Light-induced cell damage in live-cell super-resolution microscopy

Wäldchen, Sina; Lehmann, Julian; Klein, Teresa; Linde, Sebastian van de; Sauer, Markus

doi:10.1038/srep15348

Cited by 454 publications

(542 citation statements)

References 48 publications

(74 reference statements)

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“…These considerations are critical for long-term time-lapse imaging of living samples and with light-dose-intensive super-resolution techniques. Using longer wavelengths (lower energy) of light is effective [27], but careful consideration of how the light is delivered to the sample in space and time is critical in order to obtain accurate live cell information. Fortunately, discussions of the photo-toxicity of fluorescence microscopes to live samples are becoming more common and researchers are adding new ideas and protocols to lessen the problem [28].…”

Section: Resultsmentioning

confidence: 99%

Less is More: Longer Exposure Times with Low Light Intensity is Less Photo-Toxic

Mubaid

Brown

2017

Micros. Today

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Live cell imaging is commonplace across many areas of research including the physical, life, and health sciences. However, living cells are rarely exposed to light in their natural environment. Fluorescence microscopy is used extensively since fluorescent tags can target molecules very specifically in living systems and provide high signal-tonoise images to explore biological processes in situ. This study shows that apparently "gentle" imaging conditions with rapid exposures of only 15-35 ms every 2 minutes can affect cell migration speeds and cell protrusion rates. Decreasing incident light power and increasing exposure times (70-350 ms) restores cellular dynamics, maintains image quality, and thus reduces or perhaps eliminates photo-toxicity.

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Section: Resultsmentioning

confidence: 99%

Less is More: Longer Exposure Times with Low Light Intensity is Less Photo-Toxic

Mubaid

Brown

2017

Micros. Today

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“…Specimen photodamage due to exposure to light is often a critical issue in biophysical experiments 8 , resulting in photochemical changes to biological processes 8, 9 , modifying structure and growth 10,11 , and ultimately adversely affecting viability 8,11 . For instance, Ref.…”

Section: Discussionmentioning

confidence: 99%

“…These advances illustrate a near-universal feature of precision opti-cal biosensors -that increased light intensities are required to detect smaller molecules or improve spatiotemporal resolution. This increases the photodamage experienced by the specimen, which can have broad consequences on viability 8 , function 9 , structure 10 and growth 11 . It is therefore desirable to develop alternative biosensing approaches that improve sensitivity without exposing the specimen to higher optical intensities.…”

Section: Introductionmentioning

confidence: 99%

Evanescent single-molecule biosensing with quantum-limited precision

et al. 2017

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Sensors that are able to detect and track single unlabelled biomolecules are an important tool both to understand biomolecular dynamics and interactions at nanoscale, and for medical diagnostics operating at their ultimate detection limits. Recently, exceptional sensitivity has been achieved using the strongly enhanced evanescent fields provided by optical microcavities and nano-sized plasmonic resonators. However, at high field intensities photodamage to the biological specimen becomes increasingly problematic. Here, we introduce an optical nanofibre based evanescent biosensor that operates at the fundamental precision limit introduced by quantisation of light. This allows a four order-of-magnitude reduction in optical intensity whilst maintaining state-of-the-art sensitivity. It enables quantum noise limited tracking of single biomolecules as small as 3.5 nm, and surface-molecule interactions to be monitored over extended periods. By achieving quantum noise limited precision, our approach provides a pathway towards quantum-enhanced single-molecule biosensors. IntroductionEvanescent optical biosensors that operate label-free and can resolve single molecules have applications ranging from clinical diagnostics 1 , to environmental monitoring 2, 3 and the detection and manipulation of viruses 4 , proteins and antibodies [5][6][7] . Further, they offer the prospect to provide new insights into motor molecule dynamics and biophysically important conformational changes as they occur in the natural state, unmodified by the presence of fluorescent markers or nanoparticle labels 5 . Recently, the reach of evanescent techniques has been extended to single proteins with Stokes radii of a few nanometers 1,6 by concentrating the optical field using resonant structures such as optical microcavities 2, 4, 5 and plasmonic resonators 1,6,7 . These advances illustrate a near-universal feature of precision opti- cal biosensors -that increased light intensities are required to detect smaller molecules or improve spatiotemporal resolution. This increases the photodamage experienced by the specimen, which can have broad consequences on viability 8 , function 9 , structure 10 and growth 11 . It is therefore desirable to develop alternative biosensing approaches that improve sensitivity without exposing the specimen to higher optical intensities.Here we demonstrate an optical nanofibre-based approach to evanescent detection and tracking of unlabelled biomolecules that utilises a combination of heterodyne interferometry and dark field illumination. This greatly suppresses technical noise due to background scatter, vibrations and laser fluctuations that has limited previous experiments 12,13 , allowing operation at the quantum noise limit to sensitivity introduced by the quantisation of light. The increased information that is extracted per scattered photon enables state-of-the-art sensitivity to be achieved with optical intensities four orders of magnitude lower than has been possible previously 1,6 . Using the biosensor, we detect nan...

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“…In fact, time-lapse images by STED and RESOLFT are often notable for how little motion is observed, given the long acquisition times. This may be a harbinger of phototoxicity or even light-induced fixation that produces "frozen" cells (8). It is also notable that independent metrics of cell health, such as differential interference contrast (Li et al ref.…”

mentioning

confidence: 99%

“…Even so, more sophisticated measures are needed. For example, microtubule growth rates have been shown to decline significantly at a dose of 640-nm irradiation, only 3% of that at which nearly all cells still divide (8). Thus, the development of noninvasive, broadly applicable assays to measure the immediate and local effects of light on cellular physiology should be a major thrust of live-cell imaging research, for diffraction-limited as well as SR methods.…”

mentioning

confidence: 99%