SMLM relies on the ability to randomly activate only a subset of fluorescent molecules to distinguish them spatially.
By repeating the process in consecutive image acquisitions, accumulated raw data are processed to detect single molecules with a nanometric precision (down to 10 nm).
Data quantification and analysis are then performed to resolve either structures or dynamics at the nanoscale level. To reconstruct a nanoscopy image, each molecule is detected and localized by specialized algorithms.
To get information in 3D, the magnified astigmatism reveals the position of each molecule from the focus of the objective.
direct STochastic Optical Reconstruction Microscopy
dSTORM exploits the photoswitchingproperties of some fluorophores. In certain conditions, fluorophores can be sent to an intermediary state (the “dark state”) from which they randomly cycle between ON and OFF states.
PhotoActivated Localization Microscopy
PALM uses specific fluorescent fusion proteins called “photoactivatable” or “photoconvertible”. The ON state of these proteins can be randomly activated with the proper laser excitation.
Point Accumulation for Imaging in Nanoscale Topography
PAINT uses specific dyes that are ON only when they are transiently bound to their target. W ith the appropriate dye concentrations, it is possible to achieve a regime where only a few dyes are ON at the same time. DNA-PAINT uses short DNA single strands coupled to fluorophores. These probes are ON when they transiently hybridize to the complementary DNA strand coupled to an antibody targeting the structure of interest.
Single Particle Tracking
SPT in SMLM combines Single Particle Tracking with SMLM (PALM or STORM) to obtain spatially and temporally highly resolved diffusion maps of single molecules.
SPT need a precise algorithm to found and draw tracks from each molecule in the cell, state of the art algorithm are implemented in Neo Softwares.
Single-molecule imaging experiments or TIRF microscopy are in many cases limited from the range of the field of view. This is due to the use of Gaussian excitation which does not allow a homogeneous field of illumination on the samples. To solve this issue, we innovated with a very versatile illumination scheme called “Adaptative Scanning for Tunable Excitation Rendering” (ASTER). This can create a uniform field of illumination up to from 25×25μm² to150x150μm².
Illuminating with an Epifluorescence beam will lead to In-depth illumination and thus higher background. It is one of the many illuminations used in fluorescence microscopy and it allows us to visualize structures far from the coverslip such as the nucleus, thick cells, tissues …
HiLo (Highly inclined and laminated optical sheet) is an illumination technique that aims to get the angle of illuminations that will lead to the highest Signal to Noise Ratio (SNR) on the image. This will limit the background. HiLo illumination can be used with slightly in-depth samples.
TIRF (Total internal reflection fluorescence) aims to excite with the critical angle the sample leading to complete reflection of the laser beam. When doing so, an evanescence wave is created and only excites molecules that are in the first hundreds of nanometers of the coverslip. This suppresses the background in depth. This approach is used to image structures close to the coverslip: membranes, cytoskeleton, in vitro surfaces…
Multicolor super-resolution microscopy is a powerful way to reveal macromolecular structure, to measure the direct distance between molecules, and to quantify molecular interactions at the nanoscale. It’s a challenge to perform this type of data acquisition without drifts, chromatic aberrations, and with the certainty of capturing data of comparable quality in all channels. The ideal solution requires equal conditions for all fluorophores regarding their photophysical responses, the same optical path, and the same uniform illumination to capture reliable multicolor images.
In SMLM there is a different way to perform multicolor imaging. The first technique is sequential multicolor imaging which aims is to label two or more different structures with fluorophores that can be activated with different lasers wavelength without the overlay of fluorescence.
The second techniques, unique with the SAFe systems, is the spectral demixing. which the aims is explainned below.
The principle is to split the emission fluorescence into two distinct detection channels thanks to a long pass dichroic beamsplitter. By using fluorophores emitting within the same wavelength range, their partially overlapping emission is then spectrally separated by the dichroic and imaged on the two detection scientific-grade cameras (the choice of the dichroic is linked to fluorophores and the SMLM strategy employed).
Each fluorophore can be retrieved on both images provided by the two cameras and the measured photon numbers on each camera will be related to the spectral separation of the fluorophore. Thus, a characteristic intensity ratio distribution R for each fluorophore can be determined. A calibration is required, by collecting the photon distribution on each camera from samples stained with single fluorophores.