Low-dose aggregation-induced emission dye bio-imaging method and device

Abstract

This invention relates to a low-dose aggregation-induced emission dye (AIE) bioimaging method, comprising the following steps: selecting a test organism and injecting it with a low dose of AIE; stimulating the test organism with an adjustable frequency excitation light source; acquiring fluorescence signals emitted from the test organism at equal time intervals; processing the fluorescence signals and generating a fluorescence image; applying phase-locked processing to the fluorescence image to separate the fluorescence signal component with the same frequency as the excitation light source and generating a phase-locked imaging result. By convolving the amplitude signal of each pixel in the fluorescence image with a sine or cosine signal with the same frequency as the excitation source, the orthogonality of trigonometric functions is utilized to effectively suppress noise, thus preserving the fluorescence signal that changes with the same frequency as the periodic light source. This significantly improves the signal-to-noise ratio of the fluorescence image, enabling clear observation of the AIE fluorescence signal even at low doses.

Description

Technical Field

[0001] This invention relates to the field of biomedical imaging, and more particularly to a low-dose aggregation-induced emission dye bioimaging method based on phase-locked loop technology. Background Technology

[0002] Aggregation-induced emission (AIE) dyes have attracted much attention in the medical field due to their ability to provide effective imaging over long periods in vivo. AIE dyes enhance fluorescence signals through molecular aggregation, resulting in stable imaging signals and avoiding the self-quenching problem of traditional fluorescent dyes in a diluted state. Therefore, they can provide stable and clear fluorescence signals. However, this technology also has the following problems:

[0003] (i) Because fluorescence imaging requires the injection of high doses of aggregation-induced emission dyes to obtain clear fluorescence images, and high doses of aggregation-induced emission dyes have unknown toxicity to organisms, potential toxicity, and long metabolism time, which further hinders clinical application.

[0004] (ii) Traditional excitation-based fluorescence imaging is affected by a combination of factors, including focusing, light source, filters, fluorescent probes, and samples. In particular, it is difficult to obtain clear fluorescence images in the long wavelength range of 1300nm to 1700nm, especially in the range of 1400nm to 1700nm, making it impossible to accurately observe and analyze the fine structure and dynamic changes of biological samples. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a low-dose aggregation-induced emission dye bioimaging method and apparatus to solve one or more problems in the prior art.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A low-dose aggregation-induced emission dye bioimaging method includes the following steps:

[0008] Dye injection into the test organism: Select the test organism and inject it with a low dose of aggregation-induced emission dye;

[0009] Setting and adjusting the excitation source: Select an excitation light source with adjustable frequency to excite the organism being tested;

[0010] Image acquisition: Fluorescent signals emitted by the tested organism are acquired at equal time intervals;

[0011] Preprocessing: The fluorescence signal is processed and a fluorescence image is generated;

[0012] Phase-locked processing: Phase-locked processing is applied to the fluorescence image to separate the fluorescence signal component with the same frequency as the excitation light source and generate a phase-locked imaging result.

[0013] Furthermore, the modulation frequency of the excitation light source is 0.025-1000Hz, and the output intensity of the excitation light source is 1-726mW / cm². 2 .

[0014] Furthermore, the camera exposure time is set according to the modulation frequency and output intensity of the excitation light source, and the camera exposure time is 0.2ms to 10s.

[0015] Furthermore, the phase-locked loop (PLL) process includes the following steps:

[0016] The fluorescence signal component is convolved with a sinusoidal reference signal of the same frequency to obtain the sum of the sinusoidal components.

[0017] The fluorescence signal component is convolved with a cosine reference signal of the same frequency to obtain the sum of the cosine components.

[0018] The amplitude of the periodic change of each pixel with the excitation signal is calculated based on the sum of the sine and cosine components.

[0019] Furthermore, the fluorescence signal component is convolved with a sinusoidal reference signal of the same frequency, as calculated by the following formula:

[0020]

[0021] A1(x,y) represents the accumulated result of the sine components, and f(x,y,i) represents the pixel value of the pixel located at position (x,y) in the i-th image. This represents a sinusoidal reference signal.

[0022] Furthermore, the fluorescence signal component is convolved with a cosine reference signal of the same frequency, as calculated by the following formula:

[0023]

[0024] A2(x,y) represents the sum of the cosine components, and f(x,y,i) represents the pixel value of the pixel at position (x,y) in the i-th image. This represents the cosine reference signal.

[0025] Furthermore, the amplitude value of the periodic change of each pixel with the excitation signal is calculated as follows:

[0026]

[0027] Where A1(x,y) represents the accumulated result of the sine component, A2(x,y) represents the accumulated result of the cosine component, and A(x,y) represents the amplitude value of each pixel as the excitation signal changes periodically.

[0028] Accordingly, the present invention also provides an apparatus for a low-dose aggregation-induced emission dye bioimaging method, characterized in that: the apparatus comprises...

[0029] An excitation light source generates an excitation signal for measurement to excite the test organism, causing the test organism to produce fluorescence at the same frequency as the excitation light source;

[0030] A phase-locked module controls the signal modulated by the excitation light source and the image acquisition by the imaging module, so that the imaging module acquires fluorescence images at equal time intervals and applies a phase-locked algorithm to the fluorescence images to obtain the amplitude value of the periodic change of each pixel with the excitation signal.

[0031] The imaging module captures the fluorescence signal emitted by the tested organism and outputs it to the phase-locked module.

[0032] Furthermore, the phase-locked module includes a calculation module and a signal generation module, wherein...

[0033] The calculation module is used to perform phase-locked calculations on the acquired fluorescence images;

[0034] The signal generation module is used to output a square wave modulated excitation light source, so that the excitation light source periodically modulates the tested organism.

[0035] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0036] By convolving the amplitude signal of each pixel in the fluorescence image with a sine or cosine signal of the same frequency as the excitation source, the orthogonality of trigonometric functions is utilized to effectively suppress noise, thus preserving the fluorescence signal that changes at the same frequency as the periodic light source. This significantly improves the signal-to-noise ratio of the fluorescence image, enabling clear observation of the fluorescence signal of aggregation-induced emission dyes even at low doses. This not only reduces experimental costs but also minimizes the impact of dyes on organisms, facilitating safer and more environmentally friendly biological research and medical diagnosis.

[0037] Furthermore, the improved signal-to-noise ratio significantly enhances the imaging quality of fluorescence images. Fluorescent signals in the images are clearer and sharper, with reduced background noise, enabling researchers to more accurately identify and locate fluorescence sources, thereby improving the accuracy and reliability of biological research and medical diagnosis.

[0038] Furthermore, convolution processing not only suppresses noise but also enhances the contrast between the fluorescence signal and the background. This makes the detection of fluorescence signals more sensitive at low doses, allowing even very weak fluorescence signals to be clearly captured and displayed.

[0039] Thanks to the improved signal-to-noise ratio and image quality, researchers can obtain clear fluorescence images using lower doses of aggregation-induced emission dyes. This not only reduces experimental costs but also minimizes the potential impact of the dyes on organisms, facilitating safer and more environmentally friendly biological research and medical diagnostics. Attached Figure Description

[0040] Figure 1 The diagram shows a structural schematic of a low-dose aggregation-induced emission dye bioimaging method and device according to an embodiment of the present invention.

[0041] Figure 2 The diagram shows a structural schematic of a low-dose aggregation-induced emission dye bioimaging method and device according to an embodiment of the present invention.

[0042] Figure 3 The diagram shows a phase-locked module in a low-dose aggregation-induced emission dye bioimaging method and device according to an embodiment of the present invention.

[0043] Figure 4 This diagram illustrates a comparison between an image obtained by a low-dose aggregation-induced emission dye bioimaging method according to an embodiment of the present invention and the original image obtained by a conventional method.

[0044] The attached diagram is labeled as follows: 1. Biological sample; 2. Imaging module; 3. Phase-locked module; 4. Computer; 5. Excitation light source. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the following detailed description of a low-dose aggregation-induced emission dye bioimaging method and apparatus proposed by this invention, in conjunction with the accompanying drawings and specific embodiments, will provide further clarity. The advantages and features of this invention will become clearer from the following description. It should be noted that the accompanying drawings are in a very simplified form and use non-precise proportions, used only to facilitate and clearly illustrate the purpose of the embodiments of this invention. Please refer to the accompanying drawings to make the objectives, features, and advantages of this invention more apparent and understandable. It should be understood that the structures, proportions, sizes, etc., depicted in the accompanying drawings are only for illustrative purposes to aid those skilled in the art and are not intended to limit the implementation conditions of this invention. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size, without affecting the effects and objectives achieved by this invention, should still fall within the scope of the technical content disclosed in this invention.

[0046] An apparatus for implementing a low-dose aggregation-induced emission dye bioimaging method includes an imaging module 2, a phase-locked module 3, a computer 4, and an excitation light source 5.

[0047] For details, please refer to Figure 2 The excitation light source 5 is positioned between the tested organism 1 and the phase-locked module 3. The excitation light source generates an excitation signal for measurement and excites the tested organism 1. By adjustment, the tested organism 1 produces fluorescence at the same frequency as the excitation light source.

[0048] Furthermore, in this embodiment, the modulation frequency of the excitation light source is 0.025 Hz, and the output intensity of the excitation light source is 32 mW / cm². 2 The exposure time of imaging module 2 is set according to the modulation frequency and output intensity of the excitation light source, with the camera exposure time being 10 seconds. The camera sampling frequency (also known as the reciprocal of the exposure time) in the imaging module is obtained by multiplying the phase-locked frequency by the number of photos per cycle.

[0049] Furthermore, the assembly also includes an imaging module 2, which is used to capture the fluorescence signal emitted by the tested organism 1 and output it to the image-locking module 3. The output end of the imaging module 2 is connected to one of the input ends of the image-locking module 3.

[0050] Specifically, in order to ensure that the imaging module 2 can still capture clear fluorescence signals under low-dose dye conditions, in this embodiment, the imaging module 2 can be a highly sensitive InGaAs or InSb.

[0051] Furthermore, in order to achieve high sensitivity of the imaging module 2, the imaging module 2 can enhance weak signals by setting an image intensifier. The incident light passes through the objective lens and hits the photocathode of the image intensifier, and then is converted into an electronic image through the photoelectric effect. The electronic image continuously impacts in the microchannel plate, and finally produces a multiplication effect. The multiplication effect is used to enhance the weak signal and re-excite the photon image, which is transmitted to the imaging module 2 for imaging, so that a clear fluorescence image can still be captured under low-dose dye conditions.

[0052] Furthermore, the device also includes an image-locked module 3, the input of which is connected to the output of the imaging module 2. Specifically, the image-locked module is used to control the signal modulated by the excitation light source 5 and to synchronously acquire the fluorescence image collected by the imaging module 2, and to apply a phase-locked algorithm to process the fluorescence image to obtain the amplitude value of the periodic change of each pixel with the excitation signal.

[0053] For details, please refer to Figure 3The phase-locked module 3 includes a calculation module 300 and a signal generation module 301. The calculation module 300 and the signal generation module 301 are connected through a first interface. The calculation module 300 is used to perform phase-locked calculation on the acquired fluorescence image (the specific phase-locked calculation process is described in detail in the method). The signal generation module 301 is used to output a square wave modulated excitation light source 5. At the same time, the calculation module 300 receives a trigger signal or synchronization signal from the signal generation module 301 through the first interface to ensure the synchronization of fluorescence image acquisition and light source excitation. The calculation module 300 is also connected to the computer 5 through a second interface to output the results of the phase-locked calculation to the display end (e.g., a display screen) of the computer 5 for display and analysis. Furthermore, the calculation module 300 also receives the fluorescence image acquired by the imaging module 2 through a third interface.

[0054] Furthermore, the signal generation module 301 outputs a periodically changing square wave signal to modulate the light source and excite the tested organism 1. The square wave signal collects N sets of data at equal intervals within one cycle. The data is used for analysis. Specifically, the data includes fluorescence intensity data, wherein the fluorescence intensity data reflects the emission intensity of the tested organism 1.

[0055] Accordingly, please refer to Figure 1 The present invention also provides a low-dose aggregation-induced emission dye bioimaging method, comprising the following steps:

[0056] S1: Select the test organism and inject it with a low dose of aggregation-induced emission dye. Specifically, select a suitable test organism, such as a mouse, and ensure that the test organism remains stable by anesthetizing and fixing it to reduce interference with the results.

[0057] S2: Setting and Adjusting the Excitation Source: An adjustable frequency excitation light source 5 is selected to excite the test organism. The periodically changing light signal generated by this excitation light source penetrates the mouse's skin, exciting pre-injected aggregation-induced emission dye molecules in the blood vessels, causing them to emit a fluorescence signal. This fluorescence signal contains a component with the same frequency as the excitation signal, as well as components of other frequencies (such as background noise). Specifically, in the phase-locked module 3, the frequency and phase of the square wave signal are set through the signal generation module 301 to synchronize it with the periodic changes of the excitation light source. Simultaneously, the trigger conditions for image acquisition are set to ensure that N sets of fluorescence images can be acquired after the excitation light source completes one periodic change.

[0058] S3: Image Acquisition: Fluorescence signals emitted by the tested organism are acquired at equal time intervals. Specifically, when the signal generation module 301 in the phase-locked module 3 generates a synchronization signal with the same frequency as the modulation signal of the excitation light source, the synchronization signal is sent to the trigger port of the imaging module 2. When the synchronization signal reaches a preset trigger condition, the imaging module 2, for example, uses an InGaAs camera to acquire an image. Specifically, the synchronization signal generated by the signal generation module 301 can be used as a trigger signal for a timer or calculator, thus starting a new time interval when each synchronization signal arrives, and triggering the imaging module 2 to acquire an image at the end of the time interval.

[0059] S4: Preprocessing: Processing the fluorescence signal and generating a fluorescence image. Imaging module 2 uses image processing algorithms or signal processing software to extract the fluorescence signal component with the same frequency as the excitation signal from the fluorescence signal, and recombines the fluorescence signal component data and noise suppression data into an image to generate a fluorescence image.

[0060] S5: Phase-locked processing: Phase-locked processing is applied to the fluorescence image to separate the fluorescence signal component with the same frequency as the excitation light source and generate a phase-locked imaging result. Specifically, the phase-locked processing includes the following steps:

[0061] S500: Convolve the fluorescence signal component with a sinusoidal reference signal of the same frequency to obtain the summation result of the sinusoidal component;

[0062] The convolution of the fluorescence signal component with a sinusoidal reference signal of the same frequency is calculated as follows:

[0063]

[0064] A1(x,y) represents the accumulated result of the sine components, and f(x,y,i) represents the pixel value of the pixel located at position (x,y) in the i-th image. This represents a sinusoidal reference signal.

[0065] S501: Convolve the fluorescence signal component with a cosine reference signal of the same frequency to obtain the summation result of the cosine component;

[0066] The convolution of the fluorescence signal component with a cosine reference signal of the same frequency is calculated as follows:

[0067]

[0068] A2(x,y) represents the sum of the cosine components, and f(x,y,i) represents the pixel value of the pixel at position (x,y) in the i-th image. The cosine reference signal is represented by (x, y), which represents the spatial coordinates of the pixel.

[0069] S502: Calculate the amplitude value of each pixel's periodic change with the excitation signal based on the sum of the sine and cosine components. The calculation formula for the amplitude value of each pixel's periodic change with the excitation signal is as follows:

[0070]

[0071] Where A1(x,y) represents the accumulated result of the sine component, A2(x,y) represents the accumulated result of the cosine component, and A(x,y) represents the amplitude value of each pixel as the excitation signal changes periodically.

[0072] In the aforementioned phase-locked loop (PLL) process, the orthogonality of trigonometric functions allows for the separation of the intensity of a fixed-wavelength light component with the same frequency as the excitation signal. This shields against temperature drift and thermal noise from electronic components, as well as irregular fluctuations in the measurement data caused by environmental disturbances, thus significantly improving the signal-to-noise ratio (SNR) of the imaging. As the measurement time, i.e., the number of measurement cycles, increases, the SNR can be continuously improved.

[0073] S6: Traversal and Replacement: Parallel calculation is performed on each pixel in each fluorescence image. This involves replacing the original amplitude signal of the corresponding pixel in the fluorescence image with the amplitude value of the pixel after convolution processing, which changes periodically with the excitation signal. This improves the signal-to-noise ratio of the fluorescence imaging, achieving clear fluorescence imaging of aggregation-induced emission dyes at low doses. Finally, the clear fluorescence image is output to the computer and displayed on the computer screen. Please refer to [reference needed]. Figure 4 , Figure 4 The comparison between traditional imaging methods and the method of this invention is clearly displayed, with the dye dosage being 0.01 mg and the excitation light power density being 32 mW / cm². 2 Under the same dose of aggregation-induced emission dye, the structure, morphology, branching and connection of blood vessels in mice are clearer. Therefore, the structural and functional status of blood vessels can be interpreted based on the results of phase-locked imaging.

[0074] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0075] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A low-dose aggregation-induced emission dye bio-imaging method, characterized in that Includes the following steps: Dye injection into the test organism: Select the test organism and inject it with a low dose of aggregation-induced emission dye; Setting and adjusting the excitation source: Select an excitation light source with adjustable frequency to excite the organism being tested; Image acquisition: Fluorescent signals emitted by the tested organism are acquired at equal time intervals; Preprocessing: The fluorescence signal is processed and a fluorescence image is generated; Phase-locked processing: Phase-locked processing is applied to the fluorescence image to separate the fluorescence signal component with the same frequency as the excitation light source and generate a phase-locked imaging result.

2. The low-dose aggregation-induced emission dye bio-imaging method according to claim 1, wherein: The modulation frequency of the excitation light source is 0.025-1000 Hz, and the output intensity of the excitation light source is 1-726 mW / cm 2 .

3. The low-dose aggregation-induced emission dye bio-imaging method according to claim 2, wherein: The camera exposure time is set according to the modulation frequency and output intensity of the excitation light source, and the camera exposure time is 0.2ms to 10s.

4. The low-dose aggregation-induced emission dye bio-imaging method according to claim 1, wherein: The phase-locked loop process includes the following steps: The fluorescence signal component is convolved with a sinusoidal reference signal of the same frequency to obtain the sum of the sinusoidal components. The fluorescence signal component is convolved with a cosine reference signal of the same frequency to obtain the sum of the cosine components. The amplitude of the periodic change of each pixel with the excitation signal is calculated based on the sum of the sine and cosine components.

5. A low dose aggregation-induced emission dye bio-imaging method according to claim 4, wherein: The convolution of the fluorescence signal component with a sinusoidal reference signal of the same frequency is calculated as follows: ； This represents the summation result of the sinusoidal components. Indicates the location in the i-th image The pixel value at the location. This represents a sinusoidal reference signal.

6. The low-dose aggregation-induced emission dye bioimaging method as described in claim 4, characterized in that: The fluorescence signal component is convolved with a cosine reference signal of the same frequency, and the calculation formula is as follows: ； represents a cumulative result of cosine components, represents a pixel value of a pixel point located at position in the i-th image, represents a cosine reference signal.

7. The low dose aggregation-induced emission dye bio-imaging method according to claim 4, wherein: The amplitude value of the periodic change of each pixel with the excitation signal is calculated as follows: ； in This represents the summation result of the sinusoidal components. This represents the summation result of the cosine components. This represents the amplitude value of the periodic change of each pixel as the excitation signal changes.

8. The device for low-dose bio-imaging using the aggregation-induced emission dye according to any one of claims 1 to 7, wherein the device is a bio-imaging device. The device includes An excitation light source generates an excitation signal for measurement to excite the test organism, causing the test organism to produce fluorescence at the same frequency as the excitation light source; A phase-locked module controls the signal modulated by the excitation light source and the image acquisition by the imaging module, so that the imaging module acquires fluorescence images at equal time intervals and applies a phase-locked algorithm to the fluorescence images to obtain the amplitude value of the periodic change of each pixel with the excitation signal. The imaging module captures the fluorescence signal emitted by the tested organism and outputs it to the phase-locked module.

9. The apparatus as claimed in claim 8, characterized in that: The phase-locked module includes a calculation module and a signal generation module, wherein... The calculation module is used to perform phase-locked calculations on the acquired fluorescence images; The signal generation module is used to output a square wave modulated excitation light source, so that the excitation light source periodically modulates the tested organism.

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