ARUP; ISBN: 978-0-9562121-5-3 - CMBBE 2012 - Cardiff University

differentiation was induced by changing the GM to a differentiation medium (DM). The
DM consisted of the aforementioned GM, supplemented with 5μg/ml insulin (Sigma),
1μM dexamethasone (Sigma), and 0.5μM 3-isobutyl-1-methylxanthine (IBMX; Sigma).
Three days after inducing differentiation, the DM was replaced by a supporting medium
(SM) consisting of the GM supplemented with 5μg/ml insulin (Sigma). The SM was
changed every 2-3 days.
3.2 Wide field digital interferometric microscopy
17 days after differentiation was induced in the cultures, we evaluated the stiffness of
the differentiated adipocytes using wide field digital interferometric microscopy
(WFDI), described as follows (Shaked et al., 2010, 2011). Light from a coherent source
(laser 18mW HeNe) was split into reference and object beam by a beam splitter. While
the object beam was transmitted through the sample and magnified by a microscope
objective (60×, 0.85 numerical aperture), the reference beam was only transmitted
through a microscope objective that was similar to the object-beam microscope
objective. These two beams were then combined together with a small angle to each
other (off-axis holographic geometry). Using a lens, positioned in a 4f configuration
with each of the microscope objectives, the combined beams were projected onto a
digital camera (1280×1024, 5.2 μm×5.2 μm pixels). The 4f configuration allowed
projection of the amplitude and phase distributions of the sample onto the digital
camera. The digital hologram acquired by the camera was the intensity of the
summation of the object and reference waves:
(1)
where and were the sample and reference field distributions, respectively,
was the spatial phase associated with the sample, related to the angular shift between
the sample and the reference fields, and was the direction of the angular shift. The
digital processing method started with a digital two-dimensional Fourier transform. The
resulting spatial-frequency contents included reference- and sample-field
autocorrelations (as a result of transforming the first two elements of Eq. (1)) that are
located around the origin of the spatial spectrum, and cross-correlation terms (as a result
of transforming the exponential term in Eq. (1)), each located at a different side of the
spatial spectrum. The angle between the object and reference beams determined the
exact locations of the cross-correlation terms. One of the cross-correlation terms was
then isolated and centered. A digital two-dimensional inverse Fourier transform on the
result yielded . Given the transparency of the cells, weak
amplitude was assumed and the phase argument of the result was the phase
profile of the sample, given as:
(2)
where was the illumination wavelength (632.8 nm), was the spatially varying
integral refractive index (along the cell thickness, (Bolin et al., 1989),
(Curl et al., 2005)), was the medium refractive index (1.33)
(Bolin et al., 1989), was the spatially varying thickness profile of the cell, and
was the height of the cell medium. The value of was measured in places
where there were no cells located. We acquired 315 interferograms at ~21 frames/s. The
temporal changes in the cell thickness (optical fluctuations) were converted into the
optical spring factor of the cell which indicates on the cell stiffness:
(3)
where was the effective local spring constant, was Boltzmann’s constant,
3