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.
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 |
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.
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.
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.