Assessing phototoxicity in live fluorescence imaging

Laissue, Pierre Philippe; Alghamdi, Rana A.; Tomančák, Pavel; Reynaud, Emmanuel G.; Shroff, Hari

doi:10.1038/nmeth.4344

Cited by 381 publications

(365 citation statements)

References 34 publications

(64 reference statements)

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“…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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“…These combined effects offer a technique that allows the user to minimize both photobleaching and phototoxicity [10,[12][13][14], while allowing for long-term imaging studies (up to several days). This is an essential characteristic required for all sorts of developmental biology studies [15][16][17][18], such as organogenesis [19], cell migration [20,21], cardiac development [22][23][24][25], vascular development [26,27], neuro-development [28,29], and generally any kind of in vivo studies.…”

Section: 1a Lsfm Is Low Photodamage and Low Phototoxicmentioning

confidence: 99%

“…Normally, the starting point of the design is the size of the object to be imaged, which will then dictate the required size of the FOVof Eq. (12). Once the FOV has been set, a constraint on the NA ill of the illumination arm is imposed.…”

Section: Practical Examples Of Lsfm Implementationmentioning

confidence: 99%

Light-sheet microscopy: a tutorial

et al. 2018

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This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.

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“…Numerous embodiments have been suggested for OA imaging of areas located at depths ranging from superficial layers to centimeters within biological tissues . In all implementations, the optimal illumination strategy implies confining the excitation light to the region of interest, while avoiding light exposure levels that may cause cell toxicity or other damage to the imaged tissues . For depths beyond the ballistic regime of light (~1 mm in biological tissues), image formation relies on acoustic image reconstruction methods.…”

Section: Introductionmentioning

confidence: 99%

Uniform light delivery in volumetric optoacoustic tomography

Larney

Rebling

Chen

et al. 2019

Journal of Biophotonics

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Accurate image reconstruction in volumetric optoacoustic tomography implies the efficient generation and collection of ultrasound signals around the imaged object. Non‐uniform delivery of the excitation light is a common problem in optoacoustic imaging often leading to a diminished field of view, limited dynamic range and penetration, as well as impaired quantification abilities. Presented here is an optimized illumination concept for volumetric tomography that utilizes additive manufacturing via 3D printing in combination with custom‐made optical fiber illumination. The custom‐designed sample chamber ensures convenient access to the imaged object along with accurate positioning of the sample and a matrix array ultrasound transducer used for collection of the volumetric image data. Ray tracing is employed to optimize the positioning of the individual fibers in the chamber. Homogeneity of the generated light excitation field was confirmed in tissue‐mimicking agar spheres. Applicability of the system to image entire mouse organs ex vivo has been showcased. The new approach showed a clear advantage over conventional, single‐sided illumination strategies by eliminating the need to correct for illumination variances and resulting in enhancement of the effective field of view, greater penetration depth and significant improvements in the overall image quality.

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Assessing phototoxicity in live fluorescence imaging

Cited by 381 publications

References 34 publications

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

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

Light-sheet microscopy: a tutorial

Uniform light delivery in volumetric optoacoustic tomography

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