here is a theoretical limit to the reso-
lution of light microscopy. Way back
in 1873, Ernst Abbe broke the sad
news that the best resolution you can pos-
sibly get from any microscope is determined
by the diffraction properties of the light you
use. To the first approximation, the mini-
mum distance you can resolve is more or
less half the wavelength of the light. One of
Nature’s non-negotiables, it seems.
Of course that’s why we had to invent
electronmicroscopy. If wavelength is what’s
causing the problem, then let’s switch to
“light” with a really small wavelength. A
beam of electrons has a wavelength about
100,000th that of visible light and that adds
up to a big leap in resolution.
The only problem is that using an elec-
tron beam means you can only see things
that are, or have been made, electron
dense. Useless for all those amazing appli-
cations with fluorescent tags. And as for im-
aging living cells, just try putting your tissue
into an electron microscope and see what
But it turns out you can get around Ab-
be’s limit with a little bit of light trickery
and some clever computing. Selective plane
illumination microscopy (SPIM) is one such
hack that allows you to ramp up the reso-
lution of your images without running the
expense of a confocal or two-photon setup.
And because of the way SPIM works, sev-
eral extras come included as standard: you
can image large tissues (even whole living
animals) and you can image over a long pe-
riod. What is more, you can even convert
your old fluorescent scope into a SPIM set-
up using materials found in your kitchen
(provided you keep the right sort of things
in your kitchen).
Fluorescence microscopy’s downsides
So how does SPIM work? Let’s start
from the beginning. The basic problemwith
standard fluorescence microscopy is that
you illuminate the whole specimen, even
though you are only focussing on one tiny
part. Doing that introduces all sorts of prob-
lems. For one, you get a lot of background
from the out-of-focus regions above and be-
low your plane of focus. Second, irradiat-
ing a tissue with high energy electromag-
netic radiation does a good job of slowly
micro-waving the specimen, placing a se-
vere limit on how long you can image be-
fore the tissue hits medium-rare. That is a
big problem when your signal is weak and
you need a long time to overcome a poor
Sure, you can get around a lot of these
problems with confocal imaging, where a
beam of light converges on a point in the
specimen. But this comes at the expense
of a poor axial (z-plane) resolution, not to
mention great expense.
SPIM solves these problems by the sim-
ple expedient of shining an ultra-thin sheet
of light through the specimen. The sheet
lies at a 90° angle to the observing objec-
tive, so light dispersed by stained objects is
detected by the objective. The sheet of light
itself is created from a dispersed laser beam
that is focused through a cylindrical lens.
Alternatively, the equivalent of a sheet can
be achieved by scanning a circular beam. Ef-
fectively, the specimen is optically sliced.
So what does this simple approach buy
us? First, you get better contrast images.
Gone is the background that mars conven-
tional epifluorescence images. And given
the clarity of each slice, moving the sheet
through the sample, along the z-axis results
in a better-quality, reconstructed 3D image.
Then there is the speed factor. The lack
of background means you can get a good
quality image from a very rapid scan, so you
can image things that traditionally move
too quickly. Last year Jan Huisken at the
Max Planck Institute of Molecular Cell Bi-
ology and Genetics in Dresden used SPIM
to capture the first high-resolution images
of a beating heart of the zebrafish (Mick-
nmeth.3037). This was made possible not
only because of the technique’s speed but
also because of its ability to penetrate thick-
er (>1 cm) tissues.
There are other advantages, too. With
SPIM you can easily rotate the sample,
keeping the imaging hardware fixed. The
significance of this is that you can build z-
stacks from different angles, which in turn
(with a bit of computer trickery called “mul-
ti-view fusion”) gives much better-resolved
But does SPIM really give you super-res-
olution? Well, no at least not on its own.
The lateral resolution of a SPIM setup is still
limited by the diffraction limit and the nu-
merical aperture of the lens.
But some recent reports have changed
all that: it seems you can get genuine super-
resolution microscopy if you fiddle about
with the fine details of the beam. And these
Photo: EMBL Heidelberg
Bench philosophy (54): Light-sheet microscopy
Sliced by Light
Illuminating your specimen with a light-sheet means you can get a big increase in image resolution, and get deeper
penetration, quicker scans and lower toxicity with it. All with open source hardware and software.
Ernst Stelzer (l.) and Jan Huisken pioneered the SPIM technology for life science applications.