Method and platform for characterizing drug sensitivity characteristics of unmarked organoids based on photoacoustic imaging

Abstract

The invention provides an unmarked organoid drug sensitivity characteristic characterization method and platform based on a photoacoustic imaging technology, and aims at solving the defects of a traditional drug sensitivity evaluation method in the aspect of organoid three-dimensional structure evaluation through a non-invasive and unmarked imaging technology. By means of the method, the influence of the medicine on the unmarked organs can be quantified, the acting effects of the medicine at different time points can be dynamically monitored, and therefore more accurate and visual medicine sensitivity characteristic data can be provided. The platform comprises a photoacoustic module, a control and imaging module and a processing and analysis module, the three-dimensional structure change of the organoid can be monitored in real time in an unmarked mode, the drug sensitivity reaction of drugs to the organoid can be quantified, and a reliable experimental platform is provided for precise treatment and personalized medication. The method has the advantages of high efficiency, non-invasiveness, dynamic monitoring, quantitative analysis and the like, provides a more accurate and reliable technical means for drug sensitivity evaluation, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of precision medicine, and specifically relates to a label-free organoid drug sensitivity characterization method and platform based on photoacoustic imaging. Background Art

[0002] As an in vitro three-dimensional model, organoids have shown great potential in drug sensitivity assessment and disease research. They can effectively simulate the genetic characteristics, tissue structure and microenvironment of different organs, providing a more realistic and accurate platform for drug cytotoxicity, efficacy evaluation and impact on the microenvironment. However, existing drug response assessment methods mainly rely on survival rate and two-dimensional growth indicators, which fail to fully reflect the three-dimensional structural changes of organoids, and cannot accurately show the true impact of drugs on the organoid microenvironment and cell structure.

[0003] Existing 3D imaging technologies, such as confocal microscopy and light sheet microscopy, usually require the use of fluorescently labeled samples. This invasive operation may destroy the physiological state of organoids. At the same time, the scanning process is time-consuming and cumbersome, which limits the application of organoids in the evaluation of dynamic drug sensitivity characteristics. Therefore, there is an urgent need for a non-invasive, label-free 3D evaluation method that can comprehensively and accurately reflect the dynamic changes of organoids under the action of drugs, thereby providing a more accurate and intuitive evaluation method for the characterization of drug sensitivity characteristics. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a label-free organoid drug sensitivity characterization method and platform based on photoacoustic imaging. This method uses non-invasive, label-free three-dimensional imaging technology to monitor the structural changes of various organoids in real time and evaluate the effects of drugs, thereby effectively overcoming the invasive operation and sample damage problems that may be caused by traditional labeling technology. By improving the accuracy and efficiency of drug screening, the present invention can provide more reliable data support for precision drug development, and is applicable to various types of organoids, including but not limited to tumor organoids, neural organoids, liver organoids, etc.

[0005] In one aspect of the present invention, a method for characterizing drug sensitivity of label-free organoids based on photoacoustic imaging is provided, the method comprising:

[0006] Step 1: Collect organoid samples to be studied (such as tumor organoids, neural organoids, etc.), perform preliminary processing, and culture the organoids under specific culture conditions;

[0007] Step 2: Isolate the organoids from the culture system and treat them with drugs in preparation for evaluating the effects of different drugs and their combinations;

[0008] Step 3: Use a 3D photoacoustic imaging platform to perform high-resolution imaging of drug-treated organoids, and collect and process 3D image data;

[0009] Step 4: Based on the processed 3D image data, evaluate the effect of drugs on organoids and screen out effective drugs.

[0010] Furthermore, the organoids described in step one are obtained from corresponding samples of patients or animal models. After preliminary processing, the samples enter the organoid culture stage, and the cells in these samples are grown in a three-dimensional culture medium and a miniature organoid model with a certain structure and function is formed under appropriate culture conditions.

[0011] Furthermore, the culture conditions described in step 1 generally require specific culture conditions, such as appropriate culture medium, temperature, pH value, oxygen concentration, etc., to ensure that the organoids can maintain their characteristics and form structures and functions with physiological relevance.

[0012] Furthermore, the drug treatment described in step 2 involves single drug testing, combined use of two or more drugs, and drug treatment at different concentrations. The types of drugs may include chemotherapy drugs, targeted drugs, immunotherapy drugs, hormone therapy drugs, gene editing drugs, etc., covering a wide range of drug categories and treatment methods.

[0013] Furthermore, the three-dimensional photoacoustic imaging platform described in step 3 combines the advantages of optical imaging and ultrasound imaging technology, and can achieve high-resolution, deep three-dimensional imaging without destroying the structure of the organoid. The platform can not only observe the structural changes of the organoid in real time, but also monitor the changes of specific molecules or specific molecules in the organoid without any labeling, such as tumor markers, cell surface receptor expression, and molecular signaling pathways, thereby providing more accurate data support for the characterization of drug sensitivity characteristics.

[0014] Furthermore, the processing of the three-dimensional imaging data described in step 3 includes operations such as image denoising and enhancement to improve imaging accuracy and signal-to-noise ratio. Through image analysis techniques (such as segmentation, volume calculation, morphological analysis, etc.), the growth rate of organoids, morphological changes, and the effects of drugs on the organoid microenvironment can be quantified.

[0015] Furthermore, the drug screening method described in step 4 is not limited to simple volume calculations, but can also be combined with organoid morphological changes, functional performance, and multi-dimensional image analysis to reveal the effect of drugs in inhibiting organoid growth and changing the organoid microenvironment. By combining the changes in specific molecules, the most effective drugs for specific types of organoids can be finally screened out.

[0016] Another aspect of the present invention provides a label-free organoid drug sensitivity characterization platform based on photoacoustic imaging, which is characterized by comprising a photoacoustic module, a control and imaging module, and a processing and analysis module, and is applicable to label-free drug sensitivity testing of various organoids;

[0017] The photoacoustic module includes a laser source and an ultrasonic transducer. The laser source is used to emit laser pulses to excite organoid samples and produce photoacoustic effects. The ultrasonic transducer is used to receive the sound wave signals emitted by the organoids in response to the laser pulses and convert them into electrical signals to provide data support for subsequent image reconstruction.

[0018] The control and imaging module uses an imaging program to precisely control the photoacoustic module to scan along a predetermined trajectory on a displacement platform to collect three-dimensional image data of the organoid. The module supports flexible scanning path selection and can adapt to the morphology and size of different organoid samples. The advanced image reconstruction algorithm used converts acoustic wave data into a visualized three-dimensional or two-dimensional image to help construct a complete structural image of the organoid. The imaging module has the ability to adjust scanning parameters in real time to optimize the imaging effect of the organoid after treatment with different drugs, ensuring the accuracy and reliability of the data;

[0019] The processing and analysis module extracts the three-dimensional information of the organoids and calculates their volume based on the acquired three-dimensional image data, thereby quantifying the effects of drugs on organoids and analyzing their volume, morphology and structural changes. This module not only combines multi-dimensional data analysis in drug screening, but also processes information such as changes in the tumor microenvironment, expression of cell surface receptors, and activity of molecular signaling pathways. Through image analysis techniques (such as segmentation, volume calculation, morphological analysis, etc.), this module can screen out the most effective drug combination and provide valuable evaluation basis for drug screening.

[0020] Furthermore, in the photoacoustic module, the laser beam incident on the sample and the ultrasonic transducer are located on the same axis, and the imaging sample is precisely placed at the intersection of the optical focus of the laser and the acoustic focus of the ultrasonic transducer.

[0021] Furthermore, the laser source can choose to use a single wavelength laser or a combination of multiple wavelength lasers, whose wavelengths include 532nm, 355nm, 750nm and other common spectral bands to meet different experimental needs and adapt to the imaging needs of different types of organoid samples.

[0022] Furthermore, the imaging program is highly customizable and can accurately control the scanning path, instant data acquisition, data processing and real-time image reconstruction according to experimental requirements, dynamically display the three-dimensional information of organoids as well as volume changes and morphological changes during drug screening, and support simultaneous monitoring and analysis of multiple organoids.

[0023] Furthermore, the displacement platform is used to accurately adjust the three-dimensional position of the organoid sample in the imaging area, wherein the sample remains stationary and the laser beam is precisely moved and scanned under the control of a motor drive or a MEMS galvanometer, thereby achieving high-resolution imaging and data acquisition. As an alternative, the displacement platform can also be driven by a motor to accurately drive the three-dimensional position of the organoid sample in the imaging area, ensuring that the sample achieves a high degree of positioning accuracy and excellent scanning effect during the imaging process.

[0024] Furthermore, the volume calculation uses an image processing algorithm to decompose the three-dimensional image data into a large number of pixels (or voxels), quantify the value of each pixel, and sum up the volumes of all pixels, so as to accurately calculate the specific volume of the organoid and compare the changes before and after drug treatment.

[0025] The beneficial effects of the label-free organoid drug sensitivity characterization method and platform based on photoacoustic imaging provided by the present invention are as follows:

[0026] (1) The label-free organoid drug sensitivity characterization method of the present invention has the advantage of quantifying drug effects: this method quantifies the effect of drugs on organoids by measuring the three-dimensional volume changes of organoids under the action of drugs, and intuitively captures the three-dimensional structural and functional changes of organoids, thereby providing more accurate data support for drug sensitivity assessment.

[0027] (2) The label-free organoid drug sensitivity characterization method of the present invention has the advantage of dynamically monitoring the effects of drugs: by real-time monitoring of the three-dimensional structure and volume changes of organoids, the effects of drugs can be dynamically analyzed, providing an accurate basis for drug screening.

[0028] (3) The label-free organoid drug sensitivity characterization platform of the present invention has the advantage of high efficiency: the platform integrates three-dimensional imaging of organoids and drug effect analysis, simplifies the operation process and reduces the steps, effectively shortens the process time, and improves the efficiency of drug screening.

[0029] (4) The label-free organoid drug sensitivity characterization platform of the present invention has the advantages of being label-free and non-invasive: the platform uses three-dimensional photoacoustic imaging technology, which can fully display the structure and dynamic changes of organoids without interfering with their survival and development, ensuring that the natural state of organoids is not affected. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the partial structure of the label-free organoid drug sensitivity characterization platform of the present invention;

[0031] Figure 2 It is a structural block diagram of the label-free organoid drug sensitivity characterization platform of the present invention;

[0032] Figure 3 It is a flow chart of the method for characterizing drug sensitivity of label-free organoids of the present invention;

[0033] Figure 4 This is a comparison diagram of the imaging effect of the label-free organoid drug sensitivity characterization platform of the present invention and the optical microscope imaging effect;

[0034] Figure 5 Two-dimensional and three-dimensional images of images obtained by the label-free organoid drug sensitivity characterization platform of the present invention;

[0035] Figure 6 It is the correlation between the data results of the label-free organoid drug sensitivity characterization platform of the present invention and the cell viability after drug treatment. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0037] Figure 1 It is a partial structural diagram of the label-free organoid drug sensitivity characterization platform of the present invention, which shows the system structure and working principle of the platform. The platform includes key components such as laser beam 1, reflector 2, lens 3, ultrasonic transducer 4, organoid 5, culture dish 6, ultrasonic transducer fixture 7 and sample fixture 8. The laser beam 1 is focused onto the organoid 5 sample through the reflector 2 and lens 3, exciting the sample to generate photoacoustic signals. The ultrasonic transducer 4 receives these signals and converts them into electrical signals for further processing and imaging. The organoid 5 is fixed in the culture dish and stabilized by the sample fixture, and the ultrasonic transducer 4 is precisely positioned by a special fixture. These components work together to ensure that the system can perform high-resolution three-dimensional imaging, providing a reliable experimental platform for drug testing and screening.

[0038] Figure 2 It is a structural block diagram of the label-free organoid drug sensitivity characterization platform of the present invention. The platform is composed of a laser, a lens, a pinhole, an ultrasonic transducer, a motor, an amplifier, a signal generator and a computer. The laser emitted by the laser is focused by a lens and projected onto the organoid sample after filtering through a pinhole. Under the control of the motor, the scanning system can accurately adjust the sample position to ensure that the imaging area covers the entire organoid. The ultrasonic transducer receives the acoustic wave signal caused by the laser pulse and converts it into an electrical signal for subsequent processing. The signal generator and the amplifier work together to ensure the stability and clarity of the signal during transmission. Finally, the computer collects and processes these signals to generate a three-dimensional image, thereby achieving high-resolution imaging of the organoid. The design of the platform ensures efficient and accurate imaging capabilities, providing strong data support for drug screening and related research.

[0039] Figure 3 This is a flow chart of the method for characterizing the drug sensitivity of label-free organoids of the present invention. This method first collects tumor samples and performs preliminary processing, and then cultures the organoids under appropriate culture conditions to ensure that they form a good three-dimensional structure. Next, the organoids are separated from the culture system and treated with drugs to evaluate the effects of different drugs and their combinations. The drug-treated organoids are imaged using three-dimensional photoacoustic imaging technology to obtain detailed three-dimensional data. Subsequently, image reconstruction and analysis technology are used to evaluate the effects of the drugs on the organoids and quantify the drug effects. Finally, the drugs with the strongest inhibitory effects are screened out based on the evaluation results, providing effective experimental support for the development of anti-drugs and personalized treatment.

[0040] Figure 4 This is a comparison chart of the imaging effects of the label-free organoid drug sensitivity characterization platform of the present invention. The left side is an image taken by an optical microscope, and the right side is an image obtained using the platform, showing the imaging effect of tumor organoids in drug screening. Through image analysis, the volume of the tumor organoid in the box was calculated to be 382,657μm 3 , providing reliable quantitative data to support further drug screening and effect evaluation. In addition, the system's lateral resolution is about 7μm, and the axial resolution reaches about 45μm after algorithm correction, reflecting the platform's superior performance in high-resolution three-dimensional imaging.

[0041] Figure 5 These are the two-dimensional and three-dimensional images of the label-free organoid drug sensitivity characterization platform of the present invention. The left side shows the tumor organoid in the imaging area, and the right side shows the three-dimensional reconstruction image of the area, which clearly shows the structure and spatial distribution of the organoid. Through three-dimensional reconstruction, the volume of the organoid in the right image is calculated to be 2,212,401.4μm 3 , providing more accurate spatial quantitative data for subsequent drug screening and evaluation.

[0042] Figure 6 The correlation between the imaging results of this platform and the cell viability after drug treatment is demonstrated. The left figure shows the dynamic volume response of tumor organoids under different drug doses. The middle and right figures show the correlation between the viability of this platform and the traditional method after the action of cisplatin and temozolomide, respectively. Under the action of cisplatin, the correlation coefficient of the platform is r = 0.8627, while the traditional method is r = 0.8190; under the action of temozolomide, the correlation coefficient of the platform is r = 0.9069, and the traditional method is r = 0.7849. These results show that the platform has higher correlation and more accurate drug effect assessment capabilities in drug sensitivity assessment.

[0043] The present invention provides a label-free organoid drug sensitivity characterization platform based on photoacoustic imaging, which uses high-resolution imaging and precise volume calculation to provide strong experimental support for drug screening and anti-tumor research. The platform combines key components such as laser beams, ultrasonic transducers, and culture dishes, and can efficiently collect three-dimensional imaging data of organoids after drug treatment, and perform detailed image analysis and evaluation. In addition, the comparison of this platform with traditional methods verifies its higher correlation in drug effect evaluation, demonstrating its great application potential in drug screening and personalized treatment. The platform not only has high imaging accuracy, but also can realize the dynamic response analysis of drugs to organoids. It is an important tool in drug development, tumor treatment research, and personalized medicine.

[0044] The above-mentioned embodiments are only implementation methods of the present invention and are not intended to limit the scope of the present invention. It should be understood that those skilled in the art may make several modifications and improvements to the present invention without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims

1. A label-free organoid drug sensitivity characterization method based on photoacoustic imaging, characterized in that: The following steps are involved: Step 1: Collect organoid samples to be studied (such as tumor organoids, neural organoids, etc.), perform preliminary processing, and culture the organoids under specific culture conditions; Step 2: Isolate the organoids from the culture system and treat them with drugs in preparation for evaluating the effects of different drugs and their combinations; Step 3: Use a 3D photoacoustic imaging platform to perform high-resolution imaging of drug-treated organoids, and collect and process 3D image data; Step 4: Based on the processed 3D image data, evaluate the effect of drugs on organoids and screen out effective drugs.

2. The method according to claim 1, wherein: The organoid sample described in step 1 comes from a patient or an animal model. After preliminary processing, it is cultured in a three-dimensional culture medium to form a micro-organoid model.

3. The method according to claim 1, wherein: The organoids include but are not limited to tumor organoids, neural organoids, liver organoids and other types of organoids, and are suitable for drug screening and drug effect evaluation in various disease models.

4. The method according to claim 1, wherein: The drug treatment described in step 2 includes single drug testing, combined use of two or more drugs, and drug treatments at different concentrations.

5. The method according to claim 1, wherein: The three-dimensional photoacoustic imaging platform described in step three combines the advantages of optical imaging and ultrasound imaging technology to achieve high-resolution, deep three-dimensional imaging without destroying the organoid structure, and can monitor changes in specific molecules or specific molecules in the organoid.

6. The method according to claim 1, wherein: The processing of the three-dimensional imaging data described in step three includes operations such as image denoising and enhancement, and is further processed through image analysis technology to obtain organoid imaging data.

7. The method according to claim 1, wherein: The imaging data described in step 4 includes quantified volume changes, morphological changes, functional performance, and molecular changes of the organoid, wherein the volume changes also include the calculated volume.

8. The method according to claim 1, wherein the method for evaluating drug effects includes multidimensional data analysis based on organoid volume, morphological changes, functional performance and molecular level changes, revealing the effect of the drug in inhibiting organoid growth and changing the organoid microenvironment.

9. A label-free organoid drug sensitivity characterization platform based on photoacoustic imaging, characterized in that: Includes the following modules: A photoacoustic module, comprising a laser source and an ultrasonic transducer, wherein the laser source is used to emit laser pulses to excite the organoid sample and produce a photoacoustic effect, and the ultrasonic transducer is used to receive the acoustic wave signal emitted by the organoid in response to the laser pulse and convert it into an electrical signal; A control and imaging module, used to control the photoacoustic module to scan along a predetermined trajectory on the displacement platform through an imaging program to collect three-dimensional image data of the organoid; The processing and analysis module is used to extract the three-dimensional information of organoids based on three-dimensional image data, calculate their volume, and analyze the effects of drugs on organoids.

10. The platform according to claim 9, wherein: The laser beam and ultrasound transducer in the photoacoustic module are located on the same axis, and the imaging sample is precisely placed at the intersection of the optical focus of the laser and the acoustic focus of the ultrasound transducer.

11. The platform according to claim 9, wherein: The laser source can be selected to use a single wavelength laser or a multi-wavelength laser, and its wavelength range covers the ultraviolet, visible light and near-infrared bands, including but not limited to 355nm, 532nm, 750nm, 808nm, 980nm, etc., to adapt to different experimental needs and the characteristics of organoid samples.

12. The platform according to claim 9, wherein: The imaging program can accurately control the scanning path, data acquisition, data processing and real-time image reconstruction according to experimental requirements, and supports simultaneous monitoring and analysis of multiple organ types.

13. The platform according to claim 9, wherein: The displacement platform is used to accurately adjust the three-dimensional position of the organoid sample in the imaging area. The sample remains stationary, and the laser beam is precisely moved and scanned under the control of a motor or a MEMS galvanometer to achieve high-resolution imaging and data acquisition. As an alternative, the displacement platform can also be driven by a motor to drive the organoid sample to adjust its three-dimensional position in the imaging area, while the laser beam and ultrasonic transducer remain stationary, ensuring a high degree of positioning accuracy and excellent scanning effect during the imaging process.

14. The platform according to claim 9, wherein: The volume calculation uses an image processing algorithm to quantify the value of each pixel and sum up the volume of all pixels, so as to accurately calculate the specific volume of the organoid and compare the changes before and after drug treatment.

15. The platform according to claim 9, wherein: The processing and analysis module is based on image processing algorithms and combines multi-dimensional data such as organoid volume, morphological analysis, and molecular signaling pathways to analyze the changes in the organoid microenvironment caused by drugs and quantify the drug effects.

16. The platform according to claim 9, wherein: The photoacoustic module, control and imaging module, and processing and analysis module can be used in combination to realize a complete process for characterizing the drug sensitivity characteristics of label-free organoids, and provide a more efficient and accurate organoid structure and function evaluation solution.

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