Until now, research on the transport mechanism of vesicles and the analysis of interactions between vesicles and cellular organelles primarily used fluorescence microscopy. However, using fluorescence microscopy had limitations, as it allowed the observation of the transport process of fluorescently labeled specific vesicles and was restricted to a limited time frame where signals could be maintained. The Institute for Basic Science's Molecular Spectroscopy and Dynamics Research Group has developed a high-resolution label-free interference scattering microscope that selectively tracks the movement of actively moving vesicles inside living cells. It is expected to vividly reveal life phenomena from a microscopic perspective. The participating researchers will directly explain the concepts that need to be understood to grasp the significance of the research.

The intricate cellular environment and substance transport within the tiny yet complex world of cells

While a single cell may be extremely small, almost invisible to the human eye, the microscopic world inside a cell, when observed through a microscope, is not only filled with numerous substances but also comprises a highly complex realm of interactions among these substances. Cellular substance transport refers to the process of transporting essential materials for the cell's growth and development within this complex environment.

To accurately deliver the necessary substances to specific locations within the cell, the cell first encapsulates proteins, hormones, neurotransmitters, and other materials in small, pouch-like vesicles surrounded by a thin lipid membrane. Subsequently, using motor proteins that move along the microtubule and actin filament protein networks, which can be considered as cellular roadways, the cell transports these vesicles. During this process, situations may arise where vesicles are delivered to the wrong locations or transportation is delayed. Recent research results indicate that issues in the substance transport process are closely associated with the onset of neurodegenerative diseases.

Observing the transport of small vesicles within the intricate cell

As mentioned earlier, due to the exceedingly small and complex environment within the cellular world, tracking and observing only the vesicles in transport within this environment is not an easy task. Currently, the majority of researchers utilize imaging techniques with fluorescence microscopy to understand the principles and processes of vesicle transport. This method involves using fluorescent substances that selectively bind to the biomolecules constituting the vesicle of interest, rather than the vesicle itself. It tracks the fluorescent signals emitted from the fluorescent substances attached to the vesicle.

The achievements in research using fluorescence microscopy for studying cellular substance transport phenomena are too numerous to list individually. However, the limitations of this method include the ability to see only specific vesicles labeled with fluorescence and the constraint of observing within a limited time frame during which the fluorescent signal is maintained.

This situation can be explained more easily with the following analogy: Imagine a city experiencing a blackout on a dark night. In this scenario, there is a car with its headlights on, stationary at a certain location. From the perspective of an observer at a distance, the blackout situation actually helps accurately determine the current position of the car with headlights on. However, to understand why the car is stopped there, one would need to assess the surrounding circumstances. Due to the darkness caused by the blackout, it is impossible to perceive the surrounding environment. Moreover, once the car's battery is depleted, even the position of the stationary car becomes untraceable. This analogy illustrates the drawbacks of the fluorescence microscopy research method.

Observing Vesicle Transport Processes Within Living Cells Using Interference Microscopy

Interference microscopy is an imaging device capable of highly sensitive measurement of minute light signals scattered from nano-sized particles or biological molecules when exposed to light. In our research group, we have been making continuous efforts over the past 5-6 years to develop interference microscopy suitable for cellular imaging studies. Interference microscopy not only allows for the rapid and long-term tracking of vesicles transporting along the protein network within cells without the need for fluorescent labeling but also enables the simultaneous acquisition of information about the surrounding environment where the vesicles are located. Furthermore, utilizing the positional information of numerous vesicles obtained during this process, it becomes possible to reconstruct the spatial distribution of the protein network within the cell at high resolution.

[Figure 1] Traffic patterns of vesicles within the cell observed using interference microscopy. Each image is reconstructed from the moving positions of vesicles obtained from videos captured at a speed of 50 frames per second over 180 seconds. The colors in the images represent the number of vesicles observed at each fixed pixel position during the corresponding time, indicating the traffic density of vesicles on the cellular protein network.

Intracellular courier also faces traffic jam

Paper Title: "Long-term cargo tracking reveals intricate trafficking through active cytoskeletal networks in the crowded cellular environment"

Through the study of vesicle transport using interference microscopy, numerous intriguing phenomena, not previously well-documented, have been observed. One notable observation is the occurrence of traffic congestion-like transport delays in specific densely packed areas within the cell, similar to the commuting experience in a city. Concurrently, the study revealed that cells employ various interesting strategies to efficiently overcome these traffic-like conditions within the cellular environment. These strategies include collective transport, where multiple vesicles move together in the same direction, and hitchhiking transport, where vesicles attach to those already in transit. In conclusion, cells experience congestion phenomena akin to traffic congestion on city roads, and they have developed efficient transport strategies to overcome these challenges, reflecting striking similarities with transportation issues encountered in human societies.

[Figure 2] Representation of vesicle traffic within cellular networks resembling the road traffic pattern in Seoul. The observed traffic phenomenon during vesicle transport within the cell closely resembles the common road traffic experiences in large human cities. Consequently, the structure of the protein network composing the inner cellular environment was modeled to mimic the road network structure within Seoul, including both inner city and outer suburban areas. The red segments on the network represent high-traffic regions, while the green segments indicate smooth traffic flow areas.

Understanding efficient vesicle transport strategies in the complex cellular environment

Our developed interference microscopy includes a fluorescence microscopy device, allowing simultaneous observation of specific fluorescently labeled molecules within the cell. By combining high-speed, high-resolution interference imaging with chemical-selective fluorescence imaging techniques, we have enhanced the accuracy of our observations. Moving forward, our goal is to gain a more detailed understanding of the efficient transport strategies adopted by cells to overcome traffic issues. We aim to contribute to revealing how these phenomena are intricately linked to the life processes within actual cells.