In this blog post, I will provide a clear overview of the principles and background of STED microscopy, as well as its biological applications and significance.
The Importance of Microscopes and Their Existing Limitations
Microscopes have been a crucial tool in biology. The invention of the microscope led to the discovery of cells, and since then, biology has made tremendous strides. However, traditional optical microscopes have inherent physical limitations, making it difficult to observe the fine structures inside cells or arrangements at the nanoscale.
In particular, optical technologies such as fluorescence microscopy use visible light, so their resolution is limited by the wavelength of light; generally, structures smaller than half the wavelength of light (approximately 200 nm) are difficult to distinguish. Despite the advantage of being able to observe living samples, there was a problem where light from two points very close to each other caused interference as it passed through the objective lens, making them indistinguishable.
Basic Concepts of Excited States and Fluorescence
To understand the principle of STED, one must first understand the quantum mechanical concepts of the “ground state” and the “excited state.” Unless a molecule receives energy, it remains in a stable ground state. When it absorbs sufficient energy from an external source, the molecule’s energy level rises, and it enters an excited state.
A molecule in an excited state returns to the ground state within a very short time, emitting light equal to the energy difference between the excited and ground states—that is, fluorescence. Detecting this fluorescence signal to obtain an image of the sample is the basic principle of fluorescence microscopy.
How STED Microscopy Works
STED microscopy (Stimulated Emission Depletion Microscopy) is, as the name suggests, a method that artificially narrows the observation area by depleting fluorescence using stimulated emission. In short, it is a strategy that suppresses surrounding fluorescence to confine the actually observed fluorescence signal to a very small area.
The specific process is as follows. First, a first laser beam is directed at the sample, exciting the molecules in the area it strikes. At this point, the molecules in that area are ready to emit fluorescence. Immediately afterward, a second laser is superimposed on the same region; this second beam is adjusted to have a doughnut-shaped intensity distribution.
When the doughnut-shaped laser is superimposed, stimulated emission occurs in most of the region except for a very small central spot, forcing the excited molecules to return to their ground state. As a result, fluorescence is observed only in the extremely small central region (at the nanoscale), while the surrounding areas lose their fluorescence. By moving (scanning) the laser while progressively reducing the size of this central fluorescent region, a high-resolution image of the entire sample can be obtained.
To summarize, the observation area can be conceptualized in terms of three spatial regions: the entire area excited by the first laser, the surrounding region where fluorescence is quenched by the doughnut-shaped laser, and the very small central region where fluorescence is actually emitted and observed. Through the interaction of these three regions, observation beyond the conventional diffraction limit becomes possible.
Traditional fluorescence microscopy induces fluorescence using a single type of light and obtains images based solely on that fluorescence; therefore, it was difficult to resolve structures smaller than 200 nm due to the diffraction limit. In contrast, STED distinguishes itself by combining two lasers temporally and spatially to suppress surrounding fluorescence, thereby reducing the effective size of the observation point and circumventing the diffraction limit.
Hell’s Contributions and the Current Significance of STED
Dr. Stefan Hell first proposed this concept theoretically in 1994, and in 2000, he successfully built a STED microscope, producing images with significantly higher resolution than conventional optical microscopes. He reported results showing approximately threefold improved resolution in images of E. coli and contributed to expanding our understanding of synaptic structures by observing living nerve cells.
However, STED equipment is often difficult to implement and expensive, so it has not yet been widely adopted in all laboratories. Nevertheless, the achievement of nanoscale resolution using an optical microscope marked a significant turning point in basic biology and chemistry research.
