An algal experimental model for high-content imaging and construction method and application thereof

Abstract

The application discloses an algal experimental model for high-content imaging and a construction method and application thereof, relates to the technical field of environmental monitoring and biological imaging, and comprises the following steps: taking single-cell algae as an object, after gradient stress culture of heavy metals, using polyethylene glycol for fixation, using Hoechst 33342 for nuclear staining, and after resuspension, obtaining the algal experimental model, which is used for high-content imaging analysis; the algal experimental model for high-content imaging and the construction method and application thereof ensure that the morphology of the algal cells is well fixed, the nuclear fluorescence signal is strong and uniform when the algal cells are imaged, and the quality of a single image and the consistency of images between different fields of view and different experimental holes in a multi-well plate are significantly improved; the algal experimental model can be perfectly matched with the automatic sampling, focusing and multi-field scanning functions of a high-content analysis system, and high-throughput imaging of a large number of samples is realized.

Description

Technical Field

[0001] This invention relates to environmental monitoring and bioimaging technologies, specifically to an algal experimental model for high-content imaging, its construction method, and its application. Background Technology

[0002] High-content imaging (HCI) is an interdisciplinary technology integrating microscopic imaging, automated control, and intelligent analysis. Through multi-parameter, high-throughput cell imaging and analysis, it simultaneously captures diverse information such as cell morphology, molecular localization, and signaling pathway activity while maintaining the integrity of cell / tissue structure and function. Its core consists of three parts: a microscopic imaging system (such as wide-field, confocal, quantitative phase imaging (QPI)), an automated platform (multi-well plate scanning, environmental control), and image analysis software (such as MetaXpress and Harmony), supporting multi-scale observation from single cells to 3D organoids.

[0003] High-content imaging technology has been widely used in cell biology and toxicology research, but its traditional applications are mostly concentrated in animal cell or tissue culture systems, and rarely involve algae with cell wall structures.

[0004] Algae, as important indicator organisms for environmental monitoring, have significant research value in understanding their morphological and physiological responses to heavy metal stress. However, current technologies lack systematic methods for constructing imaging models tailored to the structural characteristics of algae, leading to problems such as unstable cell structures, uneven signal intensity, and poor consistency in imaging across apertures and batches during automated imaging processes.

[0005] Therefore, there is an urgent need to develop a method for constructing algal experimental models suitable for high-content imaging, so as to achieve stable, reproducible, and automated imaging analysis of algal cells under heavy metal stress. Summary of the Invention

[0006] The purpose of this invention is to provide an algal experimental model for high-content imaging, its construction method and application, to solve problems such as unstable cell structure, uneven signal intensity, and poor consistency of imaging across holes and batches in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for constructing an algal experimental model for high-content imaging, comprising the following steps:

[0008] S1. Single-celled algae were selected as experimental subjects and cultured in a culture medium until the exponential growth phase to obtain Chlorella culture medium.

[0009] S2. Pre-treat the Chlorella culture medium to obtain an algal suspension, then add heavy metal stress factor to the algal suspension and set up a stress culture system with at least two concentration gradients.

[0010] S3. Cultivate the stress culture system and take samples during the cultivation process;

[0011] S4. Fix the sampled algal cells overnight at 4°C using 4% paraformaldehyde solution. After fixation, wash the cells and incubate them overnight at 4°C in the dark using Hoechst 33342 dye for nuclear staining. After staining, wash the cells to remove unbound dye and obtain the stained algal cells.

[0012] S5. The stained algal cells were resuspended in buffer solution and homogenized to obtain an algal experimental model for high-content imaging.

[0013] Furthermore, the single-celled algae mentioned in S1 is Chlorella; the culture temperature mentioned in S1 is 22-28℃.

[0014] Furthermore, the light conditions for cultivation described in S1 consist of a 14-hour light cycle followed by a 10-hour darkness cycle; the light intensity for cultivation described in S1 is under natural light conditions, with a maximum photosynthetically active photon flux density of 3800 µE·m. -2 ·s -1 .

[0015] Furthermore, the heavy metal stress factor mentioned in S2 is selected from at least one of copper, nickel, and zinc.

[0016] Furthermore, the concentration gradient described in S2 is 2 mg / L, 10 mg / L, and 20 mg / L.

[0017] Furthermore, the sampling time points mentioned in S3 are the 3rd and 6th days after the stress treatment.

[0018] Furthermore, the concentration of the Hoechst 33342 dye described in S4 is 0.01 mg / mL.

[0019] Furthermore, the high-content imaging described in S5 uses a 40x objective lens, with each sample well acquiring no fewer than 25 fields of view; the exposure time for the bright field channel is 60ms, and the exposure time for the nuclear staining DAPI channel and the chloroplast autofluorescence channel is 1500ms.

[0020] An algal experimental model for high-content imaging is constructed using the algal experimental model construction method described above.

[0021] Application of an algal experimental model for high-content imaging as described above in environmental monitoring.

[0022] Compared with existing technologies, this invention provides an algal experimental model for high-content imaging, its construction method, and its application. Through standardized culture, gradient stress design, optimized fixation and overnight staining procedures, and sample stabilization treatment before imaging, it ensures that algal cells have good morphological fixation and strong and uniform nuclear fluorescence signals during imaging, significantly improving the quality of single images and the consistency of images between different fields of view and different experimental wells within a multi-well plate. It is suitable for standardized carriers such as 96-well plates and can be perfectly matched with the automated sample introduction, focusing, and multi-field scanning functions of high-content analysis systems, realizing high-throughput imaging of a large number of samples.

[0023] This model can be directly used for rapid and visualized biomonitoring and assessment of heavy metal pollution in environmental water bodies. It can also be used to study the specific toxicity of different heavy metals to algal cells, and has clear application value in the fields of environmental monitoring and toxicology. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0025] Figure 1 This is a schematic diagram of a grayscale image generated from a high-content algae experimental model provided in an embodiment of the present invention after color correction.

[0026] Figure 2 This is a schematic diagram illustrating the recording process during the construction of the algae experimental model provided in this embodiment of the invention. Detailed Implementation

[0027] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Example:

[0029] Please see Figure 1 and Figure 2 A method for constructing an experimental model of algae for high-content imaging includes the following steps:

[0030] S1. Single-celled algae were selected as experimental subjects and cultured in a culture medium until the exponential growth phase to obtain Chlorella culture medium.

[0031] Please see Figure 2Single-celled algae were selected as the experimental subject, with Chlorella spp. being the preferred choice. Chlorella strains were inoculated into 1000mL Erlenmeyer flasks (Gibco, catalog number A1379901) containing 500mL of culture medium to establish a pre-culture system. The cells were then cultured at room temperature (25±1℃) with continuous shaking at a constant speed (120rpm) for 7 days to obtain algal cells in the exponential growth phase. During the culture process, continuous illumination was provided using a cool white fluorescent lamp to simulate natural light conditions, thus obtaining the algal cell culture system.

[0032] The single-celled green algae *Chlorella vulgaris* was chosen as the model organism based on its multiple inherent advantages, perfectly meeting the requirements of high-content imaging technology. First, *Chlorella vulgaris* has a simple structure, mostly spherical or elliptical, with relatively uniform cell size (approximately 2-10 micrometers in diameter). This morphological simplicity and uniformity greatly facilitates the identification, segmentation, and quantification of individual cells by automated image analysis software, a prerequisite for high-throughput, high-content statistical analysis. Second, *Chlorella vulgaris* grows rapidly and has a short reproductive cycle, making it easy to synchronize its culture under laboratory conditions. This allows for the rapid acquisition of large populations of cells at the same physiological stage, providing excellent reproducibility and comparability for experiments. Finally, *Chlorella vulgaris* is sensitive to environmental stresses, including heavy metal stress, and the mechanisms underlying these stresses are representative.

[0033] Nutrient-containing culture media, preferably BG-11 medium or a media system with equivalent nutritional function, were used. The purpose of setting up a nutrient-containing medium was to ensure that the algae maintained a normal metabolic state throughout the experimental period; to eliminate non-specific morphological changes caused by nutrient limitation; and to ensure that changes observed in subsequent high-content imaging were primarily due to heavy metal stress. BG-11 medium or a media system with equivalent nutritional function was used. BG-11 medium is a synthetically produced medium specifically designed for cyanobacteria and green algae, containing nitrates, phosphates, trace elements (such as iron, manganese, zinc, copper, etc.), and bicarbonate, which can comprehensively meet the various macro- and micro-elements required for the growth of Chlorella. The core purpose of using such a medium with clearly defined components and fixed concentrations is to: ensure that the algae maintained a normal basal metabolic state throughout the experimental period; and to completely eliminate cell starvation, senescence, or non-specific morphological changes (such as cell atrophy, abnormal lipid accumulation, etc.) caused by nutrient (such as nitrogen, phosphorus) limitation. This ensures that any changes in cell morphology, number, or karyotype observed in subsequent high-content imaging can be attributed to the applied heavy metal stress to the greatest extent possible, rather than background nutrient fluctuations.

[0034] To ensure that algae exhibit a physiological state close to but highly controllable within their natural environment at the microscopic scale, the light intensity is set to: under natural light conditions, with a maximum photosynthetically active photon flux density (PPF) of 3800 µE·m. -2 ·s -1Light-dark cycle: 14 hours light (14L): 10 hours dark (10D); culture temperature: 25±1℃.

[0035] This combination of light and temperature conditions is used to ensure the growth activity of algae while avoiding cell breakdown or structural distortion caused by extreme stress, thus providing a stable prerequisite for high-content imaging.

[0036] The culture temperature was set at a constant 25±1℃, which is the optimal growth temperature for Chlorella. A shaker was continuously oscillated at a constant speed of 120 rpm to ensure uniform suspension of algal cells in the culture medium, preventing sedimentation, and promoting gas-liquid exchange, providing sufficient carbon dioxide and oxygen for photosynthesis and respiration. Light is a key energy source driving microalgal growth. During cultivation, continuous illumination was provided using cool white fluorescent lamps, employing a 14-hour light:10-hour dark (14L:10D) light-dark cycle. This cycle mimics the natural circadian rhythm, maintaining the cells' normal physiological clock and metabolic cycle.

[0037] Cells were cultured continuously for approximately 7 days under the above conditions, and cell density (optical density OD) was monitored periodically. 680 The growth curve is determined by cell counting (or cell counting), and the goal of the culture is to bring the algal cells to the mid-exponential growth phase. During this period, cell division is vigorous, metabolic activity is active, cell morphology is regular, size is uniform, and they are most sensitive to environmental influences. Using cells at this stage for subsequent stress experiments ensures high responsiveness and experimental consistency.

[0038] After approximately 7 days of standardized pre-culture, the desired "Chlorella culture medium" is obtained when the algal cells successfully enter the mid-exponential growth phase. At this point, the culture medium exhibits a uniform bright green color, high cell viability, and good population synchronicity. This culture medium will serve as the raw material for all subsequent stress experiments, and its uniformity is crucial for the reproducibility of the entire study.

[0039] S2. Pre-treat the Chlorella culture medium to obtain an algal suspension, then add heavy metal stress factor to the algal suspension and set up a stress culture system with at least two concentration gradients.

[0040] After obtaining Chlorella culture medium in its exponential growth phase, the primary task is not to immediately apply stress, but to subject it to rigorous standardized pretreatment to eliminate potential biases caused by differences in initial cell density. This is a crucial first step in ensuring comparability among all experimental groups.

[0041] Collect the culture medium of Chlorella vulgaris, gently mix to avoid cell aggregation, and transfer 100 μL of algal suspension to a 96-well plate using a microplate reader (BioTek CYT5MF) to measure its initial optical density (OD) at 450 nm. 450Select OD 450 This is because this wavelength is near the absorption peak of chlorophyll, effectively reflecting the biomass concentration of algal cells and is suitable for high-throughput microplate detection. Then, by centrifugation or dilution with fresh PBS, the algal solutions from all experimental groups were adjusted to a uniform initial OD value. 450 The value was set to 0.13-0.14 to complete the pretreatment of the Chlorella culture medium and obtain the algal suspension.

[0042] If the stock solution concentration is too high, it should be appropriately diluted using fresh, sterile PBS buffer or basal culture medium. If the concentration is too low, cells should be collected by gentle centrifugation (3000 rpm, 5 minutes) and resuspended in a smaller volume of buffer to reach the target concentration. This ensures that the cell number and physiological load of each stress experimental group (including the control group) are exactly the same before the addition of heavy metal factors. In this way, any population-level differences (such as reduced biomass, changes in the proportion of cell death) or single-cell-level changes (such as changes in cell size and nuclear morphology) observed after subsequent culture can be clearly attributed to the dose-effect of heavy metal stress, rather than the difference in the intensity of competition for nutrients or stress factors caused by uneven initial cell density.

[0043] Subsequently, 40 mL of algal suspension was dispensed into 50 mL conical tubes for various metal stress treatments. The experiment included a blank control group (Control, no metal added), a standard limit reference group (GB), and three concentration gradients (low, medium, and high) of three metals (copper, nickel, and zinc). Please refer to the table below for specific treatment concentrations.

[0044] Heavy metal stress factors Blank control group Standard Limit Reference Group low concentration group medium concentration group High concentration group copper 0 0.01 mg / L 2mg / L 10mg / L 20mg / L nickel 0 0.02 mg / L 2mg / L 10mg / L 20mg / L Zinc 0 0.05 mg / L 2mg / L 10mg / L 20mg / L

[0045] This study selected copper (Cu) 2+ ), nickel (Ni 2+ ), Zinc (Zn) 2+ Three heavy metal ions were chosen as stress factors. This choice has clear ecotoxicological significance: these three metals are all widely present and representative pollutants in the environment, originating from industrial emissions, mining, and urban wastewater. Although zinc is an essential trace element for organisms, it can also be toxic in excess. They have different cytotoxic mechanisms (e.g., copper induces oxidative stress and disrupts the photosynthetic system; nickel interferes with enzyme function; zinc affects membrane stability and ion homeostasis). Therefore, constructing such a multi-metal, multi-concentration stress model framework to simulate complex environmental stresses and explore the commonalities and characteristics of different metal toxicity mechanisms is more generalizable.

[0046] To avoid the problem of rapid destruction of algal cell structure and inability to take pictures due to a single high dose, a gradient stress design was adopted: blank control group (no heavy metals), standard limit reference group, low concentration stress group, medium concentration stress group and high concentration stress group. Through gradient design, algae can form a continuous phenotypic change range under different stress levels in which morphology is preserved, structure is not disintegrated and fluorescence signal is distinguishable. This is one of the key constraints for the construction of high-content imaging model.

[0047] Blank control group: Contains only standardized algal suspension and culture medium, without any added heavy metals. This group provides morphological and physiological "background" or "benchmark" data of algal cells growing and dividing normally under optimal conditions, serving as an absolute reference for all stress effect assessments.

[0048] Standard limit reference group: Added heavy metals at extremely low concentrations, copper 0.01 mg / L, nickel 0.02 mg / L, zinc 0.05 mg / L; establish biological response "background values" at environmentally relevant concentrations, connecting laboratory research with real-world environmental risk assessment.

[0049] Low concentration group (2 mg / L): Induces a sublethal effect. At this concentration, most cells still survive, but may have initiated a stress response, manifested as slight inhibition of growth rate, cell cycle arrest, or early morphological adaptive changes that can be observed under a microscope (such as slight cell swelling and changes in chloroplast position). Cell structure remains intact, making it ideal for high-content imaging analysis.

[0050] Medium concentration (10 mg / L): Induced significant toxic effects, possibly resulting in partial cell death (apoptosis or necrosis). Surviving cells exhibited more pronounced morphological abnormalities, nuclear condensation or fragmentation, and impaired cell membrane integrity. At this concentration, cell population differentiation occurred, providing the possibility for analyzing cell subpopulations with different resistance / sensitivity.

[0051] High concentration group (20 mg / L): produced strong stress and even lethal effects, which may lead to large-scale cell death and disintegration; on the one hand, to determine the upper limit of toxicity, and on the other hand, to observe the cell collapse pattern under extreme stress.

[0052] This gradient design avoids the problem that a single high-dose stress may cause all cells to lyse rapidly in a short period of time, making it impossible to provide any analyzable images. By setting a continuous concentration from none to some and from low to high, it is possible to force the algal cell population to form a "continuous phenotypic range" from completely normal to mildly stressed, then to moderately damaged, and finally to severely damaged.

[0053] S3. Cultivate the stress culture system and take samples during the cultivation process;

[0054] On days 3 and 6 after stress culture, 5 mL of Chlorella culture was collected from each experimental group. The samples were centrifuged at 3000×g for 5 minutes, and the supernatant was discarded. The precipitate was washed once with 1×PBS buffer.

[0055] After the addition of heavy metals, algae do not immediately enter a stable stress state. Therefore, two culture time points were set: day 3 and day 6 after stress. This sampling time point can avoid the cell rupture stage caused by acute injury; capture the stable structural state formed by algae under stress; and ensure the consistency of samples between wells and fields of view when taking pictures.

[0056] S4. Fix the sampled algal cells overnight at 4°C using 4% paraformaldehyde solution. After fixation, wash the cells and incubate them overnight at 4°C in the dark using Hoechst 33342 dye for nuclear staining. After staining, wash the cells to remove unbound dye and obtain the stained algal cells.

[0057] Subsequently, 2 mL of 4% paraformaldehyde solution was added to the sampled algal cells, and the samples were fixed overnight at 4°C. After fixation, the samples were centrifuged again at 3000×g for 5 minutes to remove the fixative, and then washed three times with PBS buffer to completely remove residual paraformaldehyde. Nuclear staining was performed using Hoechst 33342 (Invitrogen, catalog number 314945). The dye was diluted to a working concentration of 0.01 mg / mL with 1 mL of PBS buffer, added to the algal precipitate, and incubated overnight at 4°C in the dark. After staining, the algal cells were collected by centrifugation and washed three times with PBS buffer to remove unbound dye.

[0058] Fixation (for imaging): After collecting algal samples at preset time points, algal cells are fixed. The purpose is not endpoint detection, but to fix the spatial structure of cells; to prevent cell drift and deformation during imaging; and to improve the consistency of imaging within the multi-well plate.

[0059] Considering the influence of algal cell wall structure on imaging, a nuclear fluorescence staining step is introduced, preferably using DAPI or Hoechst-type dyes, to provide a stable internal reference structure for high-content systems; improve the success rate of cell recognition in autofocus and imaging; and enhance the comparability of images under different stress conditions.

[0060] S5. The stained algal cells were resuspended in buffer solution and homogenized to obtain an algal experimental model for high-content imaging.

[0061] Before formal high-content imaging, the samples were uniformly processed, including: uniform resuspension volume; uniform well plate dispensing method; and uniform settling time, so that the suspended algae were stably distributed on the imaging plane.

[0062] Through the above processing, the algae experimental model meets the following requirements: suitable for automated scanning; suitable for continuous imaging with multiple fields of view; and suitable for cross-hole and cross-batch imaging comparison.

[0063] The stained algal cells were resuspended in 2 mL of PBS buffer. 100 µL of the homogeneous suspension was transferred to a 96-well plate, and automated imaging and acquisition were performed using a high-content analysis system (PE-Operetta, PerkinElmer).

[0064] Please see Figure 1 (Image after color correction of a high-content grayscale image), using a 40x objective lens, acquiring 25 fields of view per well. Before imaging, the exposure time (60ms for bright field; 1500ms for nuclear DAPI and chloroplast autofluorescence channels), light intensity, and focal length were adjusted. The excitation wavelength and emission filter were as follows: intact cells were observed under bright field, with Hoechst staining of the cell nuclei, excitation wavelength of 350nm, and emission wavelength of 461nm. ImageJ software was used to uniformly adjust the brightness and contrast of the images.

[0065] Observations were conducted under a 40x microscope, and the exposure time for each channel was adjusted. The bright-field channel only required about 60ms due to its optical characteristics; however, for the fluorescence staining channel, where fluorescence detection and recording capabilities were relatively weak, the exposure time was extended to 1500ms for the nuclear DAPI channel and the chloroplast autofluorescence channel.

[0066] This study successfully constructed a stable and reproducible algal experimental model specifically designed for high-content imaging, tailored to the cellular structure of algae, and operating under heavy metal stress. This model addresses the challenges of using algae directly for high-content imaging, the instability of cell structure under stress conditions, and the poor consistency of photographic results in existing technologies.

[0067] An algal experimental model for high-content imaging is provided, which is constructed using the method described above. This algal experimental model for high-content imaging can be applied in environmental monitoring.

[0068] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for constructing an algal experimental model for high-content imaging, characterized in that, Includes the following steps: S1. Single-celled algae were selected as experimental subjects and cultured in a culture medium until the exponential growth phase to obtain Chlorella culture medium. S2. Pre-treat the Chlorella culture medium to obtain an algal suspension, then add heavy metal stress factor to the algal suspension and set up a stress culture system with at least two concentration gradients. S3. Cultivate the stress culture system and take samples during the cultivation process; S4. Fix the sampled algal cells overnight at 4°C using 4% paraformaldehyde solution. After fixation, wash the cells and incubate them overnight at 4°C in the dark using Hoechst 33342 dye for nuclear staining. After staining, wash the cells to remove unbound dye and obtain the stained algal cells. S5. The stained algal cells were resuspended in buffer solution and homogenized to obtain an algal experimental model for high-content imaging.

2. The method for constructing an algal experimental model for high-content imaging according to claim 1, characterized in that, The single-celled algae mentioned in S1 is Chlorella vulgaris; the culture temperature mentioned in S1 is 22-28℃.

3. The method for constructing an algal experimental model for high-content imaging according to claim 1, characterized in that, The light conditions for cultivation in S1 consist of a 14-hour light cycle followed by a 10-hour dark cycle; the light intensity for cultivation in S1 is under natural light conditions, with a maximum photosynthetically active photon flux density of 3800 µE·m. -2 ·s -1 .

4. The method for constructing an algal experimental model for high-content imaging according to claim 1, characterized in that, The heavy metal stress factor mentioned in S2 is selected from at least one of copper, nickel, and zinc.

5. The method for constructing an algal experimental model for high-content imaging according to claim 1, characterized in that, The concentration gradients described in S2 are 2 mg / L, 10 mg / L, and 20 mg / L.

6. The method for constructing an algal experimental model for high-content imaging according to claim 1, characterized in that, The sampling time points mentioned in S3 are the 3rd and 6th days after the stress treatment.

7. The method for constructing an algal experimental model for high-content imaging according to claim 1, characterized in that, The concentration of Hoechst 33342 dye described in S4 is 0.01 mg / mL.

8. The method for constructing an algal experimental model for high-content imaging according to claim 1, characterized in that, The high-content imaging described in S5 uses a 40x objective lens, with no fewer than 25 fields of view acquired per sample well; the exposure time for the bright field channel is 60ms, and the exposure time for the nuclear staining DAPI channel and the chloroplast autofluorescence channel is 1500ms.

9. An experimental model of algae for high-content imaging, characterized in that, It is constructed using the method for constructing the algae experimental model as described in any one of claims 1-8.

10. The application of the algal experimental model for high-content imaging as described in claim 9 in environmental monitoring.

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