June 16, 2016

Super-resolution microscopy reveals details eight times smaller then visible light wavelength of immune cells' surface

Researcher Lillemeier wanted more detail on how the T-cell receptors are arranged in tissue and how that arrangement might change when the T cells are activated in living hosts. The team used a super-resolution microscope developed in the laboratory of co-senior author Hu Cang, assistant professor at Salk's Waitt Advanced Biophotonics Center and holder of the Frederick B. Rentschler Developmental Chair. This microscopy approach, called light-sheet direct stochastic optical reconstruction microscopy (dSTORM), let the researchers watch T cell receptors in the membranes of T cells in mouse lymph nodes at a resolution of approximately 50 nanometers. Visible light has wavelengths of 390 to 700 nanometers.

The new imagery confirmed the previous observation that protein islands of T-cell receptors merge into larger "microclusters" when T cells are activated. But it also showed that, before cells are activated, the protein islands are already arranged in groups--dubbed "territories" by Lillemeier's team. "The pre-organization on the molecular level basically turns the T cell into a loaded gun," says Lillemeier.

The organization of surface receptors enables T cells to launch fast and effective immune response against antigens. Understanding how the molecular organization mediates the sensitivity of T cell responses could help researchers make the immune system more or less sensitive. In the case of autoimmune diseases, clinicians would like to turn down the immune system's activity, while turning up the activity could help fight infections or cancers.

The research could also have implications for understanding other receptors in the body, which have a wide range of functions both within and outside the immune system. "We think that most receptors on the surfaces of cells are organized like this," says Ying Hu, first author and postdoctoral researcher at the Salk Institute.

Salk scientists used light-sheet super-resolution imaging to capture the rearrangement of T-cell receptors from nanometer-scale protein islands (left) to micrometer-scale microclusters (right) after T-cell activation in mouse lymph nodes. CREDIT Salk Institute

Direct stochastical optical reconstruction microscopy (dSTORM) utilizes the photoswitching of a single fluorophore. In dSTORM, fluorophores are embedded in an oxidizing and reducing buffer system (ROXS) and fluorescence is excited. Sometimes, stochastically, the fluorophore will enter a triplet or some other dark state which is sensitive to the oxidation state of the buffer. As the molecules return stochastically they can be excited to fluoresce so that single molecule positions can be recorded. Development of the dSTORM method occurred in 3 independent laboratories at about the same time and was called 'Reversible photobleaching microscopy -RPM", "Ground state depletion microscopy followed by individual molecule return" -GSDIM as well as the now generally accepted moniker dSTORM.

There are two major groups of methods for functional super-resolution microscopy

Deterministic super-resolution: The most commonly used emitters in biological microscopy, fluorophores, show a nonlinear response to excitation, and this nonlinear response can be exploited to enhance resolution. These methods include STED, GSD, RESOLFT and SSIM.

Stochastic super-resolution: The chemical complexity of many molecular light sources gives them a complex temporal behavior, which can be used to make several close-by fluorophores emit light at separate times and thereby become resolvable in time. These methods include Super-resolution optical fluctuation imaging (SOFI) and all single-molecule localization methods (SMLM) such as SPDM, SPDMphymod, PALM, FPALM, STORM and dSTORM.

Another super microscopy method for 25 nanometer resolution

3 D LIMON (Light MicrOscopical nanosizing microscopy) images using the Vertico SMI microscope are made possible by the combination of SMI and SPDM, whereby first the SMI and then the SPDM process is applied.

The SMI process determines the center of particles and their spread in the direction of the microscope axis. While the center of particles/molecules can be determined with a 1–2 nm precision, the spread around this point can be determined down to an axial diameter of approx. 30–40 nm.

Subsequently, the lateral position of the individual particle/molecule is determined using SPDM, achieving a precision of a few nanometers.

As a biological application in the 3D dual color mode the spatial arrangements of Her2/neu and Her3 clusters was achieved. The positions in all three directions of the protein clusters could be determined with an accuracy of about 25 nm

3D Dual Colour Super Resolution Microscopy with Her2 and Her3 in breast cells, standard dyes: Alexa 488, Alexa 568 LIMON

SOURCES- Eurekalert, Wikipedia

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