polarization-sensitive two-photon fluorescence microcopy of bovine insulin spherulites

Characterizing Spherulites with Polarization-Sensitive Two-Photon Microscopy

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.

Author Spotlight: Non-Invasive Imaging of Complex Bio-Structures Using Polarization-Sensitive Two-Photon Microscopy 

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

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 Polarization-Sensitive Two-Photon Fluorescence Microcopy of Bovine Insulin Spherulites at JoVE.com

Polarization-Sensitive Two-Photon Fluorescence Microcopy of Bovine Insulin Spherulites  

After building and aligning the polarization-sensitive two-photon microscope system, according to the displayed schematic representation, test the optics alignment quality with an isotropic reference sample like fluorescein embodied in an amorphous polymer. To do so, switch on the excitation laser and measure the 360 degree polar graphs of the reference sample for a zero half-wave plate. Adjust the half-wave plate angle to correspond to the X and Y axes of the sample plane, and check if both photodiodes collect similar intensities of two photon-excited emission components.To analyze the sample, mount the glass slide with this spherulite specimen on the Piezoelectric Stage, ensuring the face of the slide containing the cover slip faces the high numerical aperture immersion objective. Then secure the glass slide with tape. Next, by adjusting the X, Y, and Z positions using the microscope knobs, focus the objective on the spherulite in the solution.Focus specifically on the central area of the entire structure, as spherulite diameters are usually in the range of tens of microns. Then center this spherulite in the field of view in the microscope plane. Determine the size of the XY scan by adjusting the number of Piezo Stage steps in X and Y directions to cover the entire structure's area.Adjust the Piezo Stage parameters such as scanning speed, step size, and range to cover the area of the whole spherulite. Open the shutter, turn on the photodiodes, and collect the X and Y emission components of two-photon excited fluorescence intensity for every single step of the selected scanning area for the excitation beam polarized corresponding to the X and then the Y axes. Then scan this spherulite sample, resulting in two-photon excited fluorescence.To perform full polarization analysis from the specific spot on the sample, turn off the excitation beam. Take specific coordinates of Piezo Stage corresponding to the chosen location on this spherulite where the information about the orientation of molecules is required with the submicron resolution. Adjust the Piezoelectric Stage to center the field of view at indicated X and Y coordinates.After turning on the excitation beam, turn on the 180 degree rotation of the half-wave plate to perform a full 360 degree polarization analysis of the X and Y emission components of the two-photon excited fluorescence emission. The image of the insulin spherulite obtained after performing a 2PFM raster scan was consistent with those reported for a spherulite. The position of the half-wave plate corresponding to the excitation of the sample along the X and Y axes was also verified.The polar graphs changed while performing a full polarization analysis from different selected spots. Outside the spherulite, the PLO graph looked like a collection of artifacts and random signal noise spikes. Properly measured polygraphs from highly ordered locations along the X and Y axes had different shapes and geometries depending on the local orientation and organization of fluorophores.

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197 조회수.  Wrocław University of Science and Technology. 편광에 민감한 이광자 현미경 시스템을 구축하고 정렬한 후 표시된 회로도 표현에 따라 비정질 폴리머에 구현된 플루오레세인과 같은 등방성 참조 샘플로 광학 정렬 품질을 테스트합니다. 이렇게 하려면 여기 레이저를 켜고 제로 반파장 플레이트에 대한 참조 샘플의 360도 극좌표 그래프를 측정합니다. 샘플 평면의 X축과 Y축에 대응하도록 반파장 플레이트 각도를 조정하고, ...