Name: Preparing Microscope Slide with Fully-Grown Amyloid Spherulite
Uploaded: 2023-09-08T12:20:00
Description: Watch this Scientific Journal Video about Preparing Microscope Slide with Fully-Grown Amyloid Spherulite at JoVE.com

preparing microscope slide with fully-grown amyloid spherulite

Polarization-Sensitive Two-Photon Microscopy를 이용한 구형석 특성화

Over the past decades, although there has been significant development of numerous fluorescence microscopy techniques for bioimaging of proteins and their aggregates1, only a few have been used to resolve their local ordering within the sample2,3. Fluorescence lifetime imaging microscopy4 was used to study the intrinsic structural heterogeneity of amyloid superstructures-spherulites. Moreover, quantitative determination of the local ordering inside complex and densely-packed biostructures such as spherulites could be resolved using polarization-sensitive methods3. However, standard fluorescence techniques with superficial tissue penetration are limited since using UV-VIS wavelengths to excite fluorophores in vivo leads to high tissue light scattering5. In addition, such imaging often requires designing and binding specific fluorescent probes to a targeted biomolecule, thereby increasing the cost and amount of work needed to perform imaging.
Recently, to address these issues, our team has adapted polarization-sensitive two-photon excited fluorescence microscopy (ps-2PFM) for label-free imaging of biological structures6,7. Ps-2PFM allows for the measurement of the dependence of two-photon fluorescence intensity on the direction of linear polarization of the excitation beam and analysis of the polarization of the emitted fluorescence8. The implementation of this technique requires supplementation of the standard multi-photon microscope setup excitation path (Figure 1) with a half-wave plate to control the light polarization plane. Then, polar graphs, depicting the dependence of two-photon excited fluorescence intensity on the polarization of the excitation laser beam, are created from signals collected by two avalanche photodiodes, thus collecting the two, mutually perpendicular components of fluorescence polarization.
The last step is the data analysis process, taking into account the impact of the optical elements, such as dichroic mirrors or a high numerical aperture objective, on polarization. Because of the nature of the two-photon process, this method provides both reduced angular photo-selection and enhanced axial resolution, as two-photon excitation of fluorophores outside the focal plane is probabilistically limited. It was also proven that similar methods could be successfully implemented for in vivo imaging of near-infrared probes (NIR) for deep-tissue imaging9. Ps-2PFM has been previously applied to image fluorophores in cell membranes10 and DNA11,12, as well as non-standard fluorescent markers of biological systems, such as gold nanoparticles13. However, in all these examples, the information about the organization of biomolecules was obtained indirectly and required a predefined mutual orientation between a fluorophore and a biomolecule.
In one of our recent papers, we have shown that ps-2PFM could be successfully applied for determining the local polarization of the autofluorescence of amyloid superstructures and fluorescence from Thioflavin T, an amyloid-specific dye, bound to amyloid fibrils in spherulites6. Furthermore, in another one, we have proven that ps-2PFM could be utilized to detect amyloid fibril orientation inside amyloid spherulites in a sub-micron size regime, which was confirmed by correlating it with transmission electron microscopy (TEM) imaging7. The achievement of this outcome was possible thanks to i) intrinsic autofluorescence of spherulites-amyloids, when excited with either one or two photons, exhibit intrinsic autofluorescence with emission maxima located in a range from 450 to 500 nm and two-photon absorption cross sections comparable with standard fluorescent dyes14, ii) mathematical models introduced previously to describe how ps-2PEF of dyes labeling biological membranes and DNA structures could be applied to fluorescence exhibited by spherulites and dyes bound to them8,11,15. Thus, before proceeding with the analysis, we highly recommend looking through the required theory described in both, the main text and supporting information of our first paper concerning this topic6. Here, we present the protocol for how to apply the ps-2PFM technique for a label-free amyloid structural characterization of bovine insulin spherulites.

저자 스포트라이트: 편광에 민감한 이광자 현미경을 사용한 복잡한 생체 구조의 비침습적 이미징 

Label-free Amyloid Structural Characterization을 위한 편광에 민감한 2광자 현미경

Our goal is to develop new optical markers and techniques for the detection of protein aggregates called amyloids. They are involved in a range of diseases such as Alzheimer's disease and type two diabetes. So in order to cure these diseases, we need to have techniques to visualize amyloids.We demonstrated that two-photon microscopy can be used to detect amyloid fibril orientation inside amyloid superstructures. It can be achieved not only using amyloid-specific dyes, but also by utilizing the autofluorescence of amyloids. Since our technique operates on nonlinear optical phenomena, it can achieve reduced angular photoselection, enhance axial resolution, lower light scattering, lower phototoxicity, and deeper sample penetration when compared to the one-photon fluorescence microscopic technique.Polarization-sensitive two-photon fluorescence microscopy is a promising tool to image internal structure of various complex biological structures, not only protein aggregates, but also DNA vesicles and lipid membranes.

Watch this Scientific Journal Video about Preparing Microscope Slide with Fully-Grown Amyloid Spherulite at JoVE.com

Preparing Microscope Slide with Fully-Grown Amyloid Spherulite  

완전히 성장한 아밀로이드 구형암으로 현미경 슬라이드 준비

시작하려면 현미경 유리 슬라이드를 물로 철저히 씻으십시오. 그런 다음 슬라이드를 메탄올에 담근 후 먼지가 없는 물티슈로 주변 조건에서 건조시킵니다. 다음으로, 자동 피펫을 사용하여 100 마이크로 리터의 인슐린 구형 용액을 현미경 슬라이드의 중앙 영역에 있는 웰로 옮깁니다.기포가 형성되지 않도록 커버 슬립으로 용액을 조심스럽게 덮으십시오. 그런 다음 자동 피펫을 사용하여 슬라이드의 커버 슬립 가장자리를 따라 마운턴트를 증착하여 천연 구형 용액을 밀봉합니다. 실장제가 굳을 수 있도록 샘플을 주변 조건에서 그대로 두십시오.교차 편광판이 있는 편광 광학 현미경을 사용하여 인슐린 구형석이 올바르게 형성되었는지 확인합니다. 샘플을 스캔하여 몰타 십자가로 알려진 밝은 영역의 특징적인 패턴을 찾습니다. 편광 광학 현미경은 구형석에서 몰타 십자가의 존재를 밝혔습니다.

Research

JoVE Journal

Chemistry

Preparing Microscope Slide with Fully-Grown Amyloid Spherulite

Polarization-Sensitive Two-Photon Microscopy for a Label-Free Amyloid Structural Characterization

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue ...