scholarly journals Preventing photomorbidity in long-term multi-color fluorescence imaging of S. cerevisiae and S. pombe

2017 ◽  
Author(s):  
Gregor W. Schmidt ◽  
Andreas P. Cuny ◽  
Fabian Rudolf
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Light Dose ◽  
Excitation Light ◽  
Minimal Impact ◽  
Multiple Wavelengths ◽  
Imaging Conditions

AbstractTime-lapse imaging 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 (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, no-observed-effect level). 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 a framework for how to approach photomorbidity in other organisms.

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

2020 ◽  
Vol 10 (12) ◽  
pp. 4373-4385
Author(s):  
Gregor W. Schmidt ◽  
Andreas P. Cuny ◽  
Fabian Rudolf
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Light Dose ◽  
Live Cells ◽  
Excitation Light ◽  
Minimal Impact ◽  
Imaging Conditions

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.

scholarly journals Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

2014 ◽  
Vol 25 (22) ◽  
pp. 3699-3708 ◽  
Author(s):  
Anyimilehidi Mazo-Vargas ◽  
Heungwon Park ◽  
Mert Aydin ◽  
Nicolas E. Buchler
Keyword(s):  
Gene Expression ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Photon Flux ◽  
Luminescence Microscopy ◽  
Cell Cycle Genes ◽  
Light Excitation ◽  
Better Than

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

scholarly journals A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging

Microscopy ◽  
2021 ◽  
Author(s):  
Tetsuichi Wazawa ◽  
Ryohei Noma ◽  
Shusaku Uto ◽  
Kazunori Sugiura ◽  
Takashi Washio ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Ph Dependence ◽  
Super Resolution ◽  
Time Lapse ◽  
Light Irradiation ◽  
Acquisition Time ◽  
Polarization Angle ◽  
Cellular Dynamics

Abstract Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.

scholarly journals In vivo super-resolution RESOLFT microscopy of Drosophila melanogaster

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Sebastian Schnorrenberg ◽  
Tim Grotjohann ◽  
Gerd Vorbrüggen ◽  
Alf Herzig ◽  
Stefan W Hell ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Super Resolution ◽  
Time Lapse ◽  
Live Cell ◽  
Model Organisms ◽  
Alpha Tubulin ◽  
Light Levels

Despite remarkable developments in diffraction unlimited super-resolution microscopy, in vivo nanoscopy of tissues and model organisms is still not satisfactorily established and rarely realized. RESOLFT nanoscopy is particularly suited for live cell imaging because it requires relatively low light levels to overcome the diffraction barrier. Previously, we introduced the reversibly switchable fluorescent protein rsEGFP2, which facilitated fast RESOLFT nanoscopy (<xref ref-type="bibr" rid="bib10">Grotjohann et al., 2012</xref>). In that study, as in most other nanoscopy studies, only cultivated single cells were analyzed. Here, we report on the use of rsEGFP2 for live-cell RESOLFT nanoscopy of sub-cellular structures of intact Drosophila melanogaster larvae and of resected tissues. We generated flies expressing fusion proteins of alpha-tubulin and rsEGFP2 highlighting the microtubule cytoskeleton in all cells. By focusing through the intact larval cuticle, we achieved lateral resolution of <60 nm. RESOLFT nanoscopy enabled time-lapse recordings comprising 40 images and facilitated recordings 40 µm deep within fly tissues.

scholarly journals Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

2013 ◽  
Vol 3 (5) ◽  
pp. 20130007 ◽  
Author(s):  
Masahito Yamanaka ◽  
Kenta Saito ◽  
Nicholas I. Smith ◽  
Satoshi Kawata ◽  
Takeharu Nagai ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Three Dimensions ◽  
Fluorescence Signal ◽  
Optical Transfer Function ◽  
Excitation Light ◽  
Optical Transfer ◽  
Green Fluorescent ◽  
Nonlinear Components

We report, for the first time, the saturated excitation (SAX) of fluorescent proteins for subdiffraction-limited imaging of living cells in three-dimensions. To achieve saturation, a bright yellow and green fluorescent protein (Venus and EGFP) that exhibits a strong nonlinear fluorescence response to the high excitation intensity at the laser focus is used. Harmonic demodulation of the fluorescence signal produced by a modulated excitation light extracts the nonlinear fluorescence signals. After constructing the image from the nonlinear components, we obtain fluorescence images of living cells with spatial resolution beyond the diffraction limit. We also applied linear deconvolution to SAX microscopy and found it effective in further enhancing the contrast of small intracellular structures in the SAX image, confirming the expansion of the optical transfer function in SAX microscopy.

scholarly journals Estimating numbers of fluorescent molecules in single cells by analysing fluctuations in photobleaching

2018 ◽  
Author(s):  
Elco Bakker ◽  
Peter S. Swain
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Measurement Noise ◽  
Wide Field ◽  
Stochastic Fluctuations ◽  
Biochemical Measurements ◽  
Green Fluorescent ◽  
The Impact

The impact of fluorescence microscopy has been limited by the difficulties of express-ing measurements of fluorescent proteins in numbers of molecules. Absolute numbers enable the integration of results from different laboratories, empower mathematical modelling, and are the bedrock for a quantitative, predictive biology. Here we develop a general algorithm to infer numbers of molecules from fluctuations in the photobleaching of proteins tagged with Green Fluorescent Protein. To untangle measurement noise from stochastic fluctuations, we use the linear noise approximation and Kalman filtering within a framework of Bayesian inference. Not only do our results agree with biochemical measurements for multiple proteins in budding yeast, but we also provide a statistically verified model of measurement noise for fluorescence microscopes. The experiments we require are straightforward and use only a wide-field fluorescence microscope. As such, our approach has the potential to become standard for those practising quantitative fluorescence microscopy.

Long-term Live Single Cell Quantification of Transcription Factor Dynamics

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-4-SCI-4
Author(s):  
Timm Schroeder
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Cell Fate ◽  
Fluorescent Protein ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Molecular Control ◽  
Endogenous Gene ◽  
Stem And Progenitor Cells

Abstract Hematopoiesis is highly complex and dynamic, and consist of large numbers of different cells expressing many molecules. Despite intensive research, many long-standing questions in hematopoiesis research remain disputed. One major reason is the fact that we usually only analyze populations of cells - rather than individual cells - at very few time points of an experiment. Tracking of individual cells would be an extremely powerful approach to improve our understanding of molecular cell fate control. We are therefore developing imaging systems to follow the fate of single cells over many generations. We program new software to help recording and displaying the divisional history, position, properties, interaction, etc. of all individual cells over many generations. In addition, novel microfluidics devices are designed and produced to allow improved observation and manipulation of cells. Our technologies allow continuous long-term quantification of protein expression or activity in living cells. Among other approaches, we generate knock in models expressing transcription factor to fluorescent protein fusions from endogenous gene loci. This enables non-invasive long-term live quantification of transcription factor protein dynamics in single stem and progenitor cells throughout their differentiation. The resulting novel kind of continuous quantitative single cell data is used for the generation and falsification of models describing the molecular control of hematopoietic cell fates. Disclosures No relevant conflicts of interest to declare.

scholarly journals Multiplexed labeling of cellular proteins with split fluorescent protein tags

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ryo Tamura ◽  
Fangchao Jiang ◽  
Jin Xie ◽  
Daichi Kamiyama
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Cellular Proteins ◽  
Cell Protein ◽  
Adhesion Protein ◽  
Color Palette ◽  
Focal Adhesion Protein ◽  
Fluorescent Protein Tags

AbstractSelf-complementing split fluorescent proteins (split FP1-10/11) have become an important labeling tool in live-cell protein imaging. However, current split FP systems to label multiple proteins in single cells have a fundamental limitation in the number of proteins that can be simultaneously labeled. Here, we describe an approach to expand the number of orthogonal split FP systems with spectrally distinct colors. By combining rational design and cycles of directed evolution, we expand the spectral color palette of FP1-10/11. We also circularly permutate GFP and synthesize the β-strand 7, 8, or 10 system. These split GFP pairs are not only capable of labeling proteins but are also orthogonal to the current FP1-10/11 pairs, offering multiplexed labeling of cellular proteins. Our multiplexing approach, using the new orthogonal split FP systems, demonstrates simultaneous imaging of four distinct proteins in single cells; the resulting images reveal nuclear localization of focal adhesion protein Zyxin.

scholarly journals Multiplexed live visualization of cell fate dynamics in hPSCs at single-cell resolution

2021 ◽  
Author(s):  
Sungmin Kim ◽  
Edward Ren ◽  
Paola Marco Casanova ◽  
Eugenia Piddini ◽  
Rafael Carazo Salas
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Spatial Location ◽  
Time Lapse ◽  
Strongly Coupled ◽  
Pluripotency Marker ◽  
Tightly Coupled ◽  
Differentiation Marker

ABSTRACTLive imaging can provide powerful insights into developmental and cellular processes but availability of multiplexable reporters has been limiting. Here we describe ORACLE, a cell fate reporter class in which fluorescent proteins fused with the nucleoporin POM121 are driven by promoters of transcription factors of interest. ORACLE’s nuclear rim localisation therefore enables multiplexing with conventional nuclear reporters. We applied ORACLE to investigate the dynamics of pluripotency exit at single-cell level, using human pluripotent stem cells (hPSCs) imaged by multi-day time-lapse high-content microscopy. Using an ORACLE-OCT4 pluripotency marker we reveal that G1 phase length and OCT4 level are strongly coupled and that spatial location in a colony impacts the timing of pluripotency exit. Combining ORACLE-OCT4 and an ORACLE-SOX1 early neuronal differentiation marker, we visualize in real-time the dynamics of cell fate transition between pluripotency and early neural fate, and show that pluripotency exit and differentiation onset are likely not tightly coupled in single-cells. Thus ORACLE is a powerful tool to enable quantitative studies of spatiotemporal cell fate control.

Sign in / Sign up

Export Citation Format

Share Document