Multiplexed STED nanoscopy technique reduces photobleaching and phototoxicity in live-cell imaging

A new publication from Opto-Electronic Advances; DOI 10.29026/oea.2024.240035 discusses multiplexed stimulated emission depletion nanoscopy for multi-color live-cell long-term imaging.

In the field of cell biology, an increasing number of studies are focusing on the intricate network of interactions among subcellular structures. As a powerful imaging tool, super-resolution fluorescence microscopy broke the diffraction limit, enabling biologists to observe subcellular structures with nanoscale resolution. Among super-resolution fluorescence microscopy, stimulated emission depletion microscopy (STED) is one of the leading techniques beyond the diffraction limit, and it ensures minimal artifacts by its immediate super-resolution microscopic properties without post-processing.

In the last decade, the need to study the interactions between subcellular structures has led to an increasing interest and application of multicolor live-cell STED, most conventionally achieved by using multiple excitation-depletion beam pairs. However, increasing the number of depletion beams not only makes the system more complex and dramatically increases the construction cost, but also increases the likelihood of photo-bleaching and more severe photo-cytotoxicity, which is not conducive to live-cell imaging. On the other hand, the use of a single depletion beam along with multiple excitation beams limits the range of available excitation wavelengths, and dividing the restricted band into multiple densely arranged spectral channels and reducing crosstalk between them poses a significant challenge. Currently, this approach is typically limited to two- or three-color imaging.

Consequently, researchers have employed fluorescence lifetime information to achieve multicolor imaging. Fluorescence lifetime is the average time a fluorescent molecule spends in the excited state and can be utilized to distinguish between different fluorescent molecules. However, multicolor STED based on fluorescence lifetimes is currently only applicable to fixed cells due to the difficulty of (i) screening out live-cell fluorescent markers with brightness and anti-bleaching properties suitable for STED imaging, (ii) simultaneously labeling multiple subcellular structures in living cells, and (iii) separating different fluorescent probes in the same spectral channel using appropriate analytical method. Currently, biologists face a lack of effective methods for studying the dynamics and function of subcellular structures using STED microscopy.

Based on the above challenges, the authors of this article have developed multiplexed stimulated emission depletion nanoscopy (mSTED), which allows for simultaneous observation of more structures with limited photobleaching and phototoxicity. The researchers screened a series of suitable fluorescent probe combinations capable of labeling multiple subcellular structures simultaneously. These live-cell fluorescent probes with similar spectral identity were subsequently separated by a phasor analysi. mSTED achieved 5-color live-cell STED imaging and revealed long-term interactions between different subcellular structures. The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.

The researchers first tested the performance of mSTED by two-color imaging (Fig. 1), which showed that it could successfully separate different subcellular structures and caused only 4% crosstalk. Compared to confocal microscopy, the resolution of mSTED was significantly improved (~60 nm), enabling the observation of subcellular structures in much finer detail.

To verify the performance of mSTED in photo-bleaching and photo-cytotoxicity, the researchers conducted a comparative analysis with mSTED and conventional multicolor STED methods (Fig. 2). Two depletion lasers were required in conventional two-color STED imaging. After 11 minutes of imaging, the fluorescence signal of the microtubules decreased to 13.4% of the initial value, and the mitochondria transformed into swollen and round shapes, indicating severe photo-bleaching and photo-cytotoxicity. In contrast, mSTED required only one depletion laser. After 11 minutes of imaging, the fluorescence signal of the microtubules was still 31.5% and the shape of the mitochondria did not exhibit significant changes.

Researchers employed mSTED for live-cell long-term imaging, enabling the simultaneous observation of five subcellular structures (Fig.3). The large enough information of long-term multi-color mSTED allowed us to discover interesting phenomena in cell biology. As an illustration, microtubules hold up some space beneath the nucleus for other organelles to move around, and mitochondrial fission and fusion happened in this limited space under the cooperation of ER and microtubules. These results highlight the superiority of multi-color mSTED in long-term live-cell imaging of multiple structures, enabling the systematical interpretation of organelle interaction network at the same time.