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

אפיון ספרוליטים במיקרוסקופ דו-פוטוני רגיש לקיטוב

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.

מחבר זרקור: הדמיה לא פולשנית של מבנים ביולוגיים מורכבים באמצעות מיקרוסקופ שני פוטונים רגיש לקיטוב 

מיקרוסקופ שני פוטונים רגיש לקיטוב לאפיון מבני עמילואיד ללא תוויות

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  

הכנת שקופית מיקרוסקופ עם ספרוליט עמילואיד בוגר במלואו

כדי להתחיל, לשטוף ביסודיות את שקופיות זכוכית המיקרוסקופ עם מים. לאחר מכן, לאחר טבילת המגלשות במתנול, הניחו להן להתייבש בתנאי סביבה על מגבון ללא אבק. לאחר מכן, באמצעות פיפטה אוטומטית, להעביר 100 מיקרוליטר של תמיסת אינסולין ספרוליט לבאר באזור המרכזי של שקופית המיקרוסקופ.בזהירות לכסות את הפתרון עם כיסוי להחליק, הימנעות היווצרות בועות אוויר. לאחר מכן, באמצעות פיפטה אוטומטית, הפקידו את התושבת לאורך שולי החלקת הכיסוי על המגלשה כדי לאטום את תמיסת הספרוליט המקורית. השאירו את הדגימה בתנאי סביבה כדי שהתושבת תתקשה.באמצעות מיקרוסקופ אופטי מקוטב עם מקטבים מוצלבים, לאשר אם spherulites אינסולין נוצרו כראוי. סרוק את הדגימה, מחפש דפוס אופייני של אזורים בהירים, המכונה צלב מלטזי. המיקרוסקופ האופטי המקוטב חשף את נוכחותו של הצלב המלטזי בספרוליטים.

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 ...