Advanced multiplexing techniques in single molecule localisation microscopy

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging applications. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. This thesis presents several novel techniques for multiplexed imaging, with the main focus on the DNA-PAINT super-resolution technique. At first, I demonstrate multiplexed imaging by using three different single-domain antibodies (nanobodies). They have a three times smaller size than conventional antibodies, therefore reducing the distance between the target site and the fluorophore. The used nanobodies have a high and specific binding affinity against three common fluorescent proteins: GFP, BFP and RFP. This allows me to use conventional fluorescent protein fusion for labelling cellular structures of interest, and then to use fluorescently labelled nanobodies as imager molecules in PAINT. In addition, I introduce a novel wide-field Time-Correlated Single Photon Counting (TCSPC) camera, that was successfully used for Fluorescence Lifetime Imaging (FLIM) with single-molecule sensitivity. As an additional application of Fluorescence-Lifetime Single-Molecule Localization Microscopy (FL-SMLM), I used FL-SMLM for single-molecule metal-induced energy transfer (smMIET) imaging which is a first step towards three-dimensional single-molecule localization microscopy (SMLM) with isotropic nanometer resolution. Next, I combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT (together FL-PAINT) and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) images multiple targets simultaneously, therefore shortening the total acquisition time, and requires no fluid exchange, thus both leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. FL-PAINT can be readily combined with other DNA-PAINT based techniques of multiplexed imaging, and therefore FL-PAINT has a great potential for highly multiplexed bio-imaging.2022-05-1

Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time

Exfoliation and Optical Properties of Near-Infrared Fluorescent Silicate Nanosheets

The silicates Egyptian Blue (CaCuSi4O10, EB), Han Blue (BaCuSi4O10, HB) and Han Purple (BaCuSi2O6, HP) emit in bulk bright and stable fluorescence in the near-infrared (NIR), which is of high interest for (bio)photonics due to minimal scattering, absorption and phototoxicity in this spectral range. So far the optical properties of nanosheets (NS) of these silicates are poorly understood. Here, we exfoliate them into nanosheets and report their physicochemical properties. The approach uses ball milling followed by tip sonication and centrifugation steps to exfoliate the silicates into NS with a lateral size ≈ 16-27 nm and thickness ≈ 1-4 nm. They emit at ≈ 927 nm (EB-NS), 953 nm (HB-NS) and 924 nm (HP-NS) and single NS can be resolved in the NIR. Fluorescence lifetimes decrease from ≈ 30-100 μs (bulk) to 17 μs (EB- NS), 8 μs (HB-NS) and 7 μs (HP-NS). NS of different composition/size can be imaged by fluorescence lifetime imaging, which enables lifetime-encoded multicolor imaging both on the microscopic and the macroscopic scale. Finally, remote imaging through tissue phantoms reveals the potential for bioimaging. In summary, we report a procedure to gain NIR fluorescent silicate nanosheets, characterize their photophysical properties and show their potential for NIR photonics.</div

Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells

Oleksiievets N, Sargsyan Y, Thiele JC, et al. Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells. Communications Biology . 2022;5(1): 38.DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging. © 2022. The Author(s)