easySTORM: a robust, lower‐cost approach to localisation and TIRF microscopy

Kwakwa, Kwasi; Savell, Alexander; Davies, Timothy; Munro, Ian; Parrinello, Simona; Purbhoo, Marco A.; Dunsby, Chris; Neil, Mark A. A.; French, Paul M. W.

doi:10.1002/jbio.201500324

Cited by 70 publications

(61 citation statements)

References 30 publications

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“…To illustrate the performance of the HPCS-MLM data processing, 2D and 3D STORM data were acquired using an 'easyS-TORM' microscope system described in Kwakwa et al (2016). SMLM images were acquired using dSTORM of fixed cells, in which alpha tubulin was labelled with Alexa Fluor 488 and/or Acetyl alpha-tubulin with Alexa Fluor 647.…”

Section: Experimental Single Molecule Localization Microscopymentioning

confidence: 99%

“…For many high throughput applications, it would be important to increase the numbers of cells imaged per well. This could be addressed by imaging more cells in larger FOV and we note that the use of multimode optical fibres to efficiently deliver 100 s mW of excitation power from low-cost multimode diode lasers to the sample enables dSTORM of FOV >100 × 100 µm to be routinely acquired (Kwakwa et al, 2016). Comparing this to the 20.5 × 20.5 µm FOV reported for (Beghin et al, 2017) suggests that high throughput dSTORM can be significantly accelerated if SMLM data are acquired for larger FOV (i.e.…”

Section: Introductionmentioning

confidence: 99%

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Accelerating single molecule localization microscopy through parallel processing on a high‐performance computing cluster

Munro

García

Yan

et al. 2018

Journal of Microscopy

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Summary Super‐resolved microscopy techniques have revolutionized the ability to study biological structures below the diffraction limit. Single molecule localization microscopy (SMLM) techniques are widely used because they are relatively straightforward to implement and can be realized at relatively low cost, e.g. compared to laser scanning microscopy techniques. However, while the data analysis can be readily undertaken using open source or other software tools, large SMLM data volumes and the complexity of the algorithms used often lead to long image data processing times that can hinder the iterative optimization of experiments. There is increasing interest in high throughput SMLM, but its further development and application is inhibited by the data processing challenges. We present here a widely applicable approach to accelerating SMLM data processing via a parallelized implementation of ThunderSTORM on a high‐performance computing (HPC) cluster and quantify the speed advantage for a four‐node cluster (with 24 cores and 128 GB RAM per node) compared to a high specification (28 cores, 128 GB RAM, SSD‐enabled) desktop workstation. This data processing speed can be readily scaled by accessing more HPC resources. Our approach is not specific to ThunderSTORM and can be adapted for a wide range of SMLM software. Lay Description Optical microscopy is now able to provide images with a resolution far beyond the diffraction limit thanks to relatively new super‐resolved microscopy (SRM) techniques, which have revolutionized the ability to study biological structures. One approach to SRM is to randomly switch on and off the emission of fluorescent molecules in an otherwise conventional fluorescence microscope. If only a sparse subset of the fluorescent molecules labelling a sample can be switched on at a time, then each emitter will be, on average, spaced further apart than the diffraction‐limited resolution of the conventional microscope and the separate bright spots in the image corresponding to each emitter can be localised to high precision by finding the centre of each feature using a computer program. Thus, a precise map of the emitter positions can be recorded by sequentially mapping the localisation of different subsets of emitters as they are switched on and others switched off. Typically, this approach, described as single molecule localisation microscopy (SMLM), results in large image data sets that can take many minutes to hours to process, depending on the size of the field of view and whether the SMLM analysis employs a computationally‐intensive iterative algorithm. Such a slow workflow makes it difficult to optimise experiments and to analyse large numbers of samples. Faster SMLM experiments would be generally useful and automated high throughput SMLM studies of arrays of samples, such as cells, could be applied to drug discovery and other applications. However, the time required to process the resulting data would be prohibitive on a normal comp...

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Section: Experimental Single Molecule Localization Microscopymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Accelerating single molecule localization microscopy through parallel processing on a high‐performance computing cluster

Munro

García

Yan

et al. 2018

Journal of Microscopy

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“…As a substitute for scientific-grade lasers, inexpensive laser diodes can deliver comparable laser power at a fraction of the cost (<100 euros/dollars). Such laser diodes have already been successfully used for low cost illumination in photoacoustic microscopy [6][7][8][9], pump-probe microscopy [10], stimulated emission-depletion (STED) microscopy [11] and SMLM [12]. Due to their high divergence and asymmetrical intensity profile, they are challenging to couple into single-mode optical fibers with a decent efficiency.…”

Section: Introductionmentioning

confidence: 99%

“…Perturbing the speckle pattern faster than the camera frame rate effectively averages it out. Several approaches have been explored in order to remove speckles from a multimode fiber profile, including rotating optical elements [12,16], oscillating diffusive membranes [14,17] or mechanical agitation [12,[18][19][20].…”

Section: Introductionmentioning

confidence: 99%

A cost-efficient open source laser engine for microscopy

Schroeder

Deschamps²,

Dasgupta³

et al. 2019

Preprint

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Scientific-grade lasers are costly components of modern microscopes. For high-power applications, such as single-molecule localization microscopy, their price can become prohibitive. Here, we present an open-source high-power laser engine that can be built for a fraction of the cost. It uses affordable, yet powerful laser diodes at wavelengths of 405 nm, 488 nm and 640 nm and optionally a 561 nm diode-pumped solid-state laser. The light is delivered to the microscope via an agitated multimode fiber in order to suppress speckles. We provide the part lists, CAD files and detailed descriptions, allowing any research group to build their own laser engine.

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“…To lower the cost of light source (one of the most expensive items), the industry-grade laser was proposed to replace the scientific grade laser, which has shown a decent performance in SMLM 8, 9 . Unlike SIM and STED, the imaging quality of SMLM is not sensitive to the slight degradation on the stability of the laser output and spectral linewidth.…”

Section: Introductionmentioning

confidence: 99%

A simple and cost-effective setup for super-resolution localization microscopy

et al. 2017

Sci Rep

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Single molecule localization microscopy (SMLM) has become a powerful imaging tool for biomedical research, but it is mostly available in imaging facilities and a small number of laboratories due to its high cost. Here, we evaluate the possibility of replacing high-cost components on standard SMLM with appropriate low-cost alternatives and build a simple but high-performance super-resolution SMLM setup. Through numerical simulation and biological experiments, we demonstrate that our low-cost SMLM setup can yield similar localization precision and spatial resolution compared to the standard SMLM equipped with state-of-the-art components, but at a small fraction of their cost. Our low-cost SMLM setup can potentially serve as a routine laboratory microscope with high-performance superresolution imaging capability.In the past few decades, fluorescence microscopy has significantly expanded our ability to study biological processes at the cellular and subcellular level. However, due to the diffraction-limited spatial resolution, conventional fluorescence microscopy cannot visualize biological structures smaller than ~200 nm. Until the recent decade, a number of super-resolution imaging techniques have been developed to break the diffraction barrier. These super-resolution techniques can generally be divided into two categories: the technique based on the nonlinear effects of the fluorescence that sharpens the point spread function (PSF) using modulated light, including stimulated emission depletion (STED) 1 and structured-illumination microscopy (SIM) . These super-resolution techniques have improved the spatial resolution by approximately an order of magnitude. In particular, single molecule localization microscopy (SMLM), which provides impressive spatial resolution down to 20 nm with relatively simple hardware compared to SIM and STED, has a great potential as a common laboratory tool for biomedical research. However, despite that SMLM has been commercialized for almost a decade, it remains a high-end microscopy instrument only available in the imaging facilities at major academic institutions and a small number of laboratories. Its high cost limits its widespread use as a routine microscopy system as conventional fluorescence microscope.2The key technical requirement for SMLM includes (1) a powerful laser to photo-bleach most fluorophores and activate only a small portion of sparsely distributed single fluorophores; (2) high numerical aperture (NA) objective lens to efficiently collect the limited number of photons emitted by single molecule; and (3) scientific cameras with high quantum efficiency and low noise to record the image from individual fluorescent emitters at a high signal to noise ratio (SNR). To meet these technical requirements and achieve the best performance, it is a common belief that the high-end optical and optoelectronic components have to be used, including high-power single-mode laser, high-end objectives, nano-positioning mechanical system and highly sensitive cameras, which easily drive the ...

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easySTORM: a robust, lower‐cost approach to localisation and TIRF microscopy

Cited by 70 publications

References 30 publications

Accelerating single molecule localization microscopy through parallel processing on a high‐performance computing cluster

Accelerating single molecule localization microscopy through parallel processing on a high‐performance computing cluster

A cost-efficient open source laser engine for microscopy

A simple and cost-effective setup for super-resolution localization microscopy

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