STED Microscopy

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STED Microscopy
관련코스 현대광학
소분류 물리
선행 키워드
연관 키워드

Introduction

Flourescence microscopy

Fluorescence microscopy is the optical microscopy that utilizes fluorescence phenomenon. For this microscopy, fluorophores are mainly used since they can be illuminated through absorbing light which has specific wavelengths and emitting light of longer wavelengths. Also, fluorophores binding does not change the original molecular structures of specimens. Therefore, fluorescence microscopy has played a key role in the research of cell biology because of its high specificity and non-invasive visualization of specimens.

The Abbe diffraction limit

Although fluorescence microscopy has some advantages, observation of sub-wavelength structures is limited because of the Abbe diffraction limit. The resolvable distance is determined by the equation below.

[math]d=\frac{\lambda}{2n \sin\theta}=\frac{\lambda}{2NA}[/math]
[math]\theta[/math] : maximal half-angle of light
[math]n[/math] : refractive index of a medium
[math]\lambda[/math] : wavelength [math][/math]

[math]NA=n\sin\theta[/math] is the numerical aperture of given optical system. Consider that cyan light having around 488[math]nm[/math] wavelength is used during an observation. Since [math]NA[/math] is about 1.4~1.5 for immersion oil, resolvable distance is about [math]d \simeq 160[/math]~[math]170nm[/math]. Even if most biological cells(1~100[math]\mu m[/math]) can be observed under this resolution, it is not sufficient to observe biomolecules that are much smaller than cells like many kinds of proteins(1~10[math]nm[/math]). Also, if there exist two biomolecules that are located with the distance smaller than the spatial resolution of an optical device, the point spread functions of them are overlapped, and then printed image seems like that there exists only one biomolecule. Even if there exist some kinds of microscopy that can offer a higher resolution such as electron microscopy or X-ray microscopy, these techniques are harmful to the specimens since they can make some damages to the specimens. Therefore, scientists have tried to make technical development of fluorescence microscopy to increase the resolution.

Super-resolution fluorescence microscopy

Super-resolution fluorescence microscopy is the combination of fluorescence microscopy and some techniques that can overcome the diffraction limit of optical microscopy. There are two sorts of such techniques. One way is modifying excitation areas of fluorophores which can cause the sharpening of point spread functions. There are some examples of microscopy using this way such as stimulated emission depletion(STED) microscopy, reversible saturable optically linear fluorescence transitions(RESOLFTs), saturated structured-illumination microscopy(SSIM), and so on. The other way is based on the localization of individual fluorophores by utilizing stochastical models. There are some examples such as stochastic optical reconstruction microscopy(STORM), photoactivated localization microscopy(PALM), super-resolution optical fluctuation imaging(SOFI), fluorescence photoactivation localization microscopy(FPALM), and etc.


Stimulated emission depletion(STED) microscopy

Basic principle

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STED microscopy is one of the super-resolution microscopies that uses the patterned excitation laser beam to increase the resolution. As the name suggests, STED microscopy uses the stimulated emission phenomenon to sharpen the point spread function of a specimen. For this sharpening, the second laser called STED laser is needed to selectively suppress fluorescence emission. Normal fluorescence occurs through exciting an electron by a normal excitation laser beam from the ground state S0 to an excited state S1 and then emitting a photon by spontaneous decaying of the electron from the excited state S1 to a vibrational state of S0.

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While normal fluorescence emerges at the center, the excited electrons in the periphery of the center area are stimulated by the doughnut-shaped STED laser beam which has almost zero intensity at the center area and then stimulated electrons decay from the excited state S1 to a vibrational ground-state which is located on a higher energy level than the vibrational state of S0 involving in normal fluorescence. This causes the depletion of spontaneous emission.

Resolution of STED microscopy

Because of this selective deactivation of the fluorophores, the fluorescent-activated region in the center can be made smaller than the diffraction limit of an optical system. Using this principle, an image acquired by STED microscopy is assembled through the pixel-by-pixel confocal scanning of the fluorophore-labeled specimens.

The resolution of the STED microscopy can be represented as below.

[math]\bigtriangleup r \approx \frac{\lambda}{2NA\sqrt{(1+\frac{I}{I_s})}}[/math]

[math]\bigtriangleup r[/math] is the half of the full width at half maximum(FWHM) which states the resolution. [math]NA[/math] is the numerical aperture of the objective lens in a microscope, [math]\lambda[/math] is the wavelength of excitation light, [math]I[/math] is the maximum intensity of the doughnut-shaped STED laser beam, and [math]I_s [/math] is the saturation intensity for the fluorophore.

The effect of STED clearly depends on the intensity of the STED laser. As seen above, higher STED laser intensity [math]I[/math] causes higher resolution by stronger suppression of spontaneous fluorescence emission in the periphery of the focal region. Since the STED laser intensity is almost zero at the focal point, fluorescence emission at the center is not affected. Thus, STED microscopy can give almost ~30[math]nm[/math] spatial resolution experimentally which is much better than the resolution of common optical or fluorescence microscopy. Although the theoretical resolution of STED microscopy is nearly zero, some factors such as noise of sensor in the optical system, imperfect construction of STED laser into a doughnut shape, or something else can be the cause for reducing the resolution.

Sample preparation

To use the STED microscopy, target biological specimens have to be labeled with antibodies where fluorescent dyes are attached or labeled with fluorescent proteins through the genetic modification of the specimens. Since all fluorophores undergo photobleaching during the continuous incidence of the laser beams, fluorophores with strong stability are needed to be utilized in STED microscopy. Also, both a smaller center for normal fluorescence and a larger surrounding for STED laser are preferred for better and more efficient application of STED microscopy.

Applications of STED microscopy

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Multicolor imaging

One of the valuable property of fluorescence microscopy is that multicolor imaging between different structures in cells or biomolecules are possible because every fluorophore has a specific spectrum for excitation and emission each. For example, if two fluorophores with definitely different range of excitation and emission wavelengths, such as Atto 532 and Atto647N, are chosen, then by using two sets of lasers with proper wavelengths corresponding to each fluorophore, two-color imaging between two different biomolecules or organelles can be conducted. Then, images through two-color imaging can distinguish them because of two different emitting colors.

Multicolor imaging is also possible through the proper choice of multiple fluorophores. There are many kinds of fluorophores that can be used for STED microscopy, such as Atto 532, Atto 647N, KK 114, and etc.

Live-cell imaging

Because of the high resolution of STED microscopy, it is possible to observe biomolecular functions, structures, and biomolecules in cells under the single-molecule level. For instance, live-cell imaging with STED microscopy can give more sophisticated information about actual biomolecular functions in live cells or the detailed structures such as membrane proteins moving through the cell membrane, structures of cytoskeletons that have really shallow diameters, structural modification of dendritic spines, vesicles used for synaptic transmission and so on.



References

Blom, H., & Jerker, W. (2017). Stimulated Emission Depletion Microscopy. Chemical Reviews, 117(11), 7377-7427. doi:10.1021/acs,chemrev.6b00653.

Huang, B., Bates, M., & Zhuang, X. (2009). Super resolution fluorescence microscopy. Annu Rev Biochem, 78, 993-1016. doi:101146/annurev.biochem.77.061906.092014.