MINFLUX Nanoscopy in Biological Tissue: Breaking the Resolution Barrier of Fluorescence Microscopy

Academic Background

Fluorescence microscopy plays a crucial role in biological research, but its resolution is limited by the diffraction barrier, typically around 200 nanometers. In recent years, the development of super-resolution microscopy (SR) techniques has broken this limit, enabling researchers to observe the distribution of biological molecules at the nanoscale. However, in complex biological tissues, especially in thick samples, issues such as optical aberrations, light absorption, and scattering severely affect the performance of super-resolution microscopy. To achieve nanoscale visualization of protein distributions in physiologically relevant environments, researchers have been exploring new imaging technologies.

MINFLUX (minimal photon fluxes) nanoscopy is an emerging optical imaging technique that combines the strengths of coordinate-targeted and coordinate-stochastic super-resolution microscopy, enabling nanoscale resolution fluorescence imaging with minimal photon fluxes. However, applying MINFLUX technology to thick biological tissue samples, particularly brain tissue sections, still faces numerous challenges. This study aims to explore the imaging performance of MINFLUX in biological tissues, especially its nanoscale resolution imaging in deeper tissues.

Source of the Paper

This paper was co-authored by Thea Moosmayer, Kamila A. Kiszka, and other researchers from the Max Planck Institute for Multidisciplinary Sciences, the University of Göttingen, and other institutions. The paper was published on December 20, 2024, in the Proceedings of the National Academy of Sciences (PNAS), titled “MINFLUX fluorescence nanoscopy in biological tissue.”

Research Process and Results

1. Experimental Design and Equipment Improvements

To perform MINFLUX imaging in biological tissues, the research team made several improvements to the existing MINFLUX microscope. First, they adopted a silicone oil immersion objective to improve refractive index matching and reduce aberrations. Second, they developed a depth-adaptable focus lock system to stabilize the sample position at different depths, ensuring nanometer-level localization accuracy. Additionally, the team introduced a progressive activation scheme, which gradually increases the power of the activation laser to improve the detection efficiency of fluorescent molecules.

2. Tissue Sample Preparation and Imaging

The research team used mouse brain tissue sections as experimental samples, with thicknesses ranging from 50 to 300 micrometers. They selected the cortex and hippocampus as imaging regions and performed pre-scanning with a confocal microscope to identify regions of interest. Subsequently, they used the MINFLUX microscope to perform nanoscale resolution imaging of these regions.

3. MINFLUX Imaging Performance

The research team first imaged the Caveolin-1 protein at shallow tissue depths (0 to 80 micrometers). The results showed that even at a depth of 80 micrometers, MINFLUX could achieve localization precision of less than 5 nanometers. As the depth increased, the signal-to-background ratio (SBR) gradually decreased, but MINFLUX still maintained high localization precision in deeper tissues.

Next, the team imaged the postsynaptic proteins PSD95 and actin. The results showed that MINFLUX could clearly resolve the distribution of these proteins in deeper tissues, with localization precision within 10 nanometers. Additionally, the team demonstrated dual-color MINFLUX imaging, which simultaneously observed the distribution of PSD95 and actin, further revealing the nanoscale details of synaptic structures.

4. Live Tissue Imaging

To explore the potential of MINFLUX in live tissue imaging, the research team performed imaging on acute brain tissue slices. The results showed that MINFLUX’s imaging performance in live tissue was similar to that in fixed tissue, with slightly lower localization precision but still within the nanoscale range. This indicates that MINFLUX technology has the potential for future nanoscale resolution imaging in live tissues.

5. Three-Dimensional MINFLUX Imaging

The research team also developed a new three-dimensional MINFLUX imaging scheme, enabling nanometer-level 3D localization in complex tissue environments. They used an imaging mode that combined regular focus and a donut-shaped beam, achieving high localization precision in deeper tissues. Using this technique, the team successfully performed 3D imaging of the postsynaptic proteins PSD95 and AMPA receptors, revealing the spatial distribution of these proteins in synapses.

Conclusions and Significance

This study demonstrates that MINFLUX nanoscopy can achieve nanoscale resolution imaging in thick biological tissues, particularly in brain tissue sections, with localization precision of less than 10 nanometers. This technology breaks the resolution limitations of traditional fluorescence microscopy in tissue imaging, providing a new tool for studying protein distributions in physiologically relevant environments.

The application of MINFLUX technology is not limited to fixed tissues; its potential in live tissue imaging also opens new directions for future neuroscience research. Through nanoscale resolution imaging, researchers can gain deeper insights into the molecular mechanisms of synaptic structure and function, particularly the dynamic changes of proteins in processes such as learning and memory.

Research Highlights

1. Nanoscale Resolution: MINFLUX technology achieved localization precision of less than 10 nanometers in thick biological tissues, breaking the resolution barrier of traditional fluorescence microscopy.
   
2. Deep Tissue Imaging: The research team maintained high imaging performance at tissue depths of up to 80 micrometers, enabling nanoscale resolution imaging in deep tissues.
   
3. Dual-Color and 3D Imaging: MINFLUX technology can simultaneously observe the distribution of multiple proteins and achieve nanometer-level 3D localization, providing new perspectives for studying complex biological structures.
   
4. Live Tissue Imaging: The team demonstrated the potential of MINFLUX in live tissue imaging, offering a new tool for future neuroscience research.
   

Future Prospects

With further development of MINFLUX technology, researchers are expected to perform nanoscale resolution imaging in more complex biological tissues, particularly observing the dynamic changes of proteins in live tissues. Additionally, combined with automated imaging schemes, MINFLUX technology has the potential to play a greater role in neuroscience, cell biology, and other fields, helping researchers gain deeper insights into the distribution and function of biological molecules.

This study provides new technological means for nanoscale resolution imaging of biological tissues, with significant scientific value and application prospects.