Optimizing live-cell fluorescence imaging conditions to minimize phototoxicity

Kiepas, Alexander; Voorand, Elena; Mubaid, Firas; Siegel, Peter M.; Brown, Claire M.

doi:10.1242/jcs.242834

Cited by 66 publications

(69 citation statements)

References 51 publications

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“…To verify the results of our semiautomatic analysis, a custom algorithm was created in MATLAB (35,115). Here, a spline curve was first fitted to each intensity trace to identify segments of assembly and disassembly.…”

Section: Imaging Adhesion Turnover − Nmumgmentioning

confidence: 99%

The SHCA adapter protein cooperates with lipoma-preferred partner in the regulation of adhesion dynamics and invadopodia formation

Kiepas

Voorand

Senécal

et al. 2020

Journal of Biological Chemistry

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SHC adaptor protein (SHCA) and lipoma-preferred partner (LPP) mediate transforming growth factor β (TGFβ)-induced breast cancer cell migration and invasion. Reduced expression of either protein diminishes breast cancer lung metastasis, but the reason for this effect is unclear. Here, using total internal reflection fluorescence (TIRF) microscopy, we found that TGFβ enhances the assembly and disassembly rates of paxillin-containing adhesions in an SHCA-dependent manner through the phosphorylation of the specific SHCA tyrosine residues Tyr-239, Tyr-240, and Tyr-313. Using a BioID proximity labeling approach, we show that SHCA exists in a complex with a variety of actin cytoskeletal proteins, including paxillin and LPP. Consistent with a functional interaction between SHCA and LPP, TGFβ-induced LPP localization to cellular adhesions depended on SHCA. Once localized to the adhesions, LPP was required for TGFβ-induced cell migration and adhesion dynamics. Mutations that impaired LPP localization to adhesions (mLIM1) or impeded interactions with the actin cytoskeleton via α-actinin (ΔABD) abrogated migratory responses to TGFβ. Live-cell TIRF microscopy revealed that SHCA clustering at the cell membrane precedes LPP recruitment. We therefore hypothesize that, in the presence of TGFβ, SHCA promotes the formation of small, dynamic adhesions by acting as a nucleator of focal complex formation. Finally, we defined a previously unknown function for SHCA in the formation of invadopodia, a process that also required LPP. Our results reveal that SHCA controls the formation and function of adhesions and invadopodia, two key cellular structures required for breast cancer metastasis.

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Section: Imaging Adhesion Turnover − Nmumgmentioning

confidence: 99%

The SHCA adapter protein cooperates with lipoma-preferred partner in the regulation of adhesion dynamics and invadopodia formation

Kiepas

Voorand

Senécal

et al. 2020

Journal of Biological Chemistry

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“…Some of our results contradict previous studies, especially the absence of a correlation between photomorbidity and the intensity of the excitation light ( Dixit and Cyr 2003 ; Logg et al 2009 ) as well as a lack of effects on photomorbidity and background fluorescence from pulsed illumination regimes ( Nishigaki et al 2006 ; Boudreau et al 2016 ). Some of these discrepancies are most likely explained by unaccounted for hardware delays, as has been shown recently ( Kiepas et al 2020 ). However, our inability to confirm the benefits of pulsed illumination will require further investigation.…”

Section: Discussionmentioning

confidence: 58%

“…We measured that the sample is illuminated for an additional 187 ms compared to the set exposure time in our system ( Figure S4), a hardware delay similar to previous reports (Magidson and Khodjakov 2013;Kiepas et al 2020). Next, we determined the spectra and intensity of our light sources in combination with commonly used excitation filters and beamsplitters ( Figure S5,…”

Section: Data Availabilitymentioning

confidence: 99%

Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe

Schmidt

Cuny

Rudolf

2020

G3 Genes|Genomes|Genetics

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Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth rate (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide framework for how to approach photomorbidity in other organisms.

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“…With improvements in cell viability resulting from the former, this achieves not only insightful but accurate data. 3…”

Section: Taking Speed One Step Furthermentioning

confidence: 99%

Microscopy Automation on a Budget: Capturing High-Speed Multi-Labelled Events with LED Illumination Systems

2020

BioTechniques

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Capturing fast cellular processes with widefield fluorescence microscopy and multiple fluorophores has historically required the addition of motorised components. The light source has often been overlooked as an affordable means of achieving multi-wavelength imaging, but LEDs have changed the game. Here we compare typical configurations for automated illumination systems in terms of speed, cost and signal-to-noise ratio (i.e. image contrast). We also explore how to transform a manual microscope into a powerful automated imaging system by capitalising on LED switching speeds, with lower cost filters and minimal requirement for expensive motorised components.

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Optimizing live-cell fluorescence imaging conditions to minimize phototoxicity

Cited by 66 publications

References 51 publications

The SHCA adapter protein cooperates with lipoma-preferred partner in the regulation of adhesion dynamics and invadopodia formation

The SHCA adapter protein cooperates with lipoma-preferred partner in the regulation of adhesion dynamics and invadopodia formation

Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe

Microscopy Automation on a Budget: Capturing High-Speed Multi-Labelled Events with LED Illumination Systems

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