorescent protein imaging has been widely used in life science research.

At present, the research and application of fluorescent protein imaging at the molecular and cellular levels has been extremely mature. However, deep tissue imaging and in vivo imaging still face great challenges, mainly due to the scattering of signal light during imaging, and the interference of tissue on signal light absorption and autofluorescence. For example, imaging systems based on traditional fluorescent proteins such as green fluorescent protein (GFP-like fluorescent proteins) have long been developed and perfected, but due to the limitation of their emission spectrum (only 400-600 nm), the background signal is strong when imaging tissue cells, which is very effective. not ideal. The solution to the in vivo imaging problem is to use fluorescent protein probes with emission wavelengths in the longer near-infrared region, that is, near-infrared fluorescent proteins.

At present, near-infrared fluorescent protein imaging is one of the research hotspots in bioluminescence imaging. Near-infrared fluorescent proteins use biliverdin as a chromophore to overcome the fundamental limitations of GFP-like fluorescent protein chromophores. Through directed evolution, the excitation and emission wavelengths of near-infrared fluorescent proteins are both in the near-infrared "optical window". In this wavelength band, the scattering of signal light is small and the autofluorescence of tissue is usually low, so it has better light penetration, greater penetration depth during imaging, and higher signal-to-noise ratio.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method, device and application for near-infrared second-region fluorescence imaging using near-infrared fluorescent protein derivatives or analogs in view of the deficiencies of the prior art.

The purpose of this invention is to realize through the following technical solutions:

The present invention provides a method for performing near-infrared second-region fluorescence imaging using near-infrared fluorescent protein derivatives or analogs, the near-infrared fluorescent protein derivatives or analogs have a longer near-infrared second region than GFP fluorescent proteins The near-infrared fluorescent protein derivatives or analogs have considerable fluorescence signals above 900 nm, and the near-infrared fluorescent protein derivatives or analogs can achieve fluorescence imaging in the near-infrared second region.

Preferably, the near-infrared fluorescent protein derivatives or analogs include iRFP670, iRFP682, iRFP702, iRFP720, miRFP670, miRFP703, and miRFP709.

Preferably, the wavelength of the second near-infrared region is in the range of 900-1700 nm.

The present invention provides a recombinant plasmid comprising near-infrared fluorescent protein derivatives or analogs, wherein the near-infrared fluorescent protein derivatives or analogs are iRFP670, iRFP682, iRFP702, iRFP720, miRFP670, miRFP703 or miRFP709.

The present invention provides an engineering bacterium, which is obtained by introducing the above recombinant plasmid into Escherichia coli.

The present invention provides a recombinant cell obtained by transfecting the above recombinant plasmid into HEK293 cells.

The invention provides an application of near-infrared fluorescent protein derivatives or analogs for near-infrared second-region fluorescence imaging, including cell imaging applications, intestinal flora imaging applications and tumor marker imaging applications.

Preferably, when used for cell imaging, the cells used are HEK293 cells;

When used for intestinal flora imaging, the intestinal bacteria used are Escherichia coli;

When used for tumor marker imaging, the tumor cells used were LM3 tumor cells.

The invention provides a device for near-infrared second-region fluorescence imaging using near-infrared fluorescent protein derivatives or analogs, which is characterized in that the device includes a near-infrared second-region macro imaging system and a near-infrared second-region microscopic imaging system;

The near-infrared two-zone macro imaging system includes a lens 7, a light source 3, a 35mm fixed-focus lens 6, a detector 2 and a long-pass filter 5, and the light source 3 is a laser or LED; in the macro imaging system, the lens 7 After being set in the light source 3, the outgoing light of the laser or LED is expanded to make the sample evenly excited; the fluorescent signal emitted by the sample is collected by the 35mm fixed-focus lens 6; And set it between the fixed focus lens 6 and the detector 2 to filter out the background signal;

The near-infrared two-zone microscopic imaging system includes a lens 8, a light source 3, an upright microscope epi-illuminator 11, a dichroic mirror 9, an objective lens 10, a long-pass filter 5 and a detector 2; In the system, the collimating lens 8 is arranged between the upright microscope epi-illuminator 11 and the laser or LED outgoing light; in the epi-illuminator 11, just above the objective lens 10, a long-pass short-reverse dichroic mirror 9 is arranged Reflect the excitation light; the fluorescence signal on the front focal plane of the objective lens 10 is collected by the objective lens 10 set above the sample, and passed through the dichroic mirror 9 above the objective lens 10; different settings are set between the tube lens 8 and the dichroic mirror 9 The long-pass filter 5 of the cut-off wavelength filters out the background, and the target surface of the detector 2 is set above the tube lens;

Preferably, the power density of the light source 3 is 60 mW cm ⁻² .

Preferably, the detector 2 is an InGaAs two-dimensional detector.

Preferably, the detector 2 is connected to the computer 1 .

Beneficial effects of the present invention: In the prior art, the near-infrared fluorescent protein has low signal-to-noise ratio, penetration depth and resolution in in vivo imaging in the near-infrared region, and cannot achieve a good imaging effect. The present invention discovers and confirms for the first time that the fluorescence emission of the near-infrared fluorescent protein iRFP in the near-infrared second region (900-1700 nm) is sufficient for fluorescence imaging and achieves a good imaging effect. Based on this imaging characteristic, the present invention proposes a new application of the near-infrared fluorescent protein iRFP in the near-infrared second region, thereby overcoming the low signal-to-noise ratio and low penetration depth and resolution of the near-infrared fluorescent protein in the near-infrared first region in vivo imaging. The application limitation of low rate breaks through the limitations of traditional near-infrared second-region fluorescent probes in the observation of long-term life activities; and then uses its fluorescence properties in the near-infrared second region to achieve high-sensitivity and high-penetration fluorescence. imaging technology. The present invention purifies 8 kinds of near-infrared fluorescent proteins by the method of molecular biology, and then compares their imaging effects in the near-infrared first region and near-infrared second region in detail, and selects the near-infrared fluorescent protein iRFP713 according to the test results to continue the follow-up In vivo imaging, it is proved that the NIR fluorescent protein has a significantly improved imaging effect in the NIR II region.

Description of drawings

Fig. 1 is a diagram of the detection system of Examples 2, 3, 4, and 5, wherein Fig. 1(a) is a diagram of a macroscopic imaging system, and Fig. 1(b) is a diagram of a microscopic imaging system; Figs. 1(a) and 1(b) Among them, 1 is a computer, 2 is an InGaAs two-dimensional detector, 3 is a light source, 4 is an object plane, 5 is a long pass filter, 6 is a 35mm fixed focus lens, 7 is a beam expander lens, 8 is a tube lens, and 9 10 is the objective lens, 11 is the epi-illuminator of the upright microscope;

Fig. 2 is the absorption spectrum of 8 kinds of materials in embodiment 1;

Fig. 3 is the fluorescence spectrum of 8 kinds of materials in Example 1, wherein Fig. 2(a) is a spectrum of 900-1600 nm, and Fig. 2(c) is a spectrum of 1000-1600 nm;

Figure 4 is a comparison diagram of the fluorescence intensity of the 0.3 mg mL ^-1 bright field in Example 1 and the 623 nm LED, the illumination intensity of about 60 mW cm ^-2 laser irradiation, and the imaging fluorescence intensity after passing through a 1000LP filter;

5 is a comparison diagram of the near-infrared first/second region of the simulated iRFP713 penetration depth in biological tissue in Example 2;

Fig. 6 is the near-infrared second region fluorescence imaging result diagram of the bacterial expression iRFP of Example 3;

Fig. 7 is the near-infrared second region fluorescence contrast diagram of bacterial expression GFP and iRFP713 of embodiment 3;

Fig. 8 is a near-infrared second region imaging effect diagram of the bacteria expressing iRFP713 of Example 3 in BALB/c mice; the left group in the figure is the imaging effect without abdominal cavity opening, and the right group is the imaging effect after opening the abdominal cavity, Each group from left to right, from top to bottom is the imaging results in chronological order;

9 is a single-frame image of the interception of the near-infrared second-region imaging video of the bacteria expressing iRFP713 of Example 3 in BALB/c mice;

Figure 10 is a graph of the results of fluorescence imaging in the near-infrared second region of cells stably expressing several iRFPs in Example 4;

Figure 11 is a graph of the results of near-infrared second-region imaging of LM3 cells stably expressing iRFP713 and luciferase in Example 4;

FIG. 12 is a graph showing the results of near-infrared second region and luciferase chemiluminescence imaging of tumor mice in Example 5. FIG.

detailed description

The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

Near-infrared fluorescent protein iRFP and its derivatives or analogs with larger near-infrared second-region fluorescence components can all realize fluorescence imaging applications in near-infrared second-region (900-1700 nm). Since the peak fluorescence wavelengths of iRFP fluorescent proteins all fall within 600-800 nm, the current development of this type of protein is completely limited to imaging in the near-infrared region of 700-900 nm. It has been verified that iRFP fluorescent proteins still have considerable fluorescence signals above 900 nm, especially iRFP713. This is due to the large total fluorescence emission (full band) of iRFP713 and the long fluorescence tail in the second near-infrared region. of.

This example involves two sets of laboratory-built optical systems: a near-infrared second-region macro imaging system and a near-infrared second-region microscopic imaging system. As shown in Fig. 1(a), in the macro imaging system, the lens 7 is used to expand the laser or LED light, so that the sample on the object plane 4 is uniformly excited. The fluorescent signal emitted by the sample is collected by means of a 35mm fixed-focus lens 6, and the background is filtered out by long-pass filters 5 with different cut-off wavelengths in front of the detector 2, which is connected to a computer 1. Figure 1(b), in the microscope imaging system, the laser or LED light collimated by the lens is transmitted and focused in the epi-illuminator 11 of the upright microscope, reflected by the dichroic mirror 9, and finally focused on the objective lens 10 The focal plane is uniformly illuminated on the sample on the object plane 4 through the objective lens 10 . The fluorescent signal emitted by the sample is collected by the objective lens 10, and then filtered by long-pass filters 5 with different cut-off wavelengths.

Example 1 mainly tests and compares the absorption spectra, emission spectra and fluorescence brightness of several near-infrared fluorescent protein derivatives or analog iRFPs (iRFP670, iRFP682, iRFP702, iRFP713, iRFP720, miRFP670, miRFP703 and miRFP709), indicating that iRFP713 has the highest fluorescence intensity. Example 2 mainly illustrates that the tissue penetration depth of iRFP in the second near-infrared region is better than that in the first region of near-infrared, and it can be proved that the near-infrared fluorescent protein iRFP can be used for fluorescence imaging in the second region of near-infrared (the results are shown in Figure 2); Example 3 ~5 mainly describes the near-infrared second-region fluorescence imaging of the near-infrared fluorescent protein iRFP at the level of cells, tissues and living bodies.

Example 1

The absorption spectra (300-900 nm) of iRFP670, iRFP682, iRFP702, iRFP713, iRFP720, miRFP670, miRFP703 and miRFP709 were tested using a UV-visible spectrophotometer (Shimadzu 2550 UV-vis scanning spectrophotometer). As shown in Figure 2, iRFP720 has the longest absorption peak wavelength.

The emission spectra of iRFP670, iRFP682, iRFP702, iRFP713, iRFP720, miRFP670, miRFP703 and miRFP709 in the near-infrared band were tested using a fluorescence spectrometer (FLS980, Edinburgh Instruments Ltd.). As shown in Figure 3(a), iRFP720 has the longest fluorescence peak wavelength. Figures 3(b) and 3(c) are the spectral graphs in the 900-1600 nm and 1000-1600 nm bands. It can be seen that several proteins have a certain amount of near-infrared fluorescence.

Fluorescence images of iRFP670, iRFP682, iRFP702, iRFP713, iRFP720, miRFP670, miRFP703, and miRFP709 were directly recorded using an InGaAs camera and a macroscopic imaging system (Fig. 1(a)). The protein concentration was 0.3 mg mL ^-1 , the bright field image was taken under halogen lamp illumination, and the fluorescence image was taken under 623 ^{nm LED illumination (power density: about 60 mW cm -2} ), after filtering out the background with a 1000 nm long-pass filter 5 . As shown in Figure 4, iRFP713 showed the best fluorescence intensity.

Example 2

The iRFP pure protein was diluted to 1 mg mL ^-1 . A glass capillary tube was used to pipette the pure protein solution, fill it, and tape it to the bottom of the cylindrical dish. Cylindrical dishes were filled with various volumes of 1% liposomes (lntralipid) to simulate the wavelength dependence of light scattering in biological tissues. The depth of capillaries was calculated from the bottom area of the cylindrical dish and the volume of the liposomes. During detection, the laser beam is evenly irradiated on the cylindrical dish after beam expansion. In this example, 800nm, 900nm, and 1100nm long-pass filters (Thorlabs) were used to image capillaries with depths of 0mm, 2mm, 4mm, and 6mm. After the images were uniformly enhanced, the results are shown in Figure 5. In Figure 5 The image on the left is the imaging result; the image on the right is the half-height width calculated by analyzing the fluorescence distribution of the image on the left using Image J software, and the result is presented in the form of a histogram. After the visible depth increases, the sharpness of the images using different wavelengths differs significantly, and the longer the wavelength, the smaller the scattering.

Example 3

The plasmids of iRFP670, iRFP682, iRFP702, iRFP713, iRFP720, miRFP670, miRFP703 and miRFP709 were respectively introduced into Escherichia coli and cultured in LB medium to express these fluorescent proteins. Put the bacteria together with the culture medium into the strip tube, use a 623nm LED as the excitation light source 3 to illuminate the sample, the power density is about 60mW cm ^-2 , use a 35mm fixed-focus lens 6 to collect, and filter it with a 1000nm long-pass filter 5. NIR-II fluorescence signal, detector 2 was a SW640 short-wave infrared camera (Tekwin, China). As shown in FIG. 6 and FIG. 7 , BF is an image captured in bright field.

The Escherichia coli that successfully introduced and expressed the iRFP713 near-infrared fluorescent protein were expanded and cultured. Grab BALB/c mice, suck E. coli with a gavage needle for gavage treatment, and record the time. In the experiment, a 695nm laser was used as the excitation light source 3 to irradiate the sample with a power density of about 60mW cm ^-2 , and a 35mm fixed-focus lens 6 was used to collect and filter the NIR-II fluorescence signal with a 1000nm long-pass filter 5, and the detector 2 was SW640 Gastrointestinal angiography was performed with a short-wave infrared camera (Tekwin, China). Photos were taken at 0h, 0.5h, 1h, 3h, 6h, 9h, 12h, and 24h, respectively. As shown in Figure 8, the imaging of iRFP713-E. coli in mice was clear, and the localization of the bacteria in the gastrointestinal tract changed over time. Moreover, despite the skin occlusion, NIR-II imaging can still image the details of the intestinal contour to a certain extent.

In order to prove the good real-time performance of NIR-II fluorescence imaging, during the gastrointestinal angiography, a video of gastrointestinal peristalsis was taken of the mice perfused with bacteria, and frames were extracted at 960 millisecond intervals. The results are shown in Figure 9. The peristalsis of the intestinal tract, the movement track of Escherichia coli in the intestinal tract is clearly visible.

Example 4

The 8 plasmids were transfected into HEK293 cells to express the corresponding near-infrared fluorescent protein. According to the experimental group settings, biliverdin BV (25 μM) was added 2 hours before imaging. During imaging, an epi-illumination wide-field fluorescence microscope system was used, and a 623nm LED was used as the excitation light source 3 for uniform illumination, and a 25X infrared antireflection objective (XLPLN25XWMP2, NA=1.05, Olympus) was used to collect the fluorescence signal, and the imaging camera was a SW640 short-wave infrared camera ( Tekwin, China), filtered by 1100nm long-pass filter 5 in front of the camera. The results are shown in Figure 10.

Example 5

Further, a LM3 tumor cell line stably expressing iRFP713 was constructed, and a mouse orthotopic liver cancer model was established. The results are shown in Figure 11. Taking advantage of the iRFP713 protein, mouse tumors can be continuously observed. Use a 695nm laser as the excitation light source 3 to illuminate the sample with a power density of about 60mW cm ^-2 , use a 35mm fixed-focus lens 6 to collect, and filter the NIR-II fluorescence signal with a 1000nm long-pass filter 5, and the detector 2 is SW640 short-wavelength An infrared camera (Tekwin, China) was used to photograph the back, right and front of the mice respectively. After further comparison with luciferase chemiluminescence imaging results, it can be confirmed that the NIR-II fluorescence imaging of iRFP713 protein has higher spatial resolution. The results are shown in Figure 12.

The above-described embodiments are used to explain the present invention, rather than limit the present invention. Within the scope of protection of the spirit of the present invention and claims, any modifications and changes made to the present invention all fall into the protection scope of the present invention.

Claims (12)

1. A method for near-infrared fluorescent imaging using a near-infrared fluorescent protein derivative or analog, wherein the near-infrared fluorescent protein derivative or analog has a longer near-infrared second region than a GFP fluorescent protein. The near-infrared fluorescent protein derivatives or analogs have considerable fluorescence signals above 900 nm, and the near-infrared fluorescent protein derivatives or analogs can achieve fluorescence imaging in the near-infrared second region.
2. The method for near-infrared fluorescent imaging using near-infrared fluorescent protein derivatives or analogs according to claim 1, wherein the near-infrared fluorescent protein derivatives or analogs include iRFP670, iRFP682, iRFP702, iRFP720 , miRFP670, miRFP703, miRFP709.
3. The method for near-infrared second region fluorescence imaging using near-infrared fluorescent protein derivatives or analogs according to claim 1, wherein the wavelength of the near-infrared second region is in the range of 900-1700 nm.
4. A recombinant plasmid comprising near-infrared fluorescent protein derivatives or analogs, the near-infrared fluorescent protein derivatives or analogs being iRFP670, iRFP682, iRFP702, iRFP720, miRFP670, miRFP703 or miRFP709.
5. An engineering bacterium is obtained by introducing the recombinant plasmid of claim 4 into Escherichia coli.
6. A recombinant cell obtained by transfecting the recombinant plasmid of claim 4 into HEK293 cells.
7. An application of near-infrared fluorescent protein derivatives or analogs for near-infrared second-region fluorescence imaging, including cell imaging applications, intestinal flora imaging applications, and tumor marker imaging applications.
8. The application according to claim 7, wherein when used for cell imaging, the cells used are HEK293 cells;

   When used for intestinal flora imaging, the intestinal bacteria used are Escherichia coli;

   When used for tumor marker imaging, the tumor cells used were LM3 tumor cells.
9. A device for near-infrared second-region fluorescence imaging using near-infrared fluorescent protein derivatives or analogs, characterized in that the device includes a near-infrared second-region macro imaging system and a near-infrared second-region microscopic imaging system;

   The near-infrared second-region macro imaging system includes a lens (7), a light source (3), a 35mm fixed-focus lens (6), a detector (2) and a long-pass filter (5), and the light source (3) is Laser or LED; in the macro imaging system, the lens (7) is arranged behind the light source (3) to expand the outgoing light of the laser or LED, so that the sample is uniformly excited; the sample is collected by a 35mm fixed-focus lens (6) and emitted Fluorescence signal; select long-pass filters (5) with different cut-off wavelengths as required, and set them between the fixed-focus lens (6) and the detector (2) to filter out the background signal;

   The near-infrared second-zone microscopic imaging system comprises a collimating lens (8), a light source (3), an epi-illuminator (11) for an upright microscope, a dichroic mirror (9), an objective lens (10), and a long-pass filter a light sheet (5) and a detector (2); in the microscope imaging system, a collimating lens (8) is arranged between the epi-illuminator (11) of the upright microscope and the outgoing light of the laser or LED; in the upright microscope In the epi-illuminator (11), just above the objective lens (10), a long-pass short inverse dichroic mirror (9) is arranged to reflect the excitation light; the fluorescence signal on the front focal plane of the objective lens (10) is arranged above the sample through the The objective lens (10) is collected, and passes through the dichroic mirror (9) above the objective lens (10); long-pass filters with different cut-off wavelengths are arranged between the collimating lens (8) and the dichroic mirror (9). (5) The background is filtered out, and the target surface of the detector (2) is arranged above the tube lens.
10. The device according to claim 9, characterized in that, the power density of the light source (3) is 60 mW cm ^-2 .
11. The device according to claim 9, wherein the detector (2) is an InGaAs two-dimensional detector.
12. The device according to claim 9 or 11, wherein the detector (2) is connected to a computer (1).

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