Reduction of coherent artefacts in super‐resolution fluorescence localisation microscopy

Georgiades, Pantelis; Allan, Viki; Dickinson, Mark; Waigh, Thomas A.

doi:10.1111/jmi.12453

Cited by 13 publications

(14 citation statements)

References 25 publications

(32 reference statements)

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“…Super-resolution STORM images were acquired using our custom built setup, which has undergone some minor modifications from what was described in our previous work 31 , 50 . Specifically, the four laser beams were delivered through an oscillating multimode optical fibre, instead of a free space configuration, using a dedicated speckle scrambler.…”

Section: Methodsmentioning

confidence: 99%

The flexibility and dynamics of the tubules in the endoplasmic reticulum

Georgiades

Allan

Wright

et al. 2017

Sci Rep

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The endoplasmic reticulum (ER) is a single organelle in eukaryotic cells that extends throughout the cell and is involved in a large number of cellular functions. Using a combination of fixed and live cells (human MRC5 lung cells) in diffraction limited and super-resolved fluorescence microscopy (STORM) experiments, we determined that the average persistence length of the ER tubules was 3.03 ± 0.24 μm. Removing the branched network junctions from the analysis caused a slight increase in the average persistence length to 4.71 ± 0.14 μm, and provides the tubule’s persistence length with a moderate length scale dependence. The average radius of the tubules was 44.1 ± 3.2 nm. The bending rigidity of the ER tubule membranes was found to be 10.9 ± 1.2 kT (17.0 ± 1.3 kT without branch points). We investigated the dynamic behaviour of ER tubules in live cells, and found that the ER tubules behaved like semi-flexible fibres under tension. The majority of the ER tubules experienced equilibrium transverse fluctuations under tension, whereas a minority number of them had active super-diffusive motions driven by motor proteins. Cells thus actively modulate the dynamics of the ER in a well-defined manner, which is expected in turn to impact on its many functions.

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Section: Methodsmentioning

confidence: 99%

The flexibility and dynamics of the tubules in the endoplasmic reticulum

Georgiades

Allan

Wright

et al. 2017

Sci Rep

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“…One approach that has been used to limited success in the past has been to disrupt the spatial coherence of the laser source with a rotating diffuser. 26,27 This approach was not successful in the present study, due to the process being ineffective at completely randomizing the spatial phase. Holography is another approach that has been successful in X-ray imaging methods but has yet to be attempted for coherent IR imaging and presents a promising area for future investigation, which would have a resemblance to older heterodyne methods for performing 2DIR.…”

Section: ■ Results and Discussionmentioning

confidence: 73%

Resolution Enhancement in Wide-Field IR Imaging and Time-Domain Spectroscopy Using Dielectric Microspheres

2020

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Wide-field imaging through dielectric microspheres has emerged in recent years as a simple and effective approach for generating super-resolution images at visible wavelengths. We present, to our knowledge, the first demonstration that dielectric microspheres can be used in a wide-field infrared (IR) microscope to enhance the far field resolution. We have observed a substantial improvement in resolution and magnification when images are collected through polystyrene microspheres. In addition, we demonstrate that spectroscopic imaging with a pulse-shaper based femtosecond mid-IR laser system is possible through the dielectric microspheres, which is a promising first step toward applying this technique to ultrafast IR imaging methods such as pump–probe and 2DIR microscopy.

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“…In our experiments a Digital sCMOS (scientific CMOS ORCA Flash 4.0 v2) camera (Hamamatsu C11440-22CU) was used to capture diffraction limited images at 100 frames per second and the rest of the STORM apparatus was as described previously 27 , 30 . A 100x objective lens (UAPON 100XOTIRF, Olympus) with a Numerical Aperture (NA) of 1.49 was used.…”

Section: Experiments and Methodsmentioning

confidence: 99%

Quenched Stochastic Optical Reconstruction Microscopy (qSTORM) with Graphene Oxide

Georgiades

Cox

et al. 2018

Sci Rep

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Quenched Stochastic Optical Reconstruction Microscopy (qSTORM) was demonstrated with graphene oxide sheets, peptides and bacteria; a method of contrast enhancement with super-resolution fluorescence microscopy. Individual sheets of graphene oxide (GO) were imaged with a resolution of 16 nm using the quenching of fluorescence emission by GO via its large Resonant Energy Transfer (RET) efficiency. The method was then extended to image self-assembled peptide aggregates (resolution 19 nm) and live bacterial cells (resolution 55 nm, the capsular structure of E. coli from urinary tract infections) with extremely low backgrounds and high contrasts (between one and two orders of magnitude contrast factor improvements that depended on the thickness of the graphene oxide layer used). Graphene oxide films combined with STORM imaging thus provide an extremely convenient method to image samples with large backgrounds due to non-specifically bound fluorophores (either due to excess labelling or autofluorescent molecules), which is a common occurrence in studies of both biological cells and soft-condensed matter. The GO quenches the fluorescence across a thin layer at distances of less than 15 nm. Graphene oxide films coated with thin layers (≤15 nm) of polystyrene, polymethylmethacrylate and polylysine are shown to be effective in producing high contrast qSTORM images, providing a convenient modulation of sample/substrate interactions. The GO coatings can also provide an increased image resolution and a factor of 2.3 improvement was observed with the peptide fibres using a feature of interest metric,when there was a large non-specifically bound background.

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Reduction of coherent artefacts in super‐resolution fluorescence localisation microscopy

Cited by 13 publications

References 25 publications

The flexibility and dynamics of the tubules in the endoplasmic reticulum

The flexibility and dynamics of the tubules in the endoplasmic reticulum

Resolution Enhancement in Wide-Field IR Imaging and Time-Domain Spectroscopy Using Dielectric Microspheres

Quenched Stochastic Optical Reconstruction Microscopy (qSTORM) with Graphene Oxide

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