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Microscopy and imaging science: practical approaches to applied research and education (A. Méndez-Vilas, Ed.)
Competition for the Confocal Microscope?
M. Schropp1,2, Ch. Seebacher1,2, A. Deeg2, A. Dovzhenko3, Olaf Tietz3, K. Palme3, and R. Uhl1,2
1
Bioimaging Zentrum der LMU München, Grosshadernerstrasse 2-4, D-82151 Martinsried, Germany
TILL I.D. GmbH, Am Klopferspitz 19, D-82152 Martinsried, Germany
3
Institute of Biology II, Molecular Plant Physiology, Schänzlestr. 1, D-79104 Freiburg, Germany
2
Keywords: 3-D sectioning, live cell, confocality, speed, cell viability
1. Summary
Cells are 3D-objects and their function is only fully understood if they are examined in their original 3D-context. The
gold-standard for 3D-cellular-imaging is the confocal microscope, which employs optical means for removing
unwanted out-of-focus information. However, such a confocal point scanner is slow due its sequential scanning-mode,
and it is not very gentle to cells due to the high intensities needed for obtaining a decent signal-to-noise ratio during
short pixel-dwell-times. Spinning-disk systems, on the other hand, are fast and gentle by virtue of their multipoint
scanning, but their confocality suffers at the same time.
We will demonstrate a third approach, which combines the virtues of the two and avoids their draw-backs. Our novel
3D-Structured Illumination Microscope (SIM) employs 2-D hexagonal illumination patterns and combines optical (linescanning) and mathematical means for suppressing and removing unwanted out-of-focus information. By comparing the
three 3D-imaging techniques we can show that in all but the thickest samples SIM equals resolution and confocality of
the confocal point-scanner, yet it is at least an order of magnitude faster and more gentle to live cells. Compared with a
spinning disk-system, SIM is almost as fast – under realistic conditions equally fast – but it excels with respect to its
resolution (particularly in z) and its sectioning capabilities (confocality, not to be confused with z-resolution). We feel
that our XL-SIM-approach may well become the method of choice for the majority of applications which - so far - have
been dealt using classical confocal microscopes.
2. Introduction
Over the past decades the fluorescence microscope has developed into an indispensable tool for the study of cellular
processes in live cells. This was not only due to great progress achieved on the side of instrumentation, but also due to
the discovery of fluorescent proteins and the availability of genetic tools to express these proteins anywhere in live cells
under observation [1]. In order to detect and distinguish tiny signals from nanometer-scale objects, microscope
objectives must gather as much light as possible, and this, in turn, reduces their depth of sharpness, often to such an
extent that the sought after features are completely buried in out-of-focus haze. The classical cure for this problem is the
confocal microscope [2], which uses optical means for blocking unwanted out-of-focus light. While this gold-standard
of 3D microscopy - used in thousands of central imaging facilities - provides images sufficient for most purposes, it
suffers from two major drawbacks: (i) its sequential point-scanning takes time, hence image acquisition is slow and fast
biological processes cannot be followed in real time, and (ii) the short pixel dwell-time of the process necessitates
exceedingly high excitation intensities, which are invariably harmful for live cell material: they not only bleach the
respective fluorophores, but – by means of secondary photochemistry in the cells – they damage the cells under
observation to such an extent, that often one can only study the process of deceasing cells.
To overcome this, multipoint-scanners have been employed, which illuminate the sample not with a single excitation
focus, but with many hundred of them simultaneously [3]. Emission is then recorded with an equal number of
conjugated confocal pinholes. This parallel-approach accelerates image acquisition considerably, while, at the same
time, light can be collected for much longer time from a given fluorophore, hence the danger of photodamage is greatly
reduced. Consequently multipoint-confocals (in most cases spinning- disk systems) have become the gold-standard for
fast live-cell imaging, yet for every multipoint-confocal there are still ten point-scanning confocals, which are used for
tasks they are not really suited for. So what is the drawback of the multipoint-confocal? It is cross-talk between
neighboring illumination spots. In the following we shall describe an alternative approach, which uses a combination of
mathematical and optical means to remove unwanted out-of-focus information. It unites the sectioning capabilities of a
single-point scanner with the speed of a multipoint-scanner and thus qualifies to become a new gold-standard for 3Dlive cell imaging. In the following we will compare its performance to that of a point-scanning and a spinning-disk
confocal and – based on these results – we shall arrive at the conclusion, that our novel approach of a structured
confocal microscope, by combining the virtues of both and avoiding most of their respective drawbacks, constitutes the
method of choice for most applications requiring 3D-sectioning for live cells.
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Microscopy and imaging science: practical approaches to applied research and education (A. Méndez-Vilas, Ed.)
3. Results and Discussion
The confocal microscope (a) attributes all light passing through the pinhole to the position, where its focus lies at a
given point in time. Light originating in other planes is mostly, but not completely rejected, it also contributes to the
signal as long as its direction is such that it can pass the pinhole or appears to have passed it. That is why “confocality”
is always not as good as the theoretical z-resolution of a system and why single point confocals show a residual signal at
positions where no signal ought to be. The effect becomes increasingly evident with heavily stained samples, where
more fluorophores in other planes contribute to this kind of false signal.
In a multipoint confocal (b) the excitation volumes of neighboring foci overlap and form a continuum only a few
micrometers above and below the focal plane. This “bulk excitation” diminishes with z-distance, but at the same time
more pinholes are passed by “false photons” or virtually originate from them. Thus the “false-photon-contribution” to
the “real signal” is much more prominent than in a single point-scanner. In addition, with multipoint confocals
scattering becomes an issue, because it can create an additional class of false photons. As a consequence, z-stacks
obtained with a spinning disk confocal shows photons originating from certain object planes long before the focal plane
of the objective has reached these planes.
In a “structured illumination microscope” (SIM, c) the sample is also excited with a structured illumination
pattern, but no attempt is made to eliminate photons originating from outside the pattern by optical means. Instead these
photons are also recorded and used for later mathematical removal. To allow such mathematical tricks, one needs to
know not only intensity values when a given object-point is illuminated itself (left panel in fig. 1c), but also when it
ought to be dark and only neighboring areas are illuminated (fig. 1c, right panel). Thus SIM requires shifting a pattern
and recording several phase-images, from which suitable algorithms can extract the fraction of photons arising from
planes outside the focus and can remove them completely. Sectioning achievable with a SIM microscope is not only
(significantly) better than with a multipoint confocal, but also noticeable better than that of a point-scanning confocal as
we will demonstrate below!
a
b
c
grid shi
Fig. 1: Schematic drawing of the excitation and emission beam-paths in a single-point confocal system (a), a multipoint scanner
(b) and a structured illumination microscope (SIM, c).
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However, the saying that “there is no free lunch” also holds in microscopy: While optical removal of “out-of-focus
photons” also removes their statistical photon-noise1, their mathematical removal retains these “noisy photons”! Hence,
the more unwanted photons are removed mathematically, the more the resulting desired signal is polluted with noise
originating elsewhere. In essence this means: samples, which are thin, not highly scattering or not heavily stained, i.e.
samples with little out-of-focus information, can be trimmed to yield optimal tomographic information. However, the
more unwanted information needs to be removed, the more noise spills over to the sought after signal! Rescue comes
from combining optical and mathematical means for removing unwanted out-of-focus information. A slit-shaped lightbar is modulated two-dimensionally and scanned uni-directionally over the sample. The resulting slit-image is recorded
with a rolling shutter sCMOS camera, whose electronic slit-width is adjusted so as to reject (optically) all light
originating from areas outside the slit-width, and whose position is strictly synchronized with the movement of the slitshaped light-bar. The combination of the slit-scanner with the rolling shutter forms a slit-confocal microscope, which
differs from previous slit-confocals [4] in that the slit is modulated by a pattern as needed for SIM!
In conventional SIM, a one-dimensional line-grid is used and (phase-) images are taken at 3 different grid positions.
In contrast to this, our approach employs a spatially more uniform 2-dimensional, hexagonal grid, which needs to be
phase-shifted 7 times before enough information is sampled for the calculation of well-sectioned images. We call this
heXagonal SIM-version X-SIM, and the heXagonal Line-confocal version XL-SIM.
The fillfactor of a hexagonal pattern is 1/3, 1.5x lower than that of conventional linegrid-SIM. This leads to a further
reduction of out-of-focus light and this improves the signal-to-noise ratio (S/N). Fig. 2 shows such a structured slit
illumination, which, in conjunction with a current sCMOS camera, can be swept over a sample in 10 ms. Moving the
grid between sweeps takes < 1 ms, therefore a complete set of phase-images can be recorded in 76 ms. At first sight this
is considerably slower than a spinning disk confocal, which – in theory – acquires up to 2000 fps, yet in reality the low
transfer-efficiency of microoptics-empowered spinning disk systems necessitates exposure times of 50 – 100 ms, hence
there is no real speed advantage of the spinning disk concept.
Fig. 2 modulated slit-shaped excitation pattern
With respect to sectioning, however, there is a clear performance advantage of the XL-SIM approach compared to a
spinning disk confocal! Fig. 3 shows sections of the widely used FluoCells® Prepared Slide # 3, taken with an
Andromeda Spinning Disk Confocal from FEI Munich (left) and an XL-SIM system in our lab (right). It is quite
obvious that the spinning disk images are by far not as crisp as the XL-SIM images and that the intensity-valleys
between intensity peaks never reach zero in places where it should be dark!
1
which increases with the square-root of the number of detected photons in case of Poisson distributed photon noise
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Fig. 3 Three-color image (40x, NA 0.95) of a mouse kidney section, stained with Alexa Fluor® 488 WGA, Alexa Fluor® 568
Phalloidin, and DAPI) (Molecular Probes™ slide #3). The left image (courtesy Rainer Daum, FEI Munich) was acquired with a FEI
Munich Andromeda spinning disk system, powered by 3 100 mW lasers and with exposure times of 80 ms each, whereas the right
image was taken from the same slide, using 40 mW laser-power and also 80 ms total exposure time.
The difference between the two approaches is even more striking when comparing x-z-sections of the same object
obtained with the two 3D-imaging concepts (Fig. 4). Clearly the XL-SIM image is much more well defined and better
resolved.
Fig. 4
x-z-sections of the same object as in fig. 3, left spinning disk confocal, right XL-SIM.
Fig. 5 displays a comparison of a sectioned image obtained with a conventional point-scanning confocal (Nikon C1
CLSM) and our XL-SIM approach. The better rejection of out-of-focus haze provided by XL-SIM is not as profound as
in the comparison with a spinning disk confocal, yet it is clearly visible. Note that the point-scanning system needed
more than 10x longer!
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Microscopy and imaging science: practical approaches to applied research and education (A. Méndez-Vilas, Ed.)
Fig. 5 COS7 cells, Actin GFP-labelled. Objective Nikon 40x, 1.2W. Left: LSM with 1024 x 1024 pixel scanned in 2 s. Right:
Detail of an XL-SIM image of 2048 x 1024 pixel recorded in 210 ms.
4. Summary
We have demonstrated that XL-SIM, a method combining optical and mathematical means for sectioning of 3D-objects
under the light microscope, provides images as good or better than those of a conventional point-scanning confocal
microscope, the current gold standard for 3D sectioning microscopy. With respect to imaging speed, XL-SIM
outperforms a classical confocal by a factor of 10x or more, and while we have no quantitative measures as to how
much photo-damage is reduced in XL-SIM, we are convinced that it is several orders of magnitude! This conviction
stems from the fact that a total exposure-time of 75 ms in XL-SIM means that the average time a given sample-voxel is
exposed to the excitation light amounts to 500 µs, whereas in a point-scanning confocal it can’t be longer than 1 µs,
otherwise image acquisition gets exceedingly slow! And as it is well known, that photo-damage increases with intensity
more steeply than in a linear fashion [6], a 500x longer exposure – yielding the same signal as a 1 µs exposure in pointscanning – should be 500x or more less harmful to cells!
When compared to a spinning disk confocal - the current gold standard for fast live cell imaging – we conclude that
XL-SIM can be as fast as the former, but it provides significantly better sectioning. In principle the spinning disk could
be significantly faster, however this speed-advantage can rarely be utilized under realistic measurement conditions,
since in the usual micro-lens- (Yokogawa CSU X1, Andor Dragonfly, GE Opera Phenix) or micro-mirror (FEI
Andromeda) based designs only < 10% of the laser-power is available for excitation, hence a decent S/N ratio
necessitates exposure times comparable to those of a XL-SIM system! Faster frame-rates can be achieved with XL-SIM
simply by reducing the field over which the slit is scanned. The only spinning disk system that can reduce field-size and
hence exposure-times without sacrificing the S/N-ratio is the Andor Dragonfly, which has a variable telescope in the
excitation beam.
We conclude that XL-SIM may challenge the two most popular methods for 3D-sectioning under the light
microscope!
Acknowledgements The support by BMBF (Microsystems FKZ 0316185) is gratefully acknowledged!
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