2024-07-24 04:46:23
A new publication from Optoelectronic Advances; DOI 10.29026/oea.2024.240035 discusses the nanoscale imaging of multiple stimulated emission depletion for long-term imaging of living multicolored cells.
In the field of cell biology, an increasing number of studies focus on the intricate network of interactions between sub-cellular structures. As a powerful imaging tool, super-resolution fluorescent microscopy has broken the diffraction limit, allowing biologists to observe sub-cellular structures at nanometer resolution. Among super-resolution fluorescent microscopy techniques, stimulated emission depletion (STED) microscopy is one of the leading methods that goes beyond the diffraction limit, promising minimal artifacts due to the immediate microscopic properties of super-resolution without post-processing.
In the past decade, the need to explore the interactions between sub-cellular structures has led to growing interest and application of multiple living cell STED, often conceptualized by using multiple pairs of excitation-depletion beams. However, increasing the number of depletion beams not only complicates the system and dramatically increases construction costs but also raises the likelihood of photo-bleaching and more severe photo-cytotoxicity, which does not contribute to the imaging of living cells. On the other hand, using a single depletion beam along with multiple excitation beams limits the available excitation wavelength range, and the restricted band allocation to a number of densely arranged spectral channels while reducing cross-talk among them poses significant challenges. Currently, this approach is generally limited to two- or three-color imaging.
As a result, researchers have utilized lifetime information of fluorophores to achieve multi-color imaging. The lifetime of fluorescence is the average time a fluorescent molecule spends in an excited state and can be exploited to distinguish between different fluorescent molecules. However, multi-color STED based on fluorescent lifetime is currently applicable only to fixed cells due to the difficulty of (i) filtering bright and anti-bleaching properties suitable for STED imaging of living cells, (ii) simultaneously labeling multiple sub-cellular structures in living cells, and (iii) separating different fluorescent probes within the same spectral channel using an appropriate analytical method. Currently, biologists face a shortage of efficient methods for investigating the dynamics and function of sub-cellular structures using STED microscopy.
Based on the above challenges, the authors of this article developed multiple stimulated emission depletion nanosopy (mSTED), enabling simultaneous observation of additional structures with limited phototoxicity and bleaching. The researchers tested a series of appropriate fluorescent probe combinations capable of marking multiple sub-cellular structures simultaneously. These fluorescent probes of living cells with similar spectral identities were then separated using phasor analysis. mSTED achieved STED imaging in 5 live colors, revealing long-term interactions between different sub-cellular structures. The results here provide a framework for understanding the complex and delicate interactome of sub-cellular structures in living cells.
The researchers first tested the performance of mSTED by imaging in two colors (Figure 1), demonstrating its ability to successfully separate different sub-cellular structures and resulting in only 4% cross-talk. Compared to confocal microscopy, the resolution of mSTED was significantly improved (~60 nanometers), allowing for more detailed observation of sub-cellular structures.
To validate the performance of mSTED in terms of photo-bleaching and photo-cytotoxicity, the researchers conducted a comparative analysis with mSTED and conventional multi-color STED methods (Figure 2). Two depletion lasers were required for conventional two-color STED imaging. After 11 minutes of imaging, the fluorescence signal of the microtubules dropped to 13.4% of the initial value, and the mitochondria became swollen and round, indicating severe photo-bleaching and photo-cytotoxicity. In contrast, mSTED required only a single depletion laser. After 11 minutes of imaging, the fluorescence signal of the microtubules was still at 31.5%, and the mitochondrial shape showed no significant changes.
Researchers used mSTED for long-term imaging of living cells, allowing for simultaneous observation of five sub-cellular structures (Figure 3). The large amount of mSTED multi-color data for the long term enabled us to uncover interesting phenomena in cell biology. For example, microtubules hold a specific area under the nucleus to allow other organelles to move, and mitochondrial fission and fusion occurred in this confined space in collaboration with ER and microtubules. These results highlight the superiority of mSTED in multi-color long-term imaging of living cells containing multiple structures, enabling systematic interpretation of organelle interaction networks simultaneously.