Supporting Material Polarized THG Microscopy Identifies Compositionally Different

Polarized THG Imaging of Lipid Droplets
Supporting Material
Polarized THG Microscopy Identifies Compositionally Different
Lipid Droplets in Mammalian Cells
Godofredo Bautista,†* Simon G. Pfisterer,‡§ Mikko J. Huttunen,†# Sanjeev Ranjan,‡§ Kristiina
Kanerva,‡§ Elina Ikonen,‡§ and Martti Kauranen†
†
Department of Physics, Tampere University of Technology, Korkeakoulunkatu 3, 33720
Tampere, Finland
‡
Institute of Biomedicine, Anatomy, University of Helsinki, Haartmaninkatu 8, 00290
Helsinki, Finland
§
Minerva Foundation Institute for Medical Research, Tukholmankatu 8, 00290 Helsinki,
Finland
#
COMP Centre of Excellence and Department of Applied Physics, Aalto University, P.O.
Box 15100, FI-00076 Aalto, Finland
SUPPORTING METHODS
Generation of synthetic LDs with pure and mixed TAGs
Appropriate amounts of TAG (glyceryl trioleate) and CE (cholesteryl linoleate), made in
chloroform:methanol mixture (9:1 ratio), were transferred into glass tubes. The TAG/CE
molar ratio was 1:1 for mixed artificial LDs. The solvent was removed under a stream of
nitrogen and the residue subsequently maintained under reduced pressure for at least 2 h. The
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Polarized THG Imaging of Lipid Droplets
lipid film was then hydrated at 60oC for 1 h in pure water. The final concentration of the lipid
dispersion was 100 mM. The hydrated lipid was vortexed vigorously to generate synthetic
LDs. The synthetic LDs were first imaged by an AX70 microscope (Olympus) with a digital
camera. For THG imaging the synthetic LDs were immobilized on 3% low-melting agarose
gel between a microscope slide and a coverslip with a spacer. Glyceryl trioleate, cholesteryl
linoleate and low melting agarose were obtained from Sigma-Aldrich.
THG modeling
The THG responses were calculated using Green’s function approach (1). The fundamental
tightly focused excitation field was calculated using vector diffraction theory. In our
experimental configuration the sample solutions were sandwiched between two 170 µm thick
coverglasses with a 200 µm spacing. This geometry potentially causes minor aberrations, i.e.,
focused beam distortion, which were considered in the calculations following the approach
described in Chapter 3.9 of Ref. (2). We assumed that the incident electric field experiences
an air-water interface and is focused a 270 µm distance away from the interface, to the center
of the sample solution. The LD was assumed to be spherical with a radius of approximately
0.8 µm and surrounded by an aqueous solution with a refractive index of 1.33. The refractive
index of the LD was assumed to be equal to the surrounding solution and both media were
assumed to have negligible dispersion. Therefore, only free-space Green’s functions were
considered, and further linear scattering effects at both the fundamental (ω) and the thirdharmonic (3ω) frequency were neglected. The THG responses from a focal volume of 2 × 2
× 2 µm3 were calculated using (1)
E (=
3ω, R )
∫∫∫ G ( 3ω, R − r ) P ( 3ω, r ) dr
V
(1)
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Polarized THG Imaging of Lipid Droplets
where P ( 3ω, r ) is the induced third-order nonlinear polarization at source point r,
G ( 3ω, R − r ) is the associated Green’s function and R denotes the far-field point, and the
integration extends over the sample volume V. When linear scattering effects are neglected
and only electric dipole interactions are considered, the excitation field equals the incident
field
and
nonlinear
polarization
P ( 3=
ω, r ) χ (3) ( 3ω, r ) E ( 3ω, r ) E ( 3ω, r ) E ( 3ω, r ) ,
where
is
χ (3)
given
is
the
by
third-order
susceptibility of the sample. The radiated third-harmonic field is assumed to be collected with
a condenser having a numerical aperture of 0.5 and subsequently directed on a detector. The
measured signal is then proportional to THG field intensity integrated over the surface
I ( 3ω) ∝ ∫∫ E ( 3ω,R ) dA
2
A
(2)
where A is restricted by the numerical aperture of the condenser. The resulting integrations
were numerically performed by using the Simpson’s quadrature typically discretizing the
focal volume over a 101 × 101 × 101 grid. The resulting THG field at the image plane was
typically evaluated at approximately 500 points, to ensure good convergence of calculations.
In order to understand how polarization effects influence the THG images, the
calculations were repeated while raster-scanning the excitation field over the LD in steps of
0.08 µm in x- and y-directions. The LDs were assumed to be isotropic. In consequence, the
relative susceptibility component values of Χ iiii = 3, Χ iijj = 1, where i,j={x,y,z}, were used for
the non-zero components of the third-order susceptibility tensor. As has been shown earlier,
the nonlinear responses of a weakly nonlinear surrounding medium can be taken into account
(3)
(3)
(3)
(3)
=
χ sample
− χ medium
by introducing an effective sample susceptibility of χ sample
where χ medium is
,eff
the susceptibility of the surrounding medium which in our case is water (1).
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Polarized THG Imaging of Lipid Droplets
In order to take into account the possibility that the nonlinear response of the
phospholipid shell surrounding the LD is different from that of the interior parts, we included
an outer shell with thickness of ~40 nm with different effective relative susceptibility
(3)
χ monolayer
,eff into the model. The possible molecular orientation effects in the shell to the THG
responses were neglected, and only isotropic responses were considered. The effective
(3)
relative susceptibility χ monolayer
,eff was assumed to be 100 times larger than the effective
(3)
susceptibility c core
,eff inside the LD. This factor is rather arbitrary, because no better estimate
is possible due to the large uncertainties in the literature values for the susceptibilities of
multilamellar lipid structures (3), triglycerides (4), and water (4-6). In particular, variations in
the value of water can be used to justify almost any value for this factor. In addition, the
phospholipid shell could affect the packing of the interior lipid molecules near the shell and
the exterior proteins could contribute to the effective nonlinear response of the shell.
The simulations result in a typical image shown in Fig. 2 a for orthogonal linear
polarizations. By taking line graphs of the image in the horizontal and vertical directions, the
peaks along the two directions are not identical (Fig. 2 c). More specifically, the peaks in the
line graph along the direction of linear polarization are about 10% higher than those for the
orthogonal polarization. Note that such polarization dependence is traditionally considered to
arise from orientational effects. However, in the present case it arises from the focusing
properties of the vectorial fields. The dependence of the THG signal on linear polarization is
therefore not necessarily an indication of ordering.
We repeated the simulations also for other factors between the interior and shell
susceptibilities. This only affected the difference between the peaks in the two line graphs but
not the fact that the peaks along the direction of polarization are higher.
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Polarized THG Imaging of Lipid Droplets
Confocal microscopy and image quantification
Fixed macrophage cell samples were stained with LipidTOX green neutral lipid stain (1:100,
Life Technologies) in PBS / 1 mM DAPI (4',6-diamidino-2-phenylindole) for 30 min and
imaged in PBS. Confocal imaging was performed using a Leica TCS SP8 inverted confocal
microscope (Leica Microsystems, Germany). Image stacks with 20 Z-slices per cell were
acquired using a 63× HC PL APO CS2 glycerol immersion objective (NA = 1.3). Z-Stacks
were acquired using identical laser powers and detector gains. Mean cellular intensities were
quantified using ImageJ.
Visualization of TAG and CE storage using fluorescent tracers
Raw 264.7 macrophages and Y1 adrenal cells were seeded in Lab-Tek live cell chamber
slides and incubated in DMEM with 10% FBS plus Pen/Strep [DMEM/F12(1:1) with 15%
FBS, 2% horse serum plus Pen/Strep for Y1 cells] for 24 h. Cells were washed with PBS and
incubated
in
DMEM
(DMEM/F12
for
Y1)
with
5%
LPDS
and
50
µM
cholesterol/cyclodextrin plus 400 µM oleic acid, together with 1 µM BODIPY® FL C 12
cholesteryl ester and 0.5 µM BODIPY® 558/568 C 12 (both from Life Technologies) for 24 h.
Cells were washed with PBS, and incubated with CO 2 -independent medium (Life
Technologies) for live cell confocal microscopy (Leica TCS SP8 with environmental
chamber).
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Polarized THG Imaging of Lipid Droplets
SUPPORTING FIGURES
FIGURE S1: (a) TAG- and CE-enriched LDs display different labelling efficiency with
LipidTOX green neutral lipid stain (green channel). Images were acquired using brightfield
and confocal microscopy with identical laser intensities and detector gains for each treatment.
The DAPI-stained cell nucleus is also shown (blue channel). Scale bar = 5 µm. (b) The
images were quantified to obtain mean LipidTOX intensities of LDs in individual cells.
(Control n = 60 cells, TAG-enriched n= 100 cells, CE-enriched n = 95 cells *** p ≤ 0.001.
The data are expressed as mean ± s.e.m.).
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Polarized THG Imaging of Lipid Droplets
FIGURE S2: Brightfield microscopy images of the regions of interest (ROIs) in Figs. 3 and 4.
Scale bar = 5 µm.
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Polarized THG Imaging of Lipid Droplets
FIGURE S3: Visualization of TAG and CE storage in Raw 264.7 macrophages and Y1
adrenal cells using fluorescent tracers as in (7). Both cell types were incubated with 50 µM
cholesterol/cyclodextrin and 400 µM oleic acid for 24 h together with 1 µM BODIPY® FL
C 12 cholesteryl ester (BODIPY-CE) and 0.5 µM BODIPY® 558/568 C 12 (BODIPY-FA) and
subjected to live-cell confocal microscopy. In Raw 264.7 cells, the fluorescent tracers are
targeted to the same LDs, whereas in Y1 cells, they partition preferentially into separate LDs
as in (7). In the merged composite images, yellow indicates colocalization. Scale bar = 10 µm
and 5 µm (magnified area).
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Polarized THG Imaging of Lipid Droplets
FIGURE S4: Lipid quantifications performed in parallel to THG analysis in Fig. 5. Raw
264.7 cells were treated with control medium (Control) or 400 µM oleic acid + 2 µg/ml
Sandoz-58-035 (OA+PKF; resulting in 1.3% CE), 35 µM cholesterol/cyclodextrin plus 400
µM OA (35 µM Chol+OA; 41% CE), 50 µM cholesterol/cyclodextrin 400 µM OA (50 µM
Chol+OA; 52% CE) or 50 µM cholesterol/cyclodextrin (50 µM Chol; 99% CE) in control
medium for 24 h and subjected to lipid extraction and high performance thin layer
chromatography for CE and TAG quantification. The fraction of CEs in Fig. 5 was calculated
as % of CE+TAG (ng lipid / µg protein).
SUPPORTING REFERENCES
(1)
Cheng, J., and X. S. Xie. 2002. Green's function formulation for third-harmonic
generation microscopy. J. Opt. Soc. Am. 19:1604-1610.
(2)
Novotny, L., and B. Hecht 2006. Principles of Nano-Optics. Cambridge University
Press, Cambridge.
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Polarized THG Imaging of Lipid Droplets
(3)
Zimmerley, M., P. Mahou, ..., E. Beaurepaire. 2013. Probing Ordered Lipid
Assemblies with Polarized Third-Harmonic-Generation Microscopy. Phys. Rev. X. 3:011002.
(4)
Débarre D. and E. Beaurepaire. 2007. Quantitative characterization of biological
liquids for third-harmonic generation microscopy. Biophys J. 92(2):603-12.
(5)
Kajzar, F. and J. Messier. 1985. Third-harmonic generation in liquids. Phys. Rev. A
32:2352.
(6)
Boyd, R. Nonlinear Optics. Academic Press, U.S.A.
(7)
Hsieh, K., Y. K. Lee, ..., A. R. Kimmel. 2012. Perilipin family members preferentially
sequester to either triacylglycerol-specific or cholesteryl-ester-specific intracellular lipid
storage droplets. J. Cell Sci. 125:4067-4076.
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