Academic Background and Problem Statement

Mitochondria, as crucial organelles in eukaryotic cells, are involved in cellular energy metabolism, signal transduction, and the regulation of cell survival and death. Mitochondrial dysfunction is associated with various human diseases, including neurodegenerative disorders, cardiovascular diseases, diabetes, and cancer. Therefore, studying the dynamics of mitochondria is essential for understanding their biological functions and their roles in diseases. However, traditional electron microscopy (EM), while offering extremely high spatial resolution, is limited to fixed samples and cannot capture mitochondrial dynamics. Fluorescence microscopy, although suitable for live samples, has limited resolution, especially in three-dimensional (3D) reconstruction and long-term imaging, where photobleaching severely affects the accuracy of quantitative analysis.

Photobleaching refers to the irreversible chemical alteration of fluorophores under light exposure, leading to a gradual decrease in fluorescence signal. This issue is particularly prominent in long-term imaging, hindering the dynamic study of organelles such as mitochondria. Although various methods have been proposed to correct for photobleaching, these methods often rely on complex algorithms or assumptions that may introduce artifacts. Therefore, developing a technique that enables high-resolution imaging in live samples while effectively addressing photobleaching has become a significant challenge in current research.

Paper Source and Author Information

This paper was conducted by a research team from the Department of Cell and Developmental Biology at University College London (UCL), with primary authors including Segos Ioannis, Van Eeckhoven Jens, Greig Alan, Redd Michael, Thrasivoulou Christopher, and Conradt Barbara. The paper was published in npj Imaging in 2024, titled Impact of photobleaching on quantitative, spatio-temporal, super-resolution imaging of mitochondria in live C. elegans larvae.

Research Process and Experimental Methods

1. Mechanical Immobilization of C. elegans Larvae

To perform super-resolution (SR) imaging in live samples, the research team developed a mechanical immobilization method based on polystyrene nanobeads. This method does not require drugs and effectively immobilizes C. elegans larvae, ensuring they remain stationary during imaging. The specific steps are as follows:

1. Sample Preparation: C. elegans larvae are placed on a 10% agarose pad and covered with a coverslip.
2. Immobilization Process: The larvae are immobilized on the agarose pad through the friction of nanobeads and the pressure of the coverslip, ensuring they do not move during imaging.
3. Imaging Conditions: The left side of the larvae is in contact with the coverslip, ensuring that the target cells (e.g., ql.p neuroblasts) can be imaged with super-resolution.

2. Super-Resolution Time-Series Imaging

The research team used the Airyscan SR mode of the Zeiss LSM980 microscope to achieve single-mitochondrion resolution. The specific steps are as follows:

1. Transgenic Strain Construction: A transgenic C. elegans strain expressing a mitochondrial matrix marker (mtGFP) was constructed to label mitochondria.
2. Imaging Parameters: A 63× oil immersion objective was used, with a resolution of 120 nm (xy) and 360 nm (z), and images were acquired every 1 minute.
3. Image Processing: Multi-step time-series imaging was performed to capture the dynamic changes of mitochondria during cell division.

3. Image Processing and 3D Reconstruction

To quantitatively analyze the dynamic changes of mitochondria, the research team developed an image processing pipeline, including the following steps:

1. Image Alignment: Since the larvae may still exhibit slight movements during imaging, the team corrected the position of mitochondria through image alignment.
2. Image Subtraction: To eliminate fluorescence signal overlap, the mCherry signal was subtracted from the mtGFP images.
3. Image Cropping: Regions containing the target cells were cropped to reduce the computational load of image processing.
4. Deconvolution: The Richardson-Lucy algorithm was used for deconvolution to enhance image resolution.
5. 3D Rendering: The Imaris software was used for 3D reconstruction of mitochondria, enabling quantitative analysis of their volume, morphology, and distribution.

4. Analysis of Photobleaching Effects

The research team analyzed the impact of photobleaching on mitochondrial fluorescence signals through multi-step time-series imaging. The results showed that although photobleaching gradually reduces fluorescence signals, the global thresholding-based image segmentation method is unaffected by photobleaching and can accurately quantify mitochondrial numbers.

Main Research Findings

1. Effectiveness of Mechanical Immobilization: The mechanical immobilization method developed by the research team effectively immobilizes C. elegans larvae, ensuring they remain stationary during imaging without affecting their normal development.
2. Achievement of Super-Resolution Imaging: Using the Airyscan SR mode, the team successfully captured the dynamic changes of mitochondria during cell division, achieving single-mitochondrion resolution.
3. Optimization of Image Processing Pipeline: The image processing pipeline developed by the team effectively corrects for cell movement and fluorescence signal overlap, enabling 3D reconstruction of mitochondria.
4. Impact of Photobleaching: Although photobleaching gradually reduces fluorescence signals, the global thresholding-based image segmentation method is unaffected by photobleaching and can accurately quantify mitochondrial numbers.

Research Conclusions and Significance

This study developed a technique that enables super-resolution imaging in live samples while effectively addressing photobleaching, providing a new tool for studying the dynamic changes of organelles such as mitochondria. The results demonstrate that global thresholding-based image segmentation is unaffected by photobleaching and can accurately quantify mitochondrial numbers. This finding will advance the application of fluorescence microscopy in quantitative research.

Research Highlights

1. Novel Mechanical Immobilization Method: The nanobead-based mechanical immobilization method developed by the team does not require drugs and effectively immobilizes C. elegans larvae, making it suitable for various biological studies.
2. Achievement of Super-Resolution Imaging: Using the Airyscan SR mode, the team successfully captured the dynamic changes of mitochondria during cell division, achieving single-mitochondrion resolution.
3. Solution to Photobleaching: The results show that global thresholding-based image segmentation is unaffected by photobleaching and can accurately quantify mitochondrial numbers, advancing the application of fluorescence microscopy in quantitative research.

Other Valuable Information

The research team also explored the performance of different fluorescent proteins (e.g., GFP and mKate2) under photobleaching, showing that global thresholding-based image segmentation is applicable to various fluorescent proteins and organelle markers. Additionally, the team proposed the changes in image histograms under photobleaching, providing a theoretical basis for developing new image analysis tools.

Summary

This study successfully achieved super-resolution imaging in live samples by developing a novel mechanical immobilization method and optimizing the image processing pipeline, effectively addressing the issue of photobleaching. This technique provides a new tool for studying the dynamic changes of organelles such as mitochondria, offering significant scientific and practical value.