Phototoxicity induced in living HeLa cells by focused femtosecond laser pulses: a data-driven approach

Talone, Benedetta; Bazzarelli, M; Schirato, Andrea; Vicario, Ferdinando Dello; Viola, Daniele; Jacchetti, Emanuela; Bregonzio, M; Raimondi, Manuela Teresa; Cerullo, Giulio; Polli, Dario

doi:10.1364/boe.441225

Cited by 20 publications

(11 citation statements)

References 63 publications

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“…[ 55 ] On the other hand, in a systematic photo‐damage study with an irradiance of two orders of magnitude higher than ours, cells were reported to sustain without noticeable damage for 15‐s exposure. [ 56 ] In contrast, the laser‐on time for one 3D RI image was 10 s in our study. This was a very safe value.…”

Section: Resultsmentioning

confidence: 63%

Non‐Invasive and Minute‐Frequency 3D Tomographic Imaging Enabling Long‐Term Spatiotemporal Observation of Single Cell Fate

et al. 2023

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Non-invasive and rapid imaging technique at subcellular resolution is signi cantly important for multiple biological applications such as cell fate study. Label-free refractive-index (RI) based 3D tomographic imaging constitutes an excellent candidate for 3D imaging of cellular structures, but its full potential in long-term spatiotemporal cell fate observation has been locked due to the lack of e cient system integration. Here, we report a long-term 3D RI imaging system incorporating a cutting-edge white light diffraction phase microscopy (wDPM) module with spatiotemporal stability, and an acousto uidic device to roll and culture single cells in a customized live cell culture chamber. Using this system, we conducted 3D RI imaging experiments for 250 cells, and demonstrated e cient cell identi cation with high accuracy. Importantly, long-term and frequency-ondemand 3D RI imaging of K562 and MCF-7 cancer cells revealed different characteristics during normal cell growth, drug-induced cell apoptosis, and necrosis of drug-treated cells. In particular, for the rst time, a new two-fold transform mode was observed in the apoptosis regions by 1minute-frequency imaging in the 10-minute execution phase of apoptosis, which has not been seen otherwise by labeling microscopy. Overall, we believe that the proposed 3D tomographic imaging technique opens up a new avenue for visualizing intracellular structures and will nd many applications such as disease diagnosis and nanomedicine.

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Section: Resultsmentioning

confidence: 63%

Non‐Invasive and Minute‐Frequency 3D Tomographic Imaging Enabling Long‐Term Spatiotemporal Observation of Single Cell Fate

et al. 2023

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“…Therefore, in practice, strong light irradiation is required to obtain a good signal; however, this causes photodamage to the cell. When cells are exposed to strong light, reactive oxygen species are internally produced, which can destroy DNA, mitochondria, and other organelles, thereby leading to cell death [ 13 ]. Additionally, metabolic activity may change as a result of resistance to light irradiation, which is included as false signals in the acquired Raman scattering spectrum.…”

Section: Raman Observation Of Living Cellsmentioning

confidence: 99%

Recent Advances in Raman Spectral Imaging in Cell Diagnosis and Gene Expression Prediction

Watanabe

Sasaki

Fujita

2022

Genes

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Normal and tumor regions within cancer tissue can be distinguished using various methods, such as histological analysis, tumor marker testing, X-ray imaging, or magnetic resonance imaging. Recently, new discrimination methods utilizing the Raman spectra of tissues have been developed and put into practical use. Because Raman spectral microscopy is a non-destructive and non-labeling method, it is potentially compatible for use in the operating room. In this review, we focus on the basics of Raman spectroscopy and Raman imaging in live cells and cell type discrimination, as these form the bases for current Raman scattering-based cancer diagnosis. We also review recent attempts to estimate the gene expression profile from the Raman spectrum of living cells using simple machine learning. Considering recent advances in machine learning techniques, we speculate that cancer type discrimination using Raman spectroscopy will be possible in the near future.

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“…Even when fluorescent blurring and specimen thickness as well as spherical aberration are taken into account, scattering of light by the tissue sample is another factor that must be considered. As part of their defense mechanism against photo‐toxicity, most cells and tissues are translucent, semi‐opaque structures that scatter and diffuse light very effectively (For a recent discussion of photo‐toxicity in multi‐photon microscopy, see Talone et al., 2021). Thus, when imaging tissues of any appreciable thickness, light scattering limits the extent to which the out‐of‐focus background emission signal can be restricted by the confocal pinhole and an in‐focus image can be successfully acquired.…”

Section: Introductionmentioning

confidence: 99%

Multi‐Photon Microscopy

Sanderson

2023

Current Protocols

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In this series of papers on light microscopy imaging, we have covered the fundamentals of microscopy, super-resolution microscopy, and lightsheet microscopy. This last review covers multi-photon microscopy with a brief reference to intravital imaging and Brainbow labeling. Multi-photon microscopy is often referred to as two-photon microscopy. Indeed, using two-photon microscopy is by far the most common way of imaging thick tissues; however, it is theoretically possible to use a higher number of photons, and three-photon microscopy is possible. Therefore, this review is titled "multi-photon microscopy." Another term for describing multi-photon microscopy is "non-linear" microscopy because fluorescence intensity at the focal spot depends upon the average squared intensity rather than the squared average intensity; hence, non-linear optics (NLO) is an alternative name for multi-photon microscopy. It is this non-linear relationship (or third exponential power in the case of three-photon excitation) that determines the axial optical sectioning capability of multi-photon imaging. In this paper, the necessity for two-photon or multi-photon imaging is explained, and the method of optical sectioning by multi-photon microscopy is described. Advice is also given on what fluorescent markers to use and other practical aspects of imaging thick tissues. The technique of Brainbow imaging is discussed. The review concludes with a description of intravital imaging of the mouse.

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Phototoxicity induced in living HeLa cells by focused femtosecond laser pulses: a data-driven approach

Cited by 20 publications

References 63 publications

Non‐Invasive and Minute‐Frequency 3D Tomographic Imaging Enabling Long‐Term Spatiotemporal Observation of Single Cell Fate

Non‐Invasive and Minute‐Frequency 3D Tomographic Imaging Enabling Long‐Term Spatiotemporal Observation of Single Cell Fate

Recent Advances in Raman Spectral Imaging in Cell Diagnosis and Gene Expression Prediction

Multi‐Photon Microscopy

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