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STORM-ing through the difraction limitDaniel Metcal

National Physical Laboratory, Hampton Road, Teddington, Middlesex, TW11 0LW, UK

daniel.metcal@npl.co.uk

Biological Scale

Light microscopes have a resolution limit o around 250 nanometres (nm), which

means anything smaller will appear blurred. Any improvements in microscope

resolution will give new insights into molecular details o cells and improve our

understanding and treatments o diseases.

ConclusionsThere are still many challenges or improving dSTORM super-resolution technology,not least o which are a better understanding o sample preparation, how to takeimages and super-resolution sotware algorithms. Understanding and interpretingsuper-resolution images is o critical importance to molecular cell and biomedical

scientists who are beginning to use super-resolution microscopy to investigate themolecular basis o disease and associated therapies.

The work presented here veries that the dSTORM microscope that has beendeveloped at NPL is working correctly and outlines some general principles or howimages should be acquired and displayed.

GlossaryResolution can be dened in dierent ways. Most

commonly reers to the ability to distinguish two objects as

separate (and not merged into one object).

Localisation precision is the accuracy with which each

molecule can be measured. It depends on the sensitivity o

the microscope and brightness o the uorescent dye.

Diraction is the cause o blurring, which limits resolutionin normal microscopes. It is dependent on the wavelength

o light and the numerical aperture o the objective lens.

AcknowledgementsNPL researchers involved include Rebecca Edwards, Neelam

Kumarswami, Miklos Erdelyi and Alex Knight.

The microscope has been developed in collaboration with

Clemens Kaminski and Eric Rees rom Cambridge University.

Funding has been provided by the National Measurement

Ofce and the EPSRC.

Useul discussions on samples and image interpretationhave been taken place with our collaborators at the Medical

Research Council Laboratory or Molecular Cell biology.

Microscopy o CellsCells contain hundreds o thousands o dierent

types o molecules. However, they are very small,

densely packed and mostly transparent.

Specic molecules within cells can be labelled with a uorescent

dye or protein. Increasing zoom shows details becoming blurred

as the resolution limit is being reached (~250 nm).

A human cell. Scale bar 10 m. This human cell has a DNA dye in blue and a label or a component o the cell

cytoskeleton in yellow. Scale bars 20 m, 5 m and 1 m rom let to right.

Beating the Difraction Limit

(1) Instead o having all the uorescentmolecules on, switch most o them o.

(3) Switch a dierent subset o the uorescent molecules on and repeat steps 1 and 2 until all o

them have been plotted. Typically this is done on 10000 or more rames.

(4) Put together a super-resolution image using all the plotted molecular positions rom the

previous steps. Each plotted position is c alled a localisation and is represented by a super-

resolution pixel. The brighter a pixel is the more localisations there are within that area.

(2) Measure the middle position o each uorescentmolecule and plot its position.

Super-Resolution Microscopy

Very recently, super-resolution microscopes have been developed, which can overcome thediraction limit. One method is th e dSTORM (direct stochastic optical reconstruction microscopy),

which can achieve resolutions up to 10 times better than traditional optical microscopes.

A human cell with uorescently labelled vesicles (spherical objects in cells ~50-100 nm in diameter).

Actin Filament Test SampleTo test that the dSTORM approach is working as expected it is necessary to use a known test

sample. Actin laments were selected as these have a uniorm diameter o 7 nm. A method or

assembling and staining these laments onto glass was optimised.

Fluorescently labelled actin laments. Boxed regions are shown in the images on the right. Scale bars are 200 nm.

dSTORM pixel size is 15 nm. Normal pixel size is 100 nm.

Two Colour dSTORM ImagingThe ability to label dierent molecules with dierent dyes and image them is an extremely

powerul approach used by cell and molecular biologists and biomedical researchers. To test two

colour dSTORM imaging, actin laments labelled with two dierent dyes were used to label the

same structures. Actin laments

labelled with

2 dyes (shown

in green and

magenta). Scale

bars are 200 nm.

dSTORM pixel

size is 15 nm.

Normal pixel size

is 100 nm.

dSTORM Image QualitydSTORM images are typically reconstructed rom at least 10,000 individual rames each with

multiple localisations. A large number o localisations is required to accurately reconstruct a

dSTORM image. Insufcient localisation number leads to a pointillist image.

Also, image quality depends on the calculated localisation precision, which is in turn dependent

on the sensitivity o the microscope and the brightness o the uorescent dye used. The

reconstructed super-resolution image should have a pixel size th at reects this localisation

precision to correctly represent the data.

Fluorescently labelled actin lament. Scale bars are 200 nm. dSTORM pixel size is 15 nm. Normal pixel size is 100 nm.

Graph and dSTORM images show efect o increasing rame number used to reconstruct nal image.

Fluorescently labelledactin lament. Scale

bars are 200 nm.

Normal pixel size is

100 nm. dSTORM

pixel size varies as

indicated. Mean

localisation precision

was 15.4 nm or this

image, so the 15

nm pixel size is the

most appropriate or

representing this data.