Biology subjects

Engdahl, A. K.

Publications and source records attributed to Engdahl, A. K..

3 recordsLinked to original sources

bioRxiv2024

High sensitivity cameras can lower spatial resolution in high-resolution optical microscopy

High-resolution optical fluorescence microscopies and, in particular, super-resolution fluorescence microscopy, are rapidly adopting highly sensitive cameras as their referred photodetectors. Camera-based parallel detection facilitates high-speed live cell imaging with the highest spatial resolution. Here, we show that the drive to use ever more sensitive, photon-counting image sensors in cameras can, however, have detrimental effects on spatial resolution that many researchers are not aware of. Typical parameters which influence the selection of image sensors are pixel size, quantum efficiency, signal-to-noise performance, dynamic range, and frame rate of the sensor. A parameter that is, however, often overlooked, is the sensors modulation transfer function (MTF). We have determined the wavelength-specific MTF of front- and back-illuminated image sensors and evaluated how it affects the spatial resolution that can be achieved in high fluorescence microscopy modalities. We find significant differences in image sensor performance for sensors that cause the resulting spatial resolution to vary with up to 28%. This result shows that the choice of image sensor has significant impact on the imaging performance of all camera-based optical microscopy modalities.

biophysics↗

bioRxiv2022

Dual-color single molecule localization microscopy on transparent polymer waveguide chips

Photonic waveguide chips offer near-field excitation of biological samples, which enables cost-effective, large field-of-view super-resolution microscopy without the need for high numerical aperture (NA) objective lenses. Single molecule localization based super-resolution microscopy that requires high illumination intensities is currently limited to solid state photonic waveguide chips composed of hard-coated, high NA planar waveguides deposited on opaque substrates. These platforms do not permit epi-detection of fluorescence through the substrate, which limits the use of photonic waveguide chips to the upright configuration. Additionally, the detection efficiency is reduced because the majority of the fluorescence emission is directed towards the high refractive index substrate. A low cost waveguide chip based on a polymer core material deposited on common #1.5 coverslips that is easy to produce was recently demonstrated. Here, a platform that is capable of performing single-molecule localization microscopy (SMLM) of biological samples using polymer-based photonic waveguide chips is presented, enabling super-solution microscopy in the inverted microscope configuration. Super-resolved imaging of two different structures of the cytoskeleton in primary liver sinusoidal endothelial cells (LSECs) by two popular SMLM methods, dSTORM and DNA-PAINT, down to 23 nm is demonstrated.

biophysics↗

bioRxiv2021

Sodium sulfite as a switching agent for single molecule based super-resolution optical microscopy

Photoinduced off-switching of organic fluorophores is routinely used in super-resolution microscopy to separate and localize single fluorescent molecules, but the method typically relies on the use of complex imaging buffers. The most common buffers use primary thiols to reversibly reduce excited fluorophores to a non-fluorescent dark state, but these thiols have a limited shelf life and additionally require high illumination intensities in order to efficiently switch the emission of fluorophores. Recently a high-index, thiol-containing imaging buffer emerged which used sodium sulfite as an oxygen scavenger, but the switching properties of sulfite was not reported on. Here, we show that sodium sulfite in common buffer solutions reacts with fluorescent dyes, such as Alexa Fluor 647 and Alexa Fluor 488 under low to medium intensity illumination to form a semi-stable dark state. The duration of this dark state can be tuned by adding glycerol to the buffer. This simplifies the realization of different super-resolution microscopy modalities such as direct Stochastic Reconstruction Microscopy (dSTORM) and Super-resolution Optical Fluctuation Microscopy (SOFI). We characterize sulfite as a switching agent and compare it to the two most common switching agents by imaging cytoskeleton structures such as microtubules and the actin cytoskeleton in human osteosarcoma cells.

biophysics↗