This paper describes how polarization-sensitive two-photon microscopy could be applied to characterize the local organization within label-free amyloid superstructures-spherulites. It also describes how to prepare and measure the sample, assemble the required setup, and analyze the data to obtain information about the local organization of amyloid fibrils.
Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer’s or Parkinson’s are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.
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.
1. Preparing the microscope slides with fully-grown spherulites
NOTE: See the Table of Materials for details about all materials, reagents, and equipment used in this protocol. All solutions were prepared with deionized water (18.2 MΩ·cm at 25 °C) obtained from the water purification system.
2. Building and aligning the system
NOTE: A schematic representation of a polarization-sensitive two-photon microscope can be found in Figure 1.
3. Measurement of the bovine insulin spherulites
NOTE: To perform all the described ps-2PF measurements, hand-written software was used, which controls the positions of the piezoelectric stage and half-waveplate and collects the signal from both photodiodes, allowing for plotting of the XY scans (raster scans) from selected areas on microscope slides as well as polar graphs from specific sample coordinates. Additional notes have also been added to the protocol, which will allow users to perform the measurement without it, since both the piezo-stage and rotation stage used to rotate the half-wave plate could be controlled using their own controllers or corresponding software. Still, it is strongly recommended to write an algorithm combining the rotation angle of a half-wave plate with 2PEF intensity collected with both photodiodes since this correlation (polar graphs) is crucial for proper data analysis resulting in structural information.
4. Determination of local fibril ordering inside the bovine insulin spherulites
NOTE: All numerical calculations connected to the data analysis were done using the Python programming language and based on the functions available in libraries NumPy and SciPy. Plotting the data requires the Matplotlib library. All calculations are based on formulas presented in supporting information in one of the papers by Obstarczyk et al.6.
The presented protocol provides step-by-step guidance through the preparation of amyloid superstructures for testing with ps-2PFM, construction of the microscopic system, and measurements of the proper sample. However, before the final set of measurements, it is vital to properly align the APDs with an isotropic reference, which should result in collecting a symmetrical signal of similar shape and intensity on both detectors (Figure 4C). Even minimal differences between the intensities measured on the detectors in X and Y axes should be taken into account in further measurements and especially, during the analysis as an appropriate correction factor. After mounting, the sample containing single spherulites, giving a characteristic Maltese cross on the POM as in Figure 2B, and after performing a 2PFM raster scan, the image should look similar to what is shown in Figure 5A, which is consistent with reported 2PFM raster scans of spherulites6,7 and different organized biomolecules11,12. One may observe some changes in the location of the axis with the highest brightness, which is related to the fact that fluorescence signal intensity strongly depends on the polarization of the excitation beam. Therefore, it is necessary to verify which position of the half-waveplate corresponds to the excitation of the sample along the X (Figure 5A, top) and Y (Figure 5A, bottom) axes. It is also worth noting how polar graphs change while performing a full polarization analysis from different selected spots. Outside the spherulite, the polar graph looks like a collection of artifacts and random signal noise spikes (Figure 5B, I). Such a graph may also indicate the drift of the spherulite between measurements and require finding new coordinates of the entire structure by another 2PF raster scan. Properly measured polar graphs from highly ordered locations of insulin spherulite along the X and Y axes are shown in Figure 5B II and III, respectively. They can have different shapes and geometries, depending on the local orientation and organization of fluorophores measured from the selected spot7.
The last step of the protocol is focused on the analysis of the obtained data using an algorithm based on a mathematical model combining the orientation of amyloid fibers in the XY plane Φ, the associated fluorophore transient dipole moments Ψ, and their aberrations ΔΨ (Figure 6) with intensity and
of two-photon induced fluorescence. Correctly implemented functions presented in the protocol should, after entering the appropriate input data (protocol step 4.9), produce identical polar graphs as those presented in Figure 7. Any deviations-different data orientation, intensities, or shapes of simulated
and
-indicate errors in the code. If the model works, one can proceed to the last step of the protocol-fitting the simulated data to real data obtained from the ps-2PFM measurement. Chosen values of Φ and ΔΨ with the highest correlation coefficients and convergence between the simulated intensities and measured signal should lead to similar images as shown in Figure 8. There should be the best possible fit of
and
functions to the overall orientation and shape of
and
and the values of Φ and ΔΨ should correspond to the local orientation of fibrils and fluorophores in the measured spot on spherulite. Similar models and fitting methods could also be used for the determination of the local organization of other biomolecules like DNA11.
Figure 1: Two-photon polarization-sensitive microscopy setup. Scheme showing the two-photon microscope setup used for the polarization-sensitive two-photon excited fluorescence measurements of bovine insulin spherulites. Two-photon excited emission horizontally and vertically polarized (to microscope sample plane) components are depicted with IX, and IY, respectively. Abbreviations: SP = sample plane; O = objective; DM = dichroic mirror; λ/2 = half-wave plate; LPF = long-pass filter; BE = beam expander; P = Glan polarizer; Ti: Sa = Titan: sapphire laser light source; M = Mirror; SPF = short pass filter; PBS = polarization beam splitter; L = optical lens; APD = avalanche photodiode. Please click here to view a larger version of this figure.
Figure 2: Scheme showing the slide preparation/photo. (A) Scheme showing the sealed sample preparation; (B) photographs of the subsequent steps for the sample sealing. I: a 100 µL aliquot of the spherulite solution on the microscopy slide with a well, II: coverslip dropped on top of the solution; III: tight seal formed from deposited polymer (mountant). Please click here to view a larger version of this figure.
Figure 3: Quality control of the insulin spherulites under a polarized-light microscope. (A1,B1) Brightfield mode as well as under (A2,B2) crossed polarizers. Characteristic Maltese cross pattern can be observed on the corresponding images taken with crossed polarizers. (A) Spherulites aggregates with structural distortions, (B) high-quality isolated spherulite. Scale bars = 5 µm. Please click here to view a larger version of this figure.
Figure 4: Testing the alignment of the ps-2PFM system. (A) Concentric defocused image of the fluorescein fluorescence as seen without (A1) and with (A2) dichroic mirror, (B) photodamaged spherulite with burned holes arising due to the elongated polarization analysis in selected points along x and y directions, (C) two-photon excited emission components in dependence on incident light polarization angle as registered for an isotropic sample (fluorescein solution). and denote emission components measured by avalanche photodiodes measuring the X and Y emission polarization axis, respectively. Scale bars = 5 µm (A1,A2), 10 µm (B). Please click here to view a larger version of this figure.
Figure 5: Exemplary 2PFM raster scans and polar graphs from label-free insulin spherulite. (A) 2PF intensity raster scans of label-free insulin spherulites: polarization of excitation light: (A1) horizontal polarization, (A2) vertical polarization, and emission are denoted with white arrows, and the inset shows the same spherulite imaged under a standard polarized light microscope with crossed polarizers. (B) Polar graphs derived from three spots denoted on the A1 scan (B1, I; B2, II; B3, III). Ix and Iy denote emission components measured by avalanche photodiodes measuring the X and Y emission polarization axis, respectively. Abbreviation: 2PF = two-photon fluorescence. Please click here to view a larger version of this figure.
Figure 6: Conical distribution of the emission dipole of the dye (half angle, Ψ) with respect to the long fibril axis. The dashed line shows the long fibril axis. The rotation of the fibril in the XY microscope sample plane is described by the angle. Aberrations of Ψ due to the molecular rotations in filaments are described by ΔΨ. This figure is from Obstarczyk et al6. Please click here to view a larger version of this figure.
Figure 7: Simulated intensity of polarized two-photon excited fluorescence. Fluorescence calculated for (A) Φ = 1°, Ψ = 1°, ΔΨ = 1°; (B) Φ = 1°, Ψ = 30°, ΔΨ = 1°; (C) Φ = 1°, Ψ = 30°, ΔΨ = 60°. Red, blue, and yellow lines correspond to ,
, and
+
, respectively. Φ angle describes the rotation of the fibril in the XY microscope sample plane, Ψ – conical distribution of the emission dipole, and ΔΨ– aberrations of Ψ due to the molecular rotations in filaments. All other parameters were identical in all cases: K1 = 2.945, K2 = 0.069, K3 = 1.016, γ = 0.01, and δ = 0.98845. Please click here to view a larger version of this figure.
Figure 8: Polar graphs of experimental datasets. (A,B) Exemplary polar graphs after fitting two experimental datasets collected during ps-TPFM measurements of bovine insulin spherulites. Measured and
two-photon excited emission components for X and Y emission polarization axis are presented with red and blue dots, respectively; meanwhile, solid lines present the corresponding simulated two-photon excited fluorescence emission components for X and Y emission polarization axis
and
. Please click here to view a larger version of this figure.
Polarization-sensitive two-photon microscopy is a valuable tool for studying the local ordering of fibrils inside the amyloid superstructures, requiring only small modifications of the standard multiphoton setup. Since it operates on nonlinear optical phenomena, reduced angular photo-selection and enhanced axial resolution can be achieved compared to one-photon excited fluorescence microscopy methods. In addition, it leads to lower light scattering, lower phototoxicity, and deeper sample penetration when compared to the one-photon excitation fluorescence microscopy techniques19,20. As we already proved, it is well-suited for measurements of densely-packed aggregates of proteins such as spherulites, which can be performed in a label-free manner21.
However, to effectively use the method described here, attention should be paid to several key issues. Starting with sample preparation and its quality, namely, incubation, always ensure that amyloid superstructures have grown correctly. In the case of spherulites, we used a polarized optical microscope with crossed polarizers to localize mature spherulites, which give a characteristic Maltese cross pattern, and have a radius of ~5 µm16. However, spherulites are heterogeneous, and distinctive structures can be observed within the sample. Therefore, it is crucial to pick separated and non-distorted species. As for the measurement system itself, it is crucial to control the polarization of incident light so that the polarization of the beam at the entrance to the microscope body should be precisely determined and the depolarization introduced by the optical elements used should be minimized22. To assure the best performance, we recommend using high-quality silver mirrors and optical filters matched to the excitation and fluorescence emission wavelengths. Due to the key role of light polarization also in the emission path, it is essential to measure an isotropic reference sample before the actual measurement. If the excitation path and APD detectors are aligned correctly, one should see an equally intense signal on both detectors as presented in Figure 4C. Reference should generate a strong two-photon excited fluorescence at relatively low laser power to avoid photobleaching; we used fluorescein since its two-photon absorption cross-sections for different wavelengths were already reported in the literature23.
To correctly determine the arrangement of structures inside the spherulites, a lot of attention should also be devoted to data analysis. The basic assumption of this model is the collinearity of the transition dipole moment of light absorption and emission. Under that assumption, aligning the transition dipole moment of the molecule parallel to the polarization of the incident light allows for its combination with the internal structure of the tested sample, yielding the highest emission intensity. The given model can also be used as a good approximation for small differences in the angle between the two dipole moments8 but requires further modifications for large deviations. Therefore, as described, some assumptions regarding the sample must be taken into consideration a priori to imaging and data analysis. In the model case on which the simulation and the equations used in this method are based, the main source of depolarization in the system is the dichroic mirror. Therefore, before proceeding with the data analysis, it is necessary to determine the parameters responsible for the depolarization introduced by the dichroic mirror due to their impact on the shape of the collected polar graphs and consequently, the correct determination of the organization of the molecules within the examined structure during fitting18. This becomes a key problem, especially in the case of measurements for several wavelengths, since the change in the ellipticity of the dichroic mirror is distinctively wavelength-dependent12. Induction of depolarization can strongly affect the characteristics of the collected signal11. Last but not least, when creating the script for data analysis, one should pay attention while introducing mathematical functions and variables presented in the last part of the protocol. To avoid errors, focus on features such as multiple function calling and global parameters, which will also significantly speed up the analysis time.
We should also mention the most common problems one may encounter when using the described method. The sample preparation time is very much dependent on the speed of mountant hardening. To speed this up, we recommend that the samples be prepared in a warm, dry place and the spherulite solution is sealed before mounting it in a microscope. Measurements performed too early may lead to unsealing of the slide and destruction of the used objective lens. In addition, despite proper sample preparation, small spherulites may float in the solution during the measurement due to the material stress introduced by the objective. To avoid this, we advise scanning isolated middle-size structures since the largest ones are commonly aggregates. It is recommended to check whether the scanned spherulite has moved after data acquisition, in comparison to the image before starting the measurement. In addition, despite the correct alignment of all optical elements and detectors used to measure the ps-2PFM, the measured intensities and
of the isotropic reference may still differ slightly in intensity. In such a case, it is necessary to take this into account when analyzing the data from the sample measurements by multiplying one of the intensities by the correction factor determined during the reference measurements. Last, but not least, it is possible that during the fitting procedure, the script will not be able to find any matching values of Φ and ΔΨ. This may be due to several factors-data were collected from a disorganized spot on a sample, for example, the core of a spherulite; the studied structure was not fully formed during incubation; incorrect simulation of
and
; using incorrect dichroic mirror parameters γ and δ; too large step set for Φ and ΔΨ angles between each iteration during fitting. In the case of a disorganized spot, we recommend trying to collect data from points lying closer to the perimeter of the examined structure. In the case of an incompletely formed structure, repeat the incubation or search the slide for other structures. In the case of incorrect simulations, manually check whether by changing the parameters Φ, Ψ, and ΔΨ, the simulated intensities
and
change and correct any errors in the code. In the case of incorrect dichroic mirror parameters, carefully check the γ and δ parameters of the dichroic mirror used during the measurements. In the case of a large step set for the angles during fitting, reduce the step between successive iterations to 2º or 1º. It should also be remembered that in the case of strong local disorganization of the structure, R2 will always be lower, often in the range of 0.5–0.6. This is because the presented model describes highly ordered molecules where most of the transient dipole moments are aligned in a similar direction; hence, matching amorphous or highly disordered structures leads to lower correlation coefficients. Therefore, it is crucial to possess at least some structural information on the sample before the measurement to correctly analyze the obtained data.
It is also worth noting that the presented method possesses several limitations. Data analysis could result in several values of Φ and ΔΨ angles with high enough correlation factors, requiring the experimenter to use their knowledge and experience to select appropriate values. In this case, the boundary conditions must be taken into account, such as that fluorophore cone half-angle Ψ must always be bigger than its aberration ΔΨ. In addition, when comparing our data with the literature, it is worth noting that some papers describe Ψ as a full-cone angle, while we operate on a half-cone angle. Another limitation is the measurement resolution, limited to a sub-micron resolution. Because of that, the photo-selectivity of the system and resulting local organization determination are based on the averaged signal from fibrils excited in the focal spot rather than single structures. Moreover, the sample itself needs to possess sufficient fluorescence quantum yield, as long exposure under fs incident laser power (necessary to acquire data for polar graphs) may be destructive and impact the results.
Despite these difficulties, we believe that the method presented in this paper has a wide range of applications, for label-based and label-free imaging of biostructures in hydrated and complex environments. We had previously shown that local fibril ordering calculated from ps-2PEF data correlates with information derived from transmission electron microscopy7. Therefore, we confirmed the validity of ps-2PEF in bio-imaging and further expanded the knowledge on the structural ordering of amyloid superstructures. This method can be applied to learn about the origin of intrinsic fluorescence observed in the case of various components of biological samples, such as the autofluorescence of protein aggregates—amyloids. Given relevant data about the orientation of absorption/emission dipole moment directions with respect to the long axis of amyloid fibrils, the optical properties of the fibrils can be correlated with their structure6. We indicated that for structures with an already-determined Ψ angle, this technique allows the detection of distinct structural features of amyloid superstructures and polymorphs of insulin superstructures based on the level of their organization7. As previously noted in the protocol, the presented setup could also be applied for polarization-sensitive second harmonic generation measurements, which is also a label-free two-photon microscopy technique24. This will require not only mounting different optical filters but also changing the formulas used for data analyses25. Moreover, with the addition of a properly aligned quarter waveplate, it could be used to excite the sample with circularly polarized light, which might lead to the generation of chiral nonlinear optical properties of amyloids since they are chiral biopolymers with confirmed strong chiroptical properties26. However, in such a case, the constantly rotating electric vector would lead to the constantly changing direction of the excited emission, making it impossible to determine any structural information using the equations presented in this manuscript. All in all, ps-2PEF is a promising tool for the determination of the organization within numerous complex bio-structures with ordered emission dipoles, such as protein aggregates, DNA, or tissues in a non-invasive way.
The authors have nothing to disclose.
This work was supported by Sonata Bis 9 project (2019/34/E/ST5/00276) financed by National Science Centre in Poland.
Sample preparation | |||
Coverslips, 24 x 24 mm | Chemland | 04-298.202.04 | |
DPX mountant for histology | Sigma-Aldrich | 6522 | Slide mountant |
Eppendorf Safe-Lock tubes, 1.5 mL, polypropylene | Chemland | 02-63102 | |
Eppendorf ThermoMixer C | Eppendorf | Used for spherulite incubation | |
HLP 5UV Water purification system | Hydrolab | Source of dionized water used in sample preparation | |
Hydrochloric acid (≥37%, APHA ≤10), | Sigma-Aldrich | 30721-M | |
Insulin powder from the bovine pancreas (≥25 units/mg (HPLC)) | Sigma-Aldrich | I5500 | |
Methanol (HPLC grade) | Sigma-Aldrich | 270474 | |
Microscope slides with a concave, 76 x 26 x 1 mm | Chemland | 04-296.202.09 | |
Olympus BX60 | Olympus | Polarized Optical Microscope used in Figure 2 | |
PTFE thread seal tape, 12 mm x 12 mm x 0.1 mm, 60 gm2 | Chemland | VIT131097 | |
Microscope ps-2PFM setup | |||
Chameleon Ultra II | Coherent | ||
FELH0800 – Ø25.0 mm Longpass Filter | Thorlabs | ||
FESH0700 – Ø25.0 mm Shortpass Filter | Thorlabs | ||
IDQ100 photon-counting avalanche photodiodes | ID Quantique | ||
Multiphoton short-pass emission filter 720 nm | Semrock | ||
Mounted Achromatic Half-Wave Plate, 690-1200 nm | Thorlabs | ||
Nikon Plan Apo Oil Immersion 100x/1.4 NA | Nikon | ||
piezo 3D stage | Piezosystem Jena | ||
Polarizing Beamsplitter | Thorlabs | ||
S130C – Slim Photodiode Power Sensor, Si, 400 – 1100 nm, 500 mW | Thorlabs | ||
Software | |||
LabView 2018 | National Instruments | Version 18.0.1f2 | |
Matplotlib library | Version 3.3.2 | ||
NumPy library | Version 1.19.2 | ||
SciPy library | Version 1.5.2 | ||
Spyder Python 3 IDE | Version 4.1.5 |