WHITEPAPERSINGLE-MOLECULE
LOCALISATION MICROSCOPY(SLMS)
How to overcome the Abbediffraction limit?

©2020 Novanta Corporation. All rights reserved.

Single-MoleculeLocalisation Microscopy (SMLM)
How to overcome the Abbe diffraction limit?

The Challenge
The Abbe diffraction barrier, which fundamentally restricted the lateral optical resolution to 200 – 300 nm until
the early 1990s, can be overcome by super-resolution optical fluorescence imaging techniques. These techniques
have been so groundbreaking that the Nobel Prize 2014 in Chemistry was awarded to Eric Betzig, Stefan Hell andW. E. Moerner “for the development of super-resolved fluorescence microscopy”.

Background
There are two different techniques commonly used so far; one is based on point spread function engineering, theother on single-molecule localization. Stefan Hell’s stimulated emission depletion (STED) microscopy belongs to
the first class and is a so called deterministic super-resolution technique. In contrast, Eric Betzig’s photoactivatedlocalization microscopy (PALM) is a stochastic technique.

How does SMLM work?
The fundamental principle of all SMLM implementations is the random photoactivation and subsequentlocalization of individual fluorescence emitters by detecting their photon emission at a certain position andtime in the complete absence of emission from neighboring emitters. The centroid of the diffraction-limited
distribution of photons is a measure of the location of the emitter and is far better defined than the photon
distribution itself. Thus, whereas a standard widefield image is an overlay of the photon distributions from alarge number of emitters, a super-resolved SMLM image is a map of their individual locations that can yields
lateral resolutions down to 5 nm.

Photograph of the laser compartment of a widefield microscope for SMLM with the beam paths of the gem 473 (blue) and gem 561(lime). Courtesy of G. U. Nienhaus and A. Kobitski (KIT)©2020 Novanta Corporation. All rights reserved.

SMLM ­‌| Abbe diffraction solution

But how can one ensure that only a few fluorophores emit at a certain time making individual localization
possible? Let us focus on the strategy used in PALM, which exploits specific properties of fluorophores thatspontaneously form in photoactivatable fluorescent proteins (paFPs) of the green fluorescent protein (GFP)
family. The key advantage of these GFP-like proteins is that they are genetically encodable, so that a cellendowed with this gene produces these markers itself with no further staining procedures required. Typically,
weak laser light at 405 nm is chosen to photoconvert a few paPFs from green- to red-fluorescent emitters duringacquisition of a single camera frame. Red fluorescence is typically excited with a 561 nm laser. The red emission
appears then as a small number of isolated, diffraction-limited spots in the red color channel during eachcamera frame. Photobleaching quickly removes the red-converted paFPs, so new emitters can be activatedand detected during acquisition of the next camera frame. Careful adjustment of the experimental parameters(laser powers, camera dwell times) ensures that the probability of detecting two fluorophores with spatiallyoverlapping intensity distributions is almost zero within a single camera frame.

SMLM image of a selected cell with red-converted protein; the inset shows a close-up around the cell nucleus. SMLM images wererendered from 1000 successive camera frames taken with 561 nm illumination for fluorescence excitation and an additional weak 405
nm illumination for green-to-red photoconversion of the protein. Images taken from: Lu Zhou et al.: Cytoplasmic Transport Machineryof the SPF27 Homologue Num1 in Ustilago maydis, Sci. Rep. 8 (2018) 3611, http://creativecommons.org/licenses/by/4.0/©2020 Novanta Corporation. All rights reserved.

SMLM ­‌| Abbe diffraction solution

One supplier for all wavelengths
Unlike confocal microscopy, widefield imaging techniques require illumination of a larger area with constanthigh intensity. A key aspect in SMLM are lasers emitting at suitable wavelengths (typically 405, 473, 561 and 640nm) in order to excite (and possibly photoactivate) the fluorescent molecules employed for visualization.

The Laser Quantum gem laser series with its 4 mostpopular wavelength (473, 532, 561 and 640 nm).

For fluorescence excitation of paFPs, high laser powers in the range of 500 mW are beneficial. The gem lasers
from Laser Quantum offer these or even higher powers at various wavelengths (473, 532, 561, 640, 660 and 671nm) with near perfect beam quality in a compact, cost-efficient platform. The high powers also allow advanced
beam shaping techniques since power is not the limiting factor as with diode lasers.

Novanta Benefits
Novanta is uniquely positioned to solve even the most complex challenges for OEMs, system integrators, and end-use
customers seeking to advance their manufacturing processes with high precision laser systems. With some of the most wellknown brands in the industry and in-country application and service support, Novanta delivers reliable, precise, and durable
components and sub-systems.

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