Picture of the month: May 2023

Iotm may23 ab

DeepSTORM prediction of high-density HaloTag-PAINT

High-density HT-PAINT image predicted using DeepSTORM neural network. Deep learning assisted image analysis enables increase the density of single molecule localization microscopy. By training the neural network with high-density data with accurate localization list, PAINT data with overlapping molecules can be predicted with increased acquisition speed. Here, exchangeable HaloTag ligands were introduced for PAINT imaging in U2-OS cells stably expressing TOM20 bound to a dHT7 tag and CalR-KDEL bound to a HT7 tag. TOM20-dHT7 was measured with Hy4-SiR, and CalR-KDEL was measured with S5-JF635. Both targets were measured in 5 min. (scale bar: 5µm)

Picture of the month: Januar 2023

Iotm jan23 lk

Multiplexing arcRNA in HeLa cells using STED microscopy

"NEAT1 is a long non-coding RNA transcript expressed in the nucleus that is involved in the regulation of gene expression. Paraspeckles are subnuclear structures mainly composed of NEAT1 and various proteins, playing a role in the regulation of RNA processing and transport. The combination of Fluorescence In Situ Hybridisation (FISH) and DNA-PAINT enabled the measurement of multiple positions on one RNA with STED microscopy. Shown is the simultaneous measurement of 5’ (blue), middle (purple) and 3’ (orange) regions. The scale bar is 500 nm."

Picture of the month: November 2022

Iotm3zoom nov 2022 mg

Imaging aggrephagy in HEKa cells using STED microscopy

Aggrephagy is a selective type of cellular autophagy. Characteristic is the formation of aggresomes, perinuclear compartments with high autophagic activity. We study the regulatory function of BAG3, the co-chaperone Bcl-2 associated athanogene 3, during aggrephagy. For this purpose, we co-labeled BAG3 and Vimentin using AbberiorSTAR Orange and AberriorSTAR Red labeled antibodies and applied super-resolution STED microscopy. Aggrephagy induction by MG-132 treatment leads to condensation of cytosolic BAG3 and envelopment of the Vimentin network. Imaging performed by Dongni Wang. Scale bar is 10 µm.

Picture of the month: Juli 2022

Iotm jul 2022  overview nup96

dSTORM imaging of NUP96 in U2OS cells

NUP96 is a protein in the nuclear pore complex (NPC) which is involved in the transport of molecules between the nucleus and the cytosol. Here, SNAP-tagged NUP96 was labeled with Alexa Fluor 647-benzylguanin in U2OS cell and imaged via direct stochastic optical reconstruction microscopy (dSTORM). The eight-fold symmetry of NUP96 inside the NPC is visible.

Picture of the month: November 2021

Iotm nov2021 vimentin 3d hp

Astigmatism based 3D-imaging of Vimentin in U2OS cells

Vimentin is a Type III intermediate filament and plays an important role in anchoring organelles in the cytosol. We used a DNA-PAINT based immunolabeling protocol comprising a monoclonal primary antibody against Vimentin and a docking strand labeled secondary antibody. The complementary imager strand P1-ATTO655 was added to the sample at a concentration of 300 pM. 30.000 consecutive images were acquired at the N-STORM microscope equipped with an astigmatism lens for 3D imaging. The 3D reconstructed super-resolution image shows the distribution of the filaments in the cytosol. Scale bar is 10 µm. Sample was prepared by Soohyen Jang.

Picture of the month: August 2021

Iotm aug 2021 big

Multicolor imaging with exchangeable fluorophore labels

Fluorophore labels that transiently bind their target (e.g. PAINT-labels in SMLM and STED, or "exchangeable" labels) are a powerful tool to bypass photobleaching in microscopy. They enable a time efficient experimental protocol to visualize different cellular structures in cells with a high labeling density. This image shows multiple cellular structures of a single cell visualized using exchangeable fluorophore labels and a confocal laser scanning microscope. The endoplasmic reticulum (green), actin (blue), mitochondria (red), DNA (orange) and cellular membranes (magenta) are shown.

Picture of the month: April 2021

Iotm apr 2021

DNA-PAINT imaging of the endoplasmic reticulum

The ER-membrane protein FAM134B was imaged in U2OS cells using DNA-PAINT. The membrane protein was labeled in chemically fixed cells with a primary antibody targeting the membrane protein and short DNA docking strand labeled secondary antibodies. Using complementary fluorescently labeled DNA strands (R2-ATTO655) and HILO mode for sequential image acquisition allowed reconstruction of the endoplasmic reticulum network over the whole cell area (i) and reveals fine structural details of the endomembrane system (ii, magnified region shown in (i)). Scale bar is 10 µm (i) and 1 µm (ii), spatial resolution ~ 40 nm (determined through decorrelation analysis, Descloux et al., Nat Meth 2019).

Picture of the month: December 2020

Iotm dec 2020

Synaptic cleft

A four-color super-resolution image of brain tissue.

Picture of the month: August 2020

Iotm aug 2020

PAINT imaging of E. coli

Confocal laser scanning microscopy (CLSM) enables to study cellular structures with diffraction‑limited resolution. With the help of super-resolution microscopy the membrane and the defined filigree structure of the nucleoid can be visualized with near-molecular spatial resolution even in small organisms like E. coli. (scale bar 1 μm)

Picture of the month: January 2020

Iotm jan 2020

Recent article in Science Signaling

Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells.

Picture of the month: September 2019

Iotm sep 2019

Recent Cover in Neurophotonics

View article

Picture of the month: April 2019

Iotm apr 2019

Exchange-DNA-PAINT of the receptor tyrosine kinases EGFR and MET

The receptor tyrosine kinases epidermal growth factor receptor (EGFR, magenta) and mesenchymal epithelial transition factor receptor (MET, cyan) were imaged in unstimulated HeLa cells using Exchange-DNA-PAINT. Receptors were modified with short DNA docking strands (P1 and P5) via immunostaining and imaged sequentially after addition of the corresponding imager strands labeled with ATTO 655. These multi-color images facilitate co-localization and interaction analysis of receptors on the cell membrane. Scale bar 5 µm, insets 5 x 5 µm.

Picture of the month: August 2018

Iotm aug 2018

Fixation matters

After infection, Salmonella establish a tubular network in the host cell. This network is very sensitive to osmotic changes and is destroyed upon formaldehyde fixation, leading to the formation of artificial vesicular structures (left image). Glutaraldehyde fixation preserves the network and reveals Salmonella-induced filaments with ~ 150 nm diameter (right image). Dual-color dSTORM measurements of the secreted Salmonella effector protein SseJ (Alexa Fluor 647, red hot) and the host cell lysosomal marker LAMP-1 (Alexa Fluor 532, green). Salmonella nucleoids are counterstained with DAPI (blue). Scale bars are 1 µm.

Picture of the month: March 2018

Conf sted 01

STED image of microtubuli

Comparison of confocal vs STED pictures of our new homebuilt STED setup. Microtubuli stained with Abberior Star635.

Picture of the month: November 2017

Iotm nov 2017

Recent cover in Science Signaling

View article

Picture of the month: June 2017

Iotm june 2017

Recent cover in nature microbiology

View article

(Image: Ella Maru Studio, Cover Design: Karen Moore)

Picture of the month: September 2016

Iotm sep 2016

Quantitative analysis of SMLM data with LAMA

A list of single-molecule localizations serves as input data (top) for LAMA and is post-processed for clustering (Ripley functions, DBSCAN, OPTICS), single-molecule counting, colocalization analysis and localization precision determination.

Picture of the month: June 2016

Iotm jun 2016

PAINT microscopy image of E.coli

Membrane stained with Nile Red (red) and chromosomal DNA with JF-646-Hoechst (blue).

Picture of the month: January 2016

Iotm jan 2016

Confocal fluorescence image of a dividing cell

Stained for DNA (blue), actin (red) and tubulin (green).

Picture of the month: October 2015

Iotm oct 2015

Single molecule FRET on DNA

Picture of the month: September 2015

Iotm sep 2015 lowres

Imaging neurons

Fine structure of a neuron's membrane recorded with super-resolution imaging (CB).

Picture of the month: August 2015

Iotm aug 2015 lowres

dSTORM imaging of microtubules

Image of an HeLa cell stained for microtubules.