Spinning Disk Anisotropy
A technical overview...
Anisotropy imaging can be used to elucidate dynamic molecular organization in living cell
studies. For optimum speed and dynamic range, laser spinning disk confocal imaging is the
best tool currently available. We have overcome a limitation in the standard CSU scan head
to enable use for real time confocal imaging of fluorescence anisotropy.
The technique of fluorescence anisotropy is well-known in
fluorescence spectroscopy and biochemical assays (Lakowicz 2006)
and it has been used in epi-fluorescence microscopy in living cells
(Lidke et al 2003, Sharma et al 2004, Goswami et al 2008) and even
in super-resolution imaging (Gould et al 2008). However, when
implemented in point scanning confocal instruments with live cell
specimens it has suffered from photo-bleaching, low frame rates and
temporal skew resulting from sequential point scanning.
The CSU (Ichihara et al 1996), manufactured by Yokogawa Electric
Corporation, is the leading spinning disk confocal scanner on the
market today and finds widespread application in live cell confocal
imaging. The various CSU models provide scan rates up to 2000
frames per second, with good confocality (~1 um FWHM) and low
background across the visible range (400-650 nm). Unfortunately,
the CSU cannot be used in its native form for anisotropy studies
because its excitation path degrades the polarization of laser light. We
have overcome this limitation and we present here a spinning disk
confocal anisotropy system (SpiDA) which exploits simultaneous
dual camera detection of orthogonal emission polarizations to
provide high definition spatio-temporal anisotropy imaging.
We have worked with Dr Satyajit Mayor and his team at NCBS in
Bangalore, to bring this solution to fruition and a Revolution XD
systems now resides is in his laboratory, where it is heavily used for
confocal anisotropy studies in living cells.
2. Anisotropy and Homo-FRET
Isotropic is derived from the Greek and means quite simply "equal in
all directions". While anisotropic means not equal in all directions.
Fluorescence anisotropy measurements are based on the principle of
photo-selective excitation by polarized light.
Fluorescent molecules preferentially absorb photons whose electric
vectors (polarization) are parallel to their absorption (transition)
electric dipole. The dipole has a defined orientation with respect
to the molecular axis. Thus, when polarized light is incident on
a population of molecules it is absorbed preferentially according
to orientation. Further, the resulting fluorescence emission is also
aligned relative to the molecular axis and the relative angle between
absorption and emission polarization determines anisotropy.
Fluorescence anisotropy, r is defined as follows:
r(t) = (Ip(t) – Is(t))/ (Ip(t) +2 Is(t))
where Ip(t) is the fluorescence intensity parallel to excitation and
Is(t) is fluorescence intensity perpendicular to excitation (Lackowicz
As the notation indicates fluorescence anisotropy, r is a function
of time and decays with a relaxation time, Trot. The relaxation
time is a measure of rotational diffusion which occurs during the
lifetime of the excited state (typically 1-10 ns). In fluids molecular
rotation can take place in a few tens to hundreds of ps and as result
little anisotropy is observed. The rotational diffusion rates of larger
molecules, such as proteins, are of the same order as fluorescence
lifetimes and therefore anisotropy is sensitive to factors affecting
Anisotropy can therefore be used as an indicator of the state of
biological macro-molecules within the cell and its membranes,
including molecular size, aggregation and binding state (Lidke et al
When excited fluorescence molecules (donors) come close enough
to engage in dipole interactions (0.3-0.5 nm) with unexcited
fluorescence molecules (acceptors), an effect known as resonant
energy transfer (RET) can occur. Provided the excitation spectrum
of the acceptor overlaps the emission spectrum of the donor, this
can result in transfer of energy from donor to acceptor and is
non-radiative (no photon is emitted). The newly excited molecule
can now emit a photon, but its polarization will depend on its own
orientation. When averaged over an ensemble, the result of RET is
loss of polarization and reduction in anisotropy.
When the RET interactions happen between fluorescent molecules
of the same type the process is known as "HomoFRET", and can be
used to monitor molecular interactions and binding states. Varma and
Mayor (1998) used anisotropy to monitor HomoFRET interactions in
so-called lipid rafts and GPI-anchored proteins, which are located in
the plasma membrane of living cells. These structures are important
in key cellular processes and Mayor and co-workers have made
substantial contributions to understanding these structures and their
function using anisotropy imaging.
Figure 1 provides a visual summary of the principles of selective
excitation and how rotational diffusion and HomoFRET affect
polarization and anisotropy.
3. Polarization and the CSU
Polarization measurements in the Yokogawa CSU products shipped
from the manufacturer show that laser polarization is degraded in the
excitation optics of the instrument (Table 1). This was common in 5
different units tested and included CSU-10, 22 and X1 models. A low
extinction ratio precludes use of an unmodified CSU for anisotropy
imaging. In contrast the emission path was found to maintain
polarization with high fidelity enabling confocal detection of the
polarization state in all units tested to date.
|ALC + SMP/PM Fiber
|ALC + Fiber +CSUX1
|ALC + Fiber + CSU-P – see text
Table 1 - Polarization results from laser engine and SM/PM fiber
(before the CSU-X1), standard CSU-X1scanner and the Andormodified
CSUIX1 polarization solution (CSU-P). Standard CSUscanners
degrade input laser polarization as this table shows.
We found that a good solution to the polarization problem is to
integrate a high performance polarizer into the optical path of
the CSU at a position which affects the excitation (laser) light,
but not the detected fluorescence, whose polarization contains
the useful information. A custom carrier was designed for the
polarizer as shown in Figure 2A and this is mounted on a motorized
drive for insertion and removal under computer control. In this
way conventional intensity imaging can be re-selected without
4. Confocal Dual Camera Anisotropy Imaging
The epi-illumination system described in Varma and Mayor (1998)
was limited for dynamic studies because it used a single camera
and sequential detection of p and s polarization images using a
filter wheel. This approach introduces a time-skew between the
s and p channels. To overcome this Mayor's group constructed a
dual camera solution (Goswami et al 2009) and access to dynamic
data was extended. Post acquisition image processing is required to
achieve pixel alignment of the two images prior to the calculation of
However, a remaining problem with such a system is that emission
from microscopic polarization domains is masked by out of focus
fluorescence. Hence resolution, dynamic range and signal to
background ratio of anisotropy are all compromised. The SpiDA
system greatly reduces these effects by rejecting out of focus
fluorescence, allowing anisotropy domains to be monitored in greater
detail than ever before.
To achieve maximal temporal resolution and full field of view,
SpiDA employs TuCam, our dual camera adapter. TuCam is a
third generation adapter optimized for throughput, distortion and
ease of alignment and enables simultaneous detection of p and s
polarizations onto back-illuminated EMCCD cameras i.e. iXon3
897 - see figure 2B.
An image quality polarizing beam splitter is a critical component
of the system: it must be flat and mounted with minimal stress. We
utilize laser quality components with a surface flatness of <λ per
inch and radius of curvature ≥ 30m. This minimizes distortions and
lensing effects which can lead to focus error across the field of view.
The beam splitter is mounted in a kinematic assembly to provide
precision of adjustment and stability.
Mechanical stability is the most critical quality of an image splitter
to ensure robust and repeatable measurement. Drift or creep leads
to registration errors. Even though image registration to sub-pixel
precision requires image processing in software, this process is
driven by calibration whose temporal repeatability depends on the
physical design. We have optimized image registration in our own
iQ software, but an open source solution is also available in ImageJ
The iXon cameras deliver high signal to noise anisotropy imaging
with exposures in the 10-100 ms range. This provides a dynamic
imaging tool for protein-protein interactions and microstructure
modulation during events such as endocytosis and vesicle recycling.
Figures 4 and 5 show example calibration data and live anisotropy
This technical note describes the Andor solution for real time
confocal anisotropy imaging, which has been developed to overcome
inherent constraints in the CSU scan head for polarized excitation
of fluorescence. SpiDA utilizes a high performance image splitting
dual camera adapter, TuCam and ultra-sensitive EMCCD cameras to
deliver high contrast dynamic anisotropy data for live cell studies.
We highlight cooperation with a scientific research group whose
vision had not been realized previously. This process has now
delivered a solution which is generally available to the wider research
community as the SpiDA option for Revolution XD.
We take this opportunity to thank Dr Satyajit Mayor and his team
at NCBS for their support, feedback and collaboration during this