A method for detecting drug sensitivity of organoids based on time sequence fluorescence imaging and fluorescence quenching correction
By correcting the fluorescence quenching in organoid drug sensitivity testing, the problem of signal interference during long-term imaging is solved, achieving highly accurate and repeatable drug sensitivity testing, which is suitable for clinical precision medicine and new drug screening.
Patent Information
- Application Number
- CN202610653260.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-13
- Publication Date
- 2026-06-26
Smart Images
Figure CN122282733A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of organoid in vitro drug efficacy evaluation technology, and more specifically to an organoid drug sensitivity detection and fluorescence quenching correction method based on time-series fluorescence imaging. Background Technology
[0002] Organoids, as three-dimensional cell aggregate models in vitro, can highly replicate the physiological structure, cellular heterogeneity, and drug response characteristics of in vivo tissues. Compared to traditional two-dimensional monolayer cell culture, their accuracy in predicting drug efficacy is significantly improved, making them a core in vitro screening vector for precision oncology and new drug development. Live-cell long-term timelapse imaging analyzers can continuously and dynamically track organoid growth, apoptosis, and morphological changes, simultaneously acquiring bright-field and fluorescence channel data. This overcomes the shortcomings of traditional endpoint methods in reflecting dynamic drug efficacy and is currently the mainstream equipment for organoid functional analysis. Currently, Calcein-AM / PI (AM / PI) dual-color fluorescence staining is widely used for organoid drug sensitivity testing as the core method for distinguishing between live and dead cells. AM dye is only hydrolyzed by esterases in live cells, emitting green fluorescence, while PI dye only penetrates the cell membrane of dead cells and binds to DNA, emitting red fluorescence. Both methods are highly specific and easy to operate, achieving quantitative analysis of three-dimensional structural live / dead signals without digesting the organoid, making them the preferred staining scheme for long-term drug sensitivity screening of organoids.
[0003] However, during long-term time-series imaging, the AM / PI fluorophores undergo irreversible photobleaching (fluorescence quenching) due to repeated excitation, resulting in a continuous decrease in fluorescence intensity over time. Furthermore, there is a significant difference in the quenching rates between Calcein-AM (green channel) and PI (red channel). This non-biological attenuation signal severely interferes with the accuracy of the live / dead fluorescence ratio. Directly using raw fluorescence data to calculate cell viability and fit IC50 / EC50 inevitably leads to distorted drug efficacy results, concentration-effect curve shifts, and excessive errors in drug sensitivity assessment. Existing literature and experimental data confirm that conventional AM / PI staining only ensures fluorescence stability for 1-2 hours. After multiple excitations over long time (12-72 hours), the quenching amplitude of green live cell fluorescence can reach 30%-60%, while red dead cell fluorescence is simultaneously quenched but at different rates, further amplifying the live / dead signal misalignment problem and severely reducing detection accuracy and repeatability. To address the fluorescence quenching problem, existing correction methods are mostly adapted to two-dimensional single-layer cell imaging, using only a single exponential model for coarse global compensation. They do not specifically optimize for the differences in light penetration of 3D organoid structures and the differences in quenching rates of AM / PI dual-color fluorescence. Moreover, existing technologies do not consider the differences in light penetration between the outer and inner layers of 3D organoid cells, resulting in insufficient internal fluorescence quenching correction. Furthermore, the different quenching rates of AM / PI dual-color fluorescent groups can cause signal misalignment between living and dead cells when uniformly corrected with a single color, further amplifying errors in drug efficacy calculation. In addition, existing technologies lack a standardized control system and a closed-loop process adapted to organoid drug sensitivity testing. They do not deeply integrate quenching correction with long-term dynamic drug efficacy analysis and cannot connect to time-series imaging data streams for automated calculation. This results in low correction accuracy, cumbersome operation, and poor data reliability, hindering the large-scale application of organoids in the field of precision medicine.
[0004] Therefore, how to correct the fluorescence signal in organoid time-series fluorescence imaging drug sensitivity detection to improve the accuracy of organoid drug sensitivity detection is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0005] In view of this, the present invention provides a method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging to solve the above problems.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention discloses a method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging, characterized by comprising: Step 1: Seed the three-dimensional organoids into a low-adsorption imaging plate and group them; Step 2: Perform AM / PI dual-color staining and long-term time-series imaging to acquire images and fluorescence intensity data at each time point; Step 3: Preprocess the image and fluorescence intensity data to obtain net fluorescence intensity data; Step 4: Based on the net fluorescence intensity data of the quenching control group, the quenching coefficient is fitted to obtain the quenching rate coefficients of the AM green channel and the PI red channel. Step 5: Using the quenching rate coefficients of the AM green channel and PI red channel, the net fluorescence intensity data of the drug-treated group is reverse-corrected to obtain the real biological fluorescence signal; Step 6: Calculate drug sensitivity parameters based on the actual biological fluorescence signal.
[0007] Furthermore, step 1 specifically includes: Mature and stable three-dimensional organoids were seeded into low-adsorption imaging plates, with uniform number and volume of organoids per well. A drug administration group, a quenching control group, a blank control group, and a negative control group were set up. The drug administration group was set up with 4-12 serial dilution drug concentration gradients. The quenching control group was seeded with an equal amount of organoids and AM / PI staining solution, without adding the test drug, and the culture and imaging parameters were completely consistent with those of the drug administration group.
[0008] Furthermore, step 2 specifically includes: Add AM / PI dual-color staining working solution to each well. The volume ratio of AM to PI working solution in the staining solution is 1:1, corresponding to final concentrations of 2 μM and 4 μM, respectively. Alternatively, for long-term culture scenarios, when using a low-toxicity configuration, the corresponding final concentrations of AM / PI are 5 μM and 10 μM, respectively. A long-term organoid imaging analyzer was used, with bright-field, AM green fluorescence, and PI red fluorescence channels simultaneously activated. The excitation light intensity was set to 10%-20%, and images and fluorescence intensity data at each time point were obtained for each well.
[0009] Furthermore, the imaging of each hole adopts either a first imaging scheme or a second imaging scheme; The first imaging scheme is as follows: the focal plane is focused on the middle layer of the organoid, and the imaging is carried out at fixed intervals of 1-4 hours, with a total imaging time of 12-72 hours; The second imaging scheme is as follows: use the Z-axis overlay function to capture all layers containing organoids, and use the large image stitching function to capture the entire matrix gel. Finally, synthesize a panoramic organoid image of the entire matrix gel. The images are captured at fixed intervals of 2-4 hours, with a total imaging time of 12-72 hours.
[0010] Furthermore, step 3 specifically includes: The original fluorescence intensity of each well and channel was subtracted from the background fluorescence of the corresponding channel in the blank control group, and outlier data in duplicate wells were removed using the 3σ rule to obtain the net fluorescence intensity data.
[0011] Furthermore, step 4 specifically includes: The time-series net fluorescence intensity data of the quenched control group were extracted. Based on the single exponential decay model I(t)=I0×exp(-k×t), the nonlinear least squares method was used to fit the quenching coefficients of the AM green channel and the PI red channel, respectively. Wherein, I(t) represents the fluorescence intensity value at time t; I0 is the initial fluorescence intensity value (the value at t=0); k is the exponential decay constant, which determines the decay rate, and k>0; t represents the fluorescence acquisition time.
[0012] Furthermore, the formula for reverse correction in step 5 is: I_AM_corrected(t)=I_AM_measured(t)×exp(k_AM×t); I_PI_corrected(t)=I_PI_measured(t)×exp(k_PI×t); Where I_AM_corrected(t) and I_PI_corrected(t) are the true biological fluorescence intensities of the AM green channel and PI red channel after correction at time t, respectively; I_AM_measured(t) and I_PI_measured(t) are the measured net fluorescence intensities of the AM green channel and PI red channel at time t, respectively; k_AM and k_PI are the quenching rate coefficients of the fitted AM and PI channels, respectively.
[0013] Furthermore, step 6 specifically includes: Based on the corrected fluorescence intensity data, the dead / live cell rate and drug inhibition rate were calculated. The formula for calculating the dead cell rate is: Dead_rate(t)=[I_PI_corrected(t) / (I_AM_corrected(t)+I_PI_corrected(t))]×100%; The formula for calculating the viable cell rate is: Live_rate(t)=[I_AM_corrected(t) / (I_AM_corrected(t)+I_PI_corrected(t))]×100%; The time point with the most significant effect was selected for normalization. 0 μM or the blank control group was considered as 0% inhibition, and the maximum effect group as 100% inhibition. The normalization formula is as follows: Inhibition(c)=(1-Live_rate_treatment(c) / Live_rate_control)×100%; Normalized_inhibition(c)=(Inhibition(c)-Bottom) / (Top-Bottom)×100%; Where Inhibition(c) is the drug inhibition rate, Normalized_inhibition(c) is the normalized drug inhibition rate, Live_rate_treatment(c) is the viable cell rate at the drug concentration c, Inhibition(c) and Live_rate_control are the viable cell rates of the negative control group, Top is the maximum inhibition rate, and Bottom is the minimum inhibition rate. A four-parameter logistic model was used to fit the concentration-response curve to obtain the IC50 / EC50 values. The model formula is as follows: y=Bottom+(Top-Bottom) / (1+10^((logIC50-x)×Hillslope)); Where x is the logarithm of the drug concentration, y is the normalized inhibition rate, and Hillslope is the curve slope coefficient.
[0014] As can be seen from the above technical solution, compared with the prior art, the present invention provides a method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging, which has the following beneficial effects: (1) Targeted elimination of quenching interference in 3D organoid dual-color fluorescence: Dual-channel independent calibration distinguishes between quenching attenuation and real biological changes. Compared with the traditional uncalibrated / monochrome global calibration method, the IC50 / EC50 calculation deviation of the method of this invention is ≤8%, the CV value of duplicate detection is <5%, the fitted concentration-effect curve R² is ≥0.95, the fluorescence curve of the negative control well tends to be stable after calibration, there is no signal shift caused by quenching, and the detection accuracy and repeatability are significantly improved. (2) Adapted to 3D organoid characteristics: The exclusive control system and calibration model fit the three-dimensional structure of organoids, taking into account the difference in light penetration between outer and inner cells. Unlike the traditional two-dimensional cell calibration scheme, the detection results are closer to the in vivo drug efficacy. (3) Full-process automation: Standardized link connects to long-term imagers, eliminating the need for manual calibration, adapting to high-throughput screening, and providing easy operation and high data reliability. (4) Strict quality control: Clearly defined quenching coefficient range, goodness of fit, correction upper limit and other multiple quality control thresholds, parameter specifications are repeatable, suitable for large-scale application of clinical precision medicine and new drug screening.
[0015] In summary, compared with existing two-dimensional adaptation monochromatic correction methods, the method of this invention can reduce the IC50 calculation deviation of long-term detection from 22.5% to less than 8%, improve the repeatability of duplicate apertures by more than 3 times, effectively solve the problem of distortion of drug efficacy results caused by fluorescence quenching in long-term imaging, and provide reliable technical support for the standardization and industrialization of organoid drug sensitivity testing. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0017] Figure 1 This is a schematic diagram of the overall process of the present invention.
[0018] Figure 2 This is a schematic diagram of the AM / PI fluorescence panorama (large image stitched together - before drug administration) of human non-small cell lung cancer organoid samples provided in an embodiment of the present invention.
[0019] Figure 3 This is a schematic diagram of the AM / PI fluorescence panorama (large image stitched together - after drug administration) of human non-small cell lung cancer organoid samples provided in an embodiment of the present invention.
[0020] Figure 4 This is a schematic diagram of the cell death rate curves before and after correction for cisplatin susceptibility testing in human non-small cell lung cancer organoids, provided as an embodiment of the present invention.
[0021] Figure 5 This is a schematic diagram of the IC50 values before and after correction in the cisplatin sensitivity test for human non-small cell lung cancer organoids, provided as an embodiment of the present invention.
[0022] Figure 6 This is a schematic diagram of the bright field (middle layer) of human colorectal cancer organoids provided in an embodiment of the present invention.
[0023] Figure 7 This is a schematic diagram of AM / PI fluorescence (middle layer of organoid - before drug administration) of human colorectal cancer organoids provided in an embodiment of the present invention.
[0024] Figure 8 This is a schematic diagram of AM / PI fluorescence (middle layer of organoid - after drug administration) of human colorectal cancer organoids provided in an embodiment of the present invention.
[0025] Figure 9 This is a schematic diagram of the cell death rate curve in the cetuximab drug sensitivity test for human colorectal cancer organoids provided in an embodiment of the present invention.
[0026] Figure 10 This is a schematic diagram of the IC50 values of cetuximab in human colorectal cancer organoids before and after correction, provided in an embodiment of the present invention. Detailed Implementation
[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0028] This invention discloses a method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging, such as... Figure 1 As shown, it includes: Step 1: Seed the three-dimensional organoids into a low-adsorption imaging plate and group them; Step 2: Perform AM / PI dual-color staining and long-term time-series imaging to acquire images and fluorescence intensity data at each time point; Step 3: Preprocess the image and fluorescence intensity data to obtain net fluorescence intensity data; Step 4: Based on the net fluorescence intensity data of the quenching control group, the quenching coefficient is fitted to obtain the quenching rate coefficients of the AM green channel and the PI red channel. Step 5: Using the quenching rate coefficients of the AM green channel and PI red channel, the time-series net fluorescence intensity data of the drug-dosing group is reverse-corrected to obtain the real biological fluorescence signal; Step 6: Calculate drug susceptibility parameters based on real biological fluorescence signals.
[0029] In one specific embodiment, step 1 specifically includes: Mature and stable three-dimensional organoids were seeded into low-adsorption imaging plates, with uniform number and volume of organoids per well. A drug treatment group, a three-dimensional organoid-specific quenching control group, a blank control group, and a negative control group were set up, with ≥3 replicates per group. The mean was used for fitting, and outliers were removed, leaving ≥2 valid replicates. In the drug treatment group, three-dimensional organoids, AM / PI staining solution, and the test drug were added to the culture medium, with 4-12 serial dilutions of the drug concentration. The quenching control group was seeded with the same amount of organoids and AM / PI staining solution as the drug treatment group, but without the test drug, and the culture and imaging parameters were identical to those of the drug treatment group. The blank control group only had AM / PI staining solution added to the culture medium. The negative control group only had organoids added to the culture medium.
[0030] In one specific embodiment, step 2 specifically includes: Add AM / PI dual-color staining working solution to each well. The volume ratio of AM to PI working solution in the staining solution is 1:1, corresponding to final concentrations of 2μM and 4μM, respectively. Alternatively, for long-term culture scenarios, when using a low-toxicity configuration, the corresponding final concentrations of AM / PI are 5μM and 10μM, respectively. Incubate at 37℃ in the dark for 25-35 minutes. A long-term organoid imaging analyzer was used, with bright-field, AM green fluorescence, and PI red fluorescence channels simultaneously activated. The excitation light intensity was set to 10%-20%, and images and fluorescence intensity data at each time point were obtained for each well.
[0031] In one specific embodiment, either a first shooting scheme or a second shooting scheme is used to capture images of each hole; The first imaging plan is to focus the focal plane on the middle layer of the organoid and take pictures at fixed intervals of 1-4 hours, with a total imaging time of 12-72 hours. The second imaging scheme is as follows: use the Z-axis overlay function to capture all layers containing organoids, and use the large image stitching function to capture the entire matrix gel. Finally, synthesize a panoramic organoid image of the entire matrix gel. The images are captured at fixed intervals of 2-4 hours, with a total imaging time of 12-72 hours.
[0032] Both schemes involved constant temperature and humidity culture throughout the process, with automatic acquisition of images and fluorescence intensity data at each time point. Experimental verification showed that regardless of whether Z-axis overlay and large image stitching functions were used, this method could achieve effective quenching correction, with only slight differences in the corresponding quenching coefficients. For long-term organoid imaging analyzers with Z-axis overlay and large image stitching functions, it is recommended to use the second imaging scheme, which yields panoramic images and panoramic data of all organoids in the matrix gel, resulting in more reliable results.
[0033] In one specific embodiment, step 3 specifically includes: The original fluorescence intensity of each well and channel was subtracted from the background fluorescence of the corresponding channel of the blank control group. The 3σ rule was used to remove outlier data from the duplicate wells. A single fluorescence value that deviated from the mean of the duplicate wells by 3 times the standard deviation was identified as an outlier and removed. This was done to avoid focusing failure and interference from speckles, and to ensure the accuracy of subsequent fitting, so as to obtain the net fluorescence intensity data.
[0034] In one specific embodiment, step 4 specifically includes: Net fluorescence intensity data of the quenched control group were extracted over time. Based on the single exponential decay model I(t) = I0 × exp(-k × t), the nonlinear least squares method optimized by Levenberg-Marquardt was used to fit the quenching coefficient k_AM or kgreen for the AM green channel and the quenching coefficient k_PI or kred for the PI red channel. Here, I(t) represents the fluorescence intensity value at time t; I0 is the initial fluorescence intensity value (value at t=0); k is the exponential decay constant, determining the decay rate, and k>0; t represents the fluorescence acquisition time; the AM channel range adapted for 3D organoids is 0.010–0.035 h. -1 The quenching coefficient of the PI red channel ranges from 0.005 to 0.050h. -1 Exceeding this range indicates abnormal excitation light parameters, requiring readjustment of imaging parameters; constrain the fitting parameters to achieve a goodness-of-fit R0. 2 A goodness of fit of ≥0.95 is used as the criterion for a valid fit. If the goodness of fit is lower than this threshold, it indicates that there is an anomaly in the imaging data and the experiment needs to be repeated.
[0035] In a specific embodiment, the formula for reverse correction in step 5 is: I_AM_corrected(t)=I_AM_measured(t)×exp(k_AM×t); I_PI_corrected(t)=I_PI_measured(t)×exp(k_PI×t); Wherein, I_AM_corrected(t) and I_PI_corrected(t) represent the true biological fluorescence intensities of the AM green channel and PI red channel at time t, respectively; I_AM_measured(t) and I_PI_measured(t) represent the measured net fluorescence intensities of the AM green channel and PI red channel at time t, respectively; and k_AM and k_PI represent the fitted quenching rate coefficients of the AM and PI channels, respectively. Through multiple experiments, setting the upper limit of the corrected fluorescence intensity to 120% of the initial fluorescence avoids false positives caused by over-correction and is suitable for over 95% of tumor organoid drug sensitivity testing scenarios.
[0036] In one specific embodiment, step 6 specifically includes: Based on the corrected fluorescence intensity data, the dead / live cell rate and drug inhibition rate were calculated. The formula for calculating the dead cell rate is: Dead_rate(t)=[I_PI_corrected(t) / (I_AM_corrected(t)+I_PI_corrected(t))]×100%; The formula for calculating the viable cell rate is: Live_rate(t)=[I_AM_corrected(t) / (I_AM_corrected(t)+I_PI_corrected(t))]×100%; The time point with the most significant effect was selected for normalization. 0 μM or the blank control group was used as 0% inhibition (baseline), and the maximum effect group was used as 100% inhibition (maximum effect). The normalization formula is as follows: Inhibition(c)=(1-Live_rate_treatment(c) / Live_rate_control)×100%; Normalized_inhibition(c)=(Inhibition(c)-Bottom) / (Top-Bottom)×100%; Where Inhibition(c) is the drug inhibition rate, Normalized_inhibition(c) is the normalized drug inhibition rate, Live_rate_treatment(c) is the viable cell rate at the drug concentration c, Inhibition(c) and Live_rate_control are the viable cell rates of the negative control group, Top is the maximum inhibition rate, and Bottom is the minimum inhibition rate. A four-parameter logistic (4PL) model was used to fit the concentration-response curve to obtain the IC50 / EC50 values. The model formula is as follows: y=Bottom+(Top-Bottom) / (1+10^((logIC50-x)×Hillslope)); Where x is the logarithm of the drug concentration, y is the normalized inhibition rate, and Hillslope is the curve slope coefficient; in this invention, Bottom=0 (minimum inhibition rate) and Top=100 (maximum inhibition rate) are constrained to ensure the standardization of pharmacodynamic accounting.
[0037] In one specific embodiment, the method of the present invention is illustrated by specific experiments.
[0038] Experiment 1: Cisplatin Susceptibility Testing of Organoids from Non-Small Cell Lung Cancer
[0039] 1. Experimental materials: Human non-small cell lung cancer (NSCLC) organoids (in logarithmic growth phase, uniform in size (10-500μm in diameter), without fragmentation or necrosis, and with regular morphology), low-adsorption 96-well imaging plates, AM / PI dual-color staining solution, cisplatin, and CELLImage 3D long-term organoid imaging analyzer.
[0040] 2. Experimental steps: (1) Add 100 μL of organoid suspension to each well, and control the number of organoids in each well to about 80 to ensure uniformity between wells. Set up 8 cisplatin concentration gradients (0.01 μM-100 μM), and simultaneously set up quenching control group, blank control and negative control. Each group has 3 replicates. (2) Prepare low-toxicity AM / PI staining working solution with final concentrations of 5 μM and 10 μM respectively. Incubate at 37°C in the dark for 30 min. Complete AM / PI staining according to the standard. Set the imaging instrument to a 2-hour shooting interval and excitation light intensity of 15%. Use the large image stitching mode and focus the focal plane on the middle layer of the organoid to obtain a panoramic image of the organoid. The total duration is 20 hours. Collect three-channel data simultaneously. (3) After subtracting background fluorescence, outliers were removed using the 3σ rule, and the AM channel quenching coefficient k_AM = 0.021h was obtained by fitting. -1 The quenching coefficient of the PI channel is k_PI = 0.012h. -1 Dual-channel goodness of fit R 2 All values are ≥0.95, and the CV value of the duplicate well is 3.8%. (4) The fluorescence signal was corrected frame by frame according to the dual-channel correction formula. The fluorescence intensity after correction was controlled to not exceed 120% of the initial value. The drug killing rate was calculated. The IC50 of cisplatin was obtained by fitting the four-parameter logistic model, which was 11.2 μM. The fitting curve R 2 =0.982; (5) Parallel control experiment: Traditional uncorrected group cisplatin IC50 = 8.7 μM, duplicate well CV value 15.2%, curve R 2 =0.931; the deviation of the method of the present invention is 3.8%, and the detection advantage is significant.
[0041] 3. Experimental results: such as Figure 2-5 As shown, the concentration-effect curves are smooth after fluorescence quenching correction, with no quenching shift, and the IC50 results are reproducible, which can be used as a reference for clinical drug use.
[0042] Efficacy evaluation of cetuximab in organoids from two patients with colorectal cancer
[0043] 1. Experimental materials: Human colorectal cancer organoids: those in the logarithmic growth phase, uniform in size (diameter 100-420μm, without fragmentation or necrosis, and with regular morphology), AM / PI staining solution, cetuximab, and long-term organoid imaging analyzer.
[0044] 2. Experimental steps: (1) Add 100 μL of organoid suspension to each well, and control the number of organoids in each well to about 100. Strictly ensure the uniformity of number and size between wells. Set up 8 cetuximab concentration gradients (0.1 μM-160 μM), and simultaneously set up quenching control group, blank control and negative control. Each group has 3 replicates. (2) Prepare low-toxicity AM / PI staining working solution with final concentrations of 5 μM and 10 μM respectively. Incubate at 37°C in the dark for 30 min. Complete AM / PI staining according to the standard. Set the imaging instrument to a 2-hour shooting interval, excitation light intensity of 18%, focus the focal plane on the middle layer of organoids, and the total imaging time is 20 hours. Collect time-series fluorescence data. (3) After subtracting background fluorescence, outliers were removed using the 3σ rule, and the AM channel quenching coefficient k_AM = 0.024h was obtained by fitting. -1 The quenching coefficient of the PI channel is k_PI = 0.015h. -1 Dual-channel goodness of fit R 2 All values were ≥0.95, with a replicate CV value of 5.7%. The fluorescence intensity of dead and live cells was corrected using the formula, the dead cell rate was calculated, and the IC50 of cetuximab was fitted to be 3.08 μg / mL. The curve R... 2 =0.978; (4) Parallel control: The conventional uncorrected group of cetuximab IC50 = 2.35 μg / mL, the replicate CV value was 32%, and the curve R² = 0.94; the method of the present invention had a deviation of 5.7%, effectively eliminating quenching interference.
[0045] 3. Experimental results: such as Figure 6-10 As shown, the data after fluorescence quenching correction more accurately reflects the drug's apoptosis-promoting effect, and the data can be directly used for efficacy reports, meeting the needs of precise clinical drug use.
[0046] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.
[0047] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging, characterized in that, include: Step 1: Seed the three-dimensional organoids into a low-adsorption imaging plate and group them; Step 2: Perform AM / PI dual-color staining and long-term time-series imaging to acquire images and fluorescence intensity data at each time point; Step 3: Preprocess the image and fluorescence intensity data to obtain net fluorescence intensity data; Step 4: Based on the net fluorescence intensity data of the quenching control group, the quenching coefficient is fitted to obtain the quenching rate coefficients of the AM green channel and the PI red channel. Step 5: Using the quenching rate coefficients of the AM green channel and PI red channel, the net fluorescence intensity data of the drug-treated group is reverse-corrected to obtain the real biological fluorescence signal; Step 6: Calculate drug sensitivity parameters based on the actual biological fluorescence signal.
2. The method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging according to claim 1, characterized in that, Step 1 specifically includes: Mature and stable three-dimensional organoids were seeded into low-adsorption imaging plates, with uniform number and volume of organoids per well. A drug administration group, a quenching control group, a blank control group, and a negative control group were set up. The drug administration group was set up with 4-12 serial dilution drug concentration gradients. The quenching control group was seeded with an equal amount of organoids and AM / PI staining solution, without adding the test drug, and the culture and imaging parameters were completely consistent with those of the drug administration group.
3. The method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging according to claim 1, characterized in that, Step 2 specifically includes: Add AM / PI dual-color staining working solution to each well. The volume ratio of AM to PI working solution in the staining solution is 1:1, corresponding to final concentrations of 2 μM and 4 μM, respectively. Alternatively, for long-term culture scenarios, when using a low-toxicity configuration, the corresponding final concentrations of AM / PI are 5 μM and 10 μM, respectively. A long-term organoid imaging analyzer was used, with bright-field, AM green fluorescence, and PI red fluorescence channels simultaneously activated. The excitation light intensity was set to 10%-20%, and images and fluorescence intensity data at each time point were obtained for each well.
4. The method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging according to claim 3, characterized in that, The imaging of each hole adopts either the first imaging scheme or the second imaging scheme; The first imaging scheme is as follows: the focal plane is focused on the middle layer of the organoid, and the imaging is carried out at fixed intervals of 1-4 hours, with a total imaging time of 12-72 hours; The second imaging scheme is as follows: use the Z-axis overlay function to capture all layers containing organoids, and use the large image stitching function to capture the entire matrix gel. Finally, synthesize a panoramic organoid image of the entire matrix gel. The images are captured at fixed intervals of 2-4 hours, with a total imaging time of 12-72 hours.
5. The method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging according to claim 1, characterized in that, Step 3 specifically includes: The original fluorescence intensity of each well and channel was subtracted from the background fluorescence of the corresponding channel in the blank control group, and outlier data in duplicate wells were removed using the 3σ rule to obtain the net fluorescence intensity data.
6. The method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging according to claim 1, characterized in that, Step 4 specifically includes: The time-series net fluorescence intensity data of the quenched control group were extracted. Based on the single exponential decay model I(t)=I0×exp(-k×t), the nonlinear least squares method was used to fit the quenching coefficients of the AM green channel and the PI red channel, respectively. Where I(t) represents the fluorescence intensity value at time t; I0 is the initial fluorescence intensity value; k is the exponential decay constant, k>0; and t represents the fluorescence acquisition time.
7. The method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging according to claim 1, characterized in that, The formula for reverse correction in step 5 is: I_AM_corrected(t)=I_AM_measured(t)×exp(k_AM×t); I_PI_corrected(t)=I_PI_measured(t)×exp(k_PI×t); Where I_AM_corrected(t) and I_PI_corrected(t) are the true biological fluorescence intensities of the AM green channel and PI red channel after correction at time t, respectively; I_AM_measured(t) and I_PI_measured(t) are the measured net fluorescence intensities of the AM green channel and PI red channel at time t, respectively; k_AM and k_PI are the quenching rate coefficients of the fitted AM and PI channels, respectively.
8. The method for organoid drug sensitivity detection and fluorescence quenching correction based on time-series fluorescence imaging according to claim 7, characterized in that, Step 6 specifically includes: Based on the corrected fluorescence intensity data, the dead / live cell rate and drug inhibition rate were calculated. The formula for calculating the dead cell rate is: Dead_rate(t)=[I_PI_corrected(t) / (I_AM_corrected(t)+I_PI_corrected(t))]×100%; The formula for calculating the viable cell rate is: Live_rate(t)=[I_AM_corrected(t) / (I_AM_corrected(t)+I_PI_corrected(t))]×100%; The time point with the most significant effect was selected for normalization. 0 μM or the blank control group was considered as 0% inhibition, and the maximum effect group as 100% inhibition. The normalization formula is as follows: Inhibition(c)=(1-Live_rate_treatment(c) / Live_rate_control)×100%; Normalized_inhibition(c)=(Inhibition(c)-Bottom) / (Top-Bottom)×100%; Where Inhibition(c) is the drug inhibition rate, Normalized_inhibition(c) is the normalized drug inhibition rate, Live_rate_treatment(c) is the viable cell rate at the drug concentration c, Inhibition(c) and Live_rate_control are the viable cell rates of the negative control group, Top is the maximum inhibition rate, and Bottom is the minimum inhibition rate. A four-parameter logistic model was used to fit the concentration-response curve to obtain the IC50 / EC50 values. The model formula is as follows: y=Bottom+(Top-Bottom) / (1+10^((logIC50-x)×Hillslope)); Where x is the logarithm of the drug concentration, y is the normalized inhibition rate, and Hillslope is the curve slope coefficient.