Super Resolution Microscopy (SRM)

What is super-resolution microscopy?

Conventional light microscopy is limited by the physics of light diffraction (known as the Abbe diffraction limit). When light emitted from a fluorophore passes through the sample medium and lens, it is diffracted and when two points or structures are situated closer than the diffraction limit, they cause a blur. The size of the blur is described as a point spread function (PSF).

Super-resolution microscopy techniques work by effectively reducing the point spread function or ensuring that the sampled fluorophores are not too close in either time or space, thus preventing blurring. Super-resolution microscopy techniques can image to a resolution 20 times greater than is possible through light microscopy.

Stochastic Optical Reconstruction Microscopy (STORM) and direct STORM (dSTORM), PhotoActivated Localization Microscopy (PALM) and STimulated Emission Depletion (STED) microscopy are three of the most commonly used super-resolution microscopy techniques. Collectively these techniques can produce super-resolution images in the low nanometer range and allow single molecule localization and single particle tracking.

Stochastic Optical Reconstruction Microscopy (STORM) and direct STORM (dSTORM)

Established in 2006, STORM is the most widely used super-resolution microscopy technique. Samples labeled with suitable fluorophores are stochastically excited, causing subsets of fluorophores to randomly switch on and off - referred to as "blinking" or "photoswitching". The blinking process is repeated many times and the emitted photon location is captured. Using specialized software, the data is combined resulting in high-resolution images (Figure 1). The principals of dSTORM are in essence the same as STORM, but there is no need for activator dyes, as this technique uses fluorophores that are inherently capable of stochastic activation (e.g. HM Janelia Fluor^® 526, SE, Cat. No. 7312).

Figure 1: STORM and dSTORM microscopy principles. Fluorophores are stochastically activated and blink over a period of time. The location of the emitted photons from each fluorophore is captured and a detailed high-resolution image is created.

Photoactivated Localization Microscopy (PALM) and Fluorescence Photoactivation Localization Microscopy (FPALM)

With similar principles to STORM and dSTORM, PALM and FPALM stochastically activate fluorophores in small subsets to prevent blur. PALM and FPALM employ photoactivatable fluorophores, which remain in the "off" state until activated by a laser. Photoactivatable dyes such as PA Janelia Fluor^® 646, SE (Cat. No. 6150) and PA Janelia Fluor^® 549, SE (Cat. No. 6149) are ideal for this technique as they can be used on live or fixed cells and can also be multiplexed to perform two-color sptPALM in live cells.

Stimulated Emission Depletion (STED) Microscopy

In stimulated emission depletion or STED microscopy, both an excitation and STED laser are used to reduce the blur. The STED laser selectively 'depletes' photon emissions around the periphery of the area excited by the excitation laser, which creates a "depletion donut" allowing only a small central portion of fluorophore emissions to be collected, thus overcoming Abbe's diffraction limit and producing high resolution images.

Finding the Best Fluorophores for Super-Resolution Microscopy

For super-resolution techniques, fluorophores should ideally be:

• very bright
• highly photostable (exhibiting minimal photobleaching especially in SRM thiol-containing buffers)
• capable of stochastic photoswitching with a low duty cycle

Check for absorbance/emission spectra, extinction coefficient, quantum yield, closest laser line and suitable applications on individual product pages.

At Tocris we are delighted to offer Janelia Fluor^® Dyes for Super-Resolution Microscopy.

They are supplied with a range of reactive groups, including:

• Amine-Reactive (these dyes can be converted to relevant substrates for use in self-labeling tag systems such as HaloTag^® and SNAP-tag^®)
• Click-Reactive (azide and tetrazine)
• Sulfhydryl-Reactive (maleimide)
• Free Acid

Fluorescent Dye Conjugation protocols are available to view and download.

We also offer Janelia Fluor^® conjugated antibodies and custom conjugation services with our sister brand Novus Biologicals.

Figure 2: dSTORM images acquired using cardiac tissue labeled with Janelia Fluor^® Dyes. Left: dSTORM image displaying collagen VI in the interstitial space. Labeled with a primary ab against collagen VI and a secondary ab conjugated to Janelia Fluor^® 549 (JF549, Cat.No. 6147). Scale: 50 μm. Right: Super-dSTORM image of ventricle muscle tissue from a pig. Janelia Fluor^® 646 (JF646, Cat.No. 6148) was used to label collagen VI, imaged with a 642 nm excitation laser. Top right inset: widefield image. Scale bar 2 μm.

Images kindly provided by Professor. C. Soeller, University of Exeter; images acquired by A. Clowsley and A. Meletiou

HaloTag is a trademark of Promega Corporation, and SNAP-tag is a trademark of New England BioLabs, Inc.