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44

Lab Times

1-2015

Methods

T

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

happens.

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

signal-to-noise ratio.

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-

oleit

et al

.

Nature Methods

doi:10.1038/

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.

Image stacks

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

reconstructions.

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.