Benchmarking Smartphone Fluorescence-Based Microscopy with DNA Origami Nanobeads: Reducing the Gap toward Single-Molecule Sensitivity

Vietz, Carolin; Schütte, Max L.; Wei, Qingshan; Richter, Lars; Lalkens, Birka; Ozcan, Aydogan; Tinnefeld, Philip; Acuna, Guillermo P.

doi:10.1021/acsomega.8b03136

Cited by 53 publications

(56 citation statements)

References 35 publications

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“…Recently, the detection of only 10–16 ATTO 542 molecules was demonstrated using a simple table top setup with a monochrome smartphone camera as detector and a consumer product lens for light collection 16 . This inspired us that single-molecule detection might be possible on a portable smartphone microscope with non-specialized low-NA optics 2 , 22 – 24 (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These moderate fluorescence enhancement values were not sufficient for detecting single fluorescence molecules with low numerical aperture(NA) optics. For example, our previous work on benchmarking the sensitivity of smartphone-based detection systems suggested that a signal equivalent to at least 16 single emitters is required for detection on a smartphone-based low-NA microscope 16 . Therefore, a diagnostic single-molecule assay fully exploiting the signal amplification potential of DNA origami nanoantennas has not been presented to date and remained highly desirable to enable detection of single molecules with affordable low-NA optics.…”

Section: Introductionmentioning

confidence: 99%

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Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope

et al. 2021

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The advent of highly sensitive photodetectors and the development of photostabilization strategies made detecting the fluorescence of single molecules a routine task in many labs around the world. However, to this day, this process requires cost-intensive optical instruments due to the truly nanoscopic signal of a single emitter. Simplifying single-molecule detection would enable many exciting applications, e.g., in point-of-care diagnostic settings, where costly equipment would be prohibitive. Here, we introduce addressable NanoAntennas with Cleared HOtSpots (NACHOS) that are scaffolded by DNA origami nanostructures and can be specifically tailored for the incorporation of bioassays. Single emitters placed in NACHOS emit up to 461-fold (average of 89 ± 7-fold) brighter enabling their detection with a customary smartphone camera and an 8-US-dollar objective lens. To prove the applicability of our system, we built a portable, battery-powered smartphone microscope and successfully carried out an exemplary single-molecule detection assay for DNA specific to antibiotic-resistant Klebsiella pneumonia on the road.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope

et al. 2021

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“…Recently, the detection of only 10-16 ATTO 542 molecules was demonstrated using a simple table top setup with a monochrome smartphone camera as detector and a consumer product lens for light collection. 26 This inspired us that single-molecule detection might be possible on a portable smartphone microscope with non-specialized low-NA optics [27][28][29] (see Fig. 3a and 3b).…”

Section: Figmentioning

confidence: 99%

Addressable Nanoantennas with Cleared Hotspots for Single-Molecule Detection on a Portable Smartphone Microscope

Trofymchuk

Glembockyte

Grabenhorst

et al. 2020

Preprint

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† These authors contributed equallyThe advent of highly sensitive photodetectors 1,2 and the development of photostabilization strategies 3 made detecting the fluorescence of a single molecule a routine task in many labs around the world. However, to this day, this process requires cost-intensive optical instruments due to the truly nanoscopic signal of a single emitter. Simplifying single-molecule detection would enable many exciting applications, e.g. in point-of-care diagnostic settings, where costly equipment would be prohibitive. 4 Here, we introduce addressable NanoAntennas with Cleared HOtSpots (NACHOS) that are scaffolded by DNA origami nanostructures and can be specifically tailored for the incorporation of bioassays. Single emitters placed in the NACHOS emit up to 461-fold brighter enabling their detection with a customary smartphone camera and an 8-US-dollar objective lens. To prove the applicability of our system, we built a portable, battery-powered smartphone microscope and successfully carried out an exemplary single-molecule detection assay for DNA specific to antibiotic-resistant Klebsiella pneumonia "on the road ".Early detection of disease biomarkers generally requires high sensitivity enabled by molecular amplification mechanisms 5-9 or physical signal enhancement of commonly used fluorescence signals. [10][11][12][13] Physical fluorescence signal enhancement could enable sensitivity improvement, detection of singlemolecules on cost-effective and mobile devices and therefore help to distinguish specific signals against an unavoidable background of impurities even in low-resource settings. Fluorescence from emitters such as fluorescent dyes can be enhanced using plasmonic nanoantennas, [14][15][16] and the challenge of placing quantum emitters in their hotspots was overcome using DNA origami as constructing material. 17,18 The immense requirements for small, defined and rigid gaps between the gold or silver nanoparticles forming the gap in the nanoantenna aggravated the usability of the space between the nanoparticles for a biosensing assay that could thus far only be realized with mono-particle antennas and low enhancement values. 19 In this work, we introduce NACHOS that enable high fluorescence signal amplification and use them for a single-molecule diagnostic assay on a portable and inexpensive smartphone microscope. A novel threedimensional DNA origami structure was designed ( Fig. 1a) and folded from an M13mp18-derived scaffold strand and complementary staple strands ( Supplementary Tables 1-3). The NACHOS origami design uses two pillars to attach silver nanoparticles and creates the plasmonic hotspot at the bifurcation in the gap between the two pillars and the nanoparticles (see DNA origami sketches in Fig. 1a and full NACHOS structure in Fig.

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“…In our previous study cell STORM we identify unpredictable post-processing of image data by the post-processing performed by the smartphone firmware leading to a reduced optical resolution or artefacts in the final reconstructed image [45,16] as a serious problem of smart phone-based super-resolution microscopy. To give an example, low signal blinking events may accidentally be removed by the denoiser before the adjacent video compression alters the data by finding a sparse representation in the spatial and temporal domain.…”

Section: Improving Cellphone-based Sri Using Raw Imaging Data In Srrfmentioning

confidence: 99%

Nanoscopy on the Chea(i)p

Diederich

Helle

Then

et al. 2020

Preprint

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Super-resolution microscopy allows for stunning images with a resolution well beyond the optical diffraction limit, but the imaging techniques are demanding in terms of instrumentation and software. Using scientific-grade cameras, solid-state lasers and top-shelf microscopy objective lenses drives the price and complexity of the system, limiting its use to well-funded institutions. However, by harnessing recent developments in CMOS image sensor technology and low-cost illumination strategies, super-resolution microscopy can be made available to the mass-markets for a fraction of the price. Here, we present a 3D printed, self-contained super-resolution microscope with a price tag below 1000 $ including the objective and a cellphone. The system relies on a cellphone to both acquire and process images as well as control the hardware, and a photonic-chip enabled illumination. The system exhibits 100nm optical resolution using single-molecule localization microscopy and can provide live super-resolution imaging using light intensity fluctuation methods. Furthermore, due to its compactness, we demonstrate its potential use inside bench-top incubators and high biological safety level environments imaging SARS-CoV-2 viroids. By the development of low-cost instrumentation and by sharing the designs and manuals, the stage for democratizing super-resolution imaging is set.

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Benchmarking Smartphone Fluorescence-Based Microscopy with DNA Origami Nanobeads: Reducing the Gap toward Single-Molecule Sensitivity

Cited by 53 publications

References 35 publications

Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope

Addressable nanoantennas with cleared hotspots for single-molecule detection on a portable smartphone microscope

Addressable Nanoantennas with Cleared Hotspots for Single-Molecule Detection on a Portable Smartphone Microscope

Nanoscopy on the Chea(i)p

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