Multidimensional fluorescence microscopy in live cell imaging – A mini review

2012 ◽  
Vol 1 (1) ◽  
Author(s):  
Herbert Schneckenburger ◽  
Petra Weber ◽  
Michael Wagner ◽  
Thomas Bruns ◽  
Verena Richter ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Proteins ◽  
Light Exposure ◽  
Single Cells ◽  
Three Dimensional ◽  
Fluorescence Lifetime Imaging ◽  
Single Plane ◽  
Fluorescent Markers ◽  
And Function ◽  
Multidimensional Fluorescence

AbstractFluorescence microscopy methods are described with high spatial, spectral, and temporal resolution. In addition to three-dimensional (3D) microscopy, based on confocal, structured, or single-plane illumination, spectral imaging and fluorescence lifetime imaging microscopy are used to probe the conformation of fluorescent molecules as well as their interaction with the microenvironment. In addition to single cells or cell monolayers, 3D cell cultures are used increasingly, as they are more representative for tissue morphology and function. All methods are discussed in the context of controlled light exposure, which is regarded as a key parameter to maintain cell viability. The applications presented in this mini review include autofluorescence measurements of glioblastoma cells as well as various fluorescent markers or fluorescent proteins.

scholarly journals Monitoring microbial communities using light sheet fluorescence microscopy

2017 ◽  
Author(s):  
Raghuveer Parthasarathy
Keyword(s):  
Fluorescence Microscopy ◽  
Microbial Communities ◽  
Three Dimensional ◽  
Light Sheet ◽  
Rapid Acquisition ◽  
History Of ◽  
The Many ◽  
And Function ◽  
Microbial Systems ◽  
Light Sheet Fluorescence Microscopy

Microbes often live in dense, dynamic, multi-species communities whose architecture and function are intimately intertwined. Imaging these complex, three-dimensional ensembles presents considerable technical challenges, however. In this review, I describe light sheet fluorescence microscopy, a technique that enables rapid acquisition of three-dimensional images over large fields of view and over long durations, and I highlight recent applications of this method to microbial systems that include artificial closed ecosystems, bacterial biofilms, and gut microbiota. I comment also on the history of light sheet imaging and the many variants of the method. Light sheet techniques have tremendous potential for illuminating the workings of microbial communities, a potential that is just beginning to be realized.

scholarly journals Monitoring microbial communities using light sheet fluorescence microscopy

2017 ◽  
Author(s):  
Raghuveer Parthasarathy
Keyword(s):  
Fluorescence Microscopy ◽  
Microbial Communities ◽  
Three Dimensional ◽  
Light Sheet ◽  
Rapid Acquisition ◽  
History Of ◽  
The Many ◽  
And Function ◽  
Microbial Systems ◽  
Light Sheet Fluorescence Microscopy

Microbes often live in dense, dynamic, multi-species communities whose architecture and function are intimately intertwined. Imaging these complex, three-dimensional ensembles presents considerable technical challenges, however. In this review, I describe light sheet fluorescence microscopy, a technique that enables rapid acquisition of three-dimensional images over large fields of view and over long durations, and I highlight recent applications of this method to microbial systems that include artificial closed ecosystems, bacterial biofilms, and gut microbiota. I comment also on the history of light sheet imaging and the many variants of the method. Light sheet techniques have tremendous potential for illuminating the workings of microbial communities, a potential that is just beginning to be realized.

scholarly journals Monitoring microbial communities using light sheet fluorescence microscopy

2017 ◽  
Author(s):  
Raghuveer Parthasarathy
Keyword(s):  
Fluorescence Microscopy ◽  
Microbial Communities ◽  
Three Dimensional ◽  
Light Sheet ◽  
Rapid Acquisition ◽  
History Of ◽  
The Many ◽  
And Function ◽  
Microbial Systems ◽  
Light Sheet Fluorescence Microscopy

Microbes often live in dense, dynamic, multi-species communities whose architecture and function are intimately intertwined. Imaging these complex, three-dimensional ensembles presents considerable technical challenges, however. In this review, I describe light sheet fluorescence microscopy, a technique that enables rapid acquisition of three-dimensional images over large fields of view and over long durations, and I highlight recent applications of this method to microbial systems that include artificial closed ecosystems, bacterial biofilms, and gut microbiota. I comment also on the history of light sheet imaging and the many variants of the method. Light sheet techniques have tremendous potential for illuminating the workings of microbial communities, a potential that is just beginning to be realized.

scholarly journals Three-dimensional imaging reveals endo(sarco)plasmic reticulum-containing invaginations within the nucleoplasm of muscle

2018 ◽  
Vol 314 (3) ◽  
pp. C257-C267 ◽  
Author(s):  
Shin-Haw Lee ◽  
Sina Hadipour-Lakmehsari ◽  
Tetsuaki Miyake ◽  
Anthony O. Gramolini
Keyword(s):  
Regulation Of Gene Expression ◽  
Three Dimensional ◽  
Hek293 Cells ◽  
Complex Nature ◽  
Cross Sectional ◽  
Single Plane ◽  
Plasmic Reticulum ◽  
Primary Mouse ◽  
Store Operated Calcium Entry ◽  
And Function

The mammalian nucleus has invaginations from the cytoplasm, termed nucleoplasmic reticulum (NR). With increased resolution of cellular imaging, progress has been made in understanding the formation and function of NR. In fact, nucleoplasmic Ca2+ homeostasis has been implicated in the regulation of gene expression, DNA repair, and cell death. However, the majority of studies focus on cross-sectional or single-plane analyses of NR invaginations, providing an incomplete assessment of its distribution and content. Here, we provided advanced imaging and three-dimensional reconstructive analyses characterizing the molecular constituents of nuclear invaginations in the nucleoplasm in HEK293 cells, murine C2C12 muscle cells, and cardiac myocytes. We demonstrated the presence of critical Ca2+ regulatory channels, including sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), stromal interaction molecule 1 (STIM1), and Ca2+ release-activated Ca2+ channel protein 1 (ORAI1), in the nucleoplasm in isolated primary mouse cardiomyocytes. We have shown for the first time the presence of STIM1 and ORAI1 in the nucleoplasm, suggesting the presence of store-operated calcium entry (SOCE) mechanism in nucleoplasmic Ca2+ regulation. These results show that nucleoplasmic invaginations contain continuous endoplasmic reticulum components, mitochondria, and intact nuclear membranes, highlighting the extremely detailed and complex nature of this organellar structure.

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.

scholarly journals Daily mitochondrial dynamics in cone photoreceptors

2020 ◽  
Vol 117 (46) ◽  
pp. 28816-28827
Author(s):  
Michelle M. Giarmarco ◽  
Daniel C. Brock ◽  
Brian M. Robbings ◽  
Whitney M. Cleghorn ◽  
Kristine A. Tsantilas ◽  
...  
Keyword(s):  
Light Exposure ◽  
Mitochondrial Dynamics ◽  
Three Dimensional ◽  
Cone Photoreceptors ◽  
Energy Demands ◽  
Mitochondrial Turnover ◽  
And Function ◽  
Extracellular Structures ◽  
Endoplasmic Reticula ◽  
Daily Changes

Cone photoreceptors in the retina are exposed to intense daylight and have higher energy demands in darkness. Cones produce energy using a large cluster of mitochondria. Mitochondria are susceptible to oxidative damage, and healthy mitochondrial populations are maintained by regular turnover. Daily cycles of light exposure and energy consumption suggest that mitochondrial turnover is important for cone health. We investigated the three-dimensional (3D) ultrastructure and metabolic function of zebrafish cone mitochondria throughout the day. At night retinas undergo a mitochondrial biogenesis event, corresponding to an increase in the number of smaller, simpler mitochondria and increased metabolic activity in cones. In the daytime, endoplasmic reticula (ER) and autophagosomes associate more with mitochondria, and mitochondrial size distribution across the cluster changes. We also report dense material shared between cone mitochondria that is extruded from the cell at night, sometimes forming extracellular structures. Our findings reveal an elaborate set of daily changes to cone mitochondrial structure and function.

scholarly journals Optoacoustic flow-cytometry with light scattering referencing

2020 ◽  
Author(s):  
Markus Seeger ◽  
Andre C. Stiel ◽  
Vasilis Ntziachristos
Keyword(s):  
Light Scattering ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Optoacoustic Imaging ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Ultrasound Backscatter ◽  
Molecular Specificity ◽  
And Function

AbstractIn analogy to the development of fluorescent proteins, innovative tools for screening optoacoustic cell labels could lead to tailored protein labels for OA, imparting novel ways to visualize biological structure and function. Optoacoustic imaging emerges towards a highly promising modality for life sciences and medical practise with advantageous capabilities such as great accessible depth, and 3D studying of living tissue. The development of novel labels with molecular specificity could significantly enhance the optoacoustic contrast, specificity, and sensitivity and allow optoacoustic to interrogate tissues not amenable to the fluorescence method. We report on an optoacoustic flow cytometer (OAFC) prototype, developed for screening optoacoustic reporter genes. The cytometer concurrently records light scattering for referencing purposes. Since recording light scattering is completely independent from OA, we believe it to be a more reliable referencing method than e.g. fluorescence or ultrasound-backscatter. Precise characterization of our OAFC prototype showcases its ability to optoacoustically characterize objects in-flow that are in the size range of single cells. We apply the OAFC to distinguish individual E. coli cells based on optoacoustic properties of their expressed chromoproteins read in-flow using microfluidic arrangements and achieved precisions over 90%. We discuss how the light scattering referenced OAFC method offers a critical step towards routine measurement of optoacoustic properties of single-cells and could pave the way for identifying genetically encoded optoacoustic reporters, by transferring working concepts of the fluorescence field.

scholarly journals A versatile compressed sensing scheme for faster and less phototoxic 3D fluorescence microscopy

2017 ◽  
Author(s):  
Maxime Woringer ◽  
Xavier Darzacq ◽  
Christophe Zimmer ◽  
Mustafa Mir
Keyword(s):  
Fluorescence Microscopy ◽  
Compressed Sensing ◽  
Light Exposure ◽  
Three Dimensional ◽  
Light Sheet ◽  
Computational Imaging ◽  
Acquisition Time ◽  
Trade Offs ◽  
Fold Reduction ◽  
3D Fluorescence Microscopy

AbstractThree-dimensional fluorescence microscopy based on Nyquist sampling of focal planes faces harsh trade-offs between acquisition time, light exposure, and signal-to-noise. We propose a 3D compressed sensing approach that uses temporal modulation of the excitation intensity during axial stage sweeping and can be adapted to fluorescence microscopes without hardware modification. We describe implementations on a lattice light sheet microscope and an epifluorescence microscope, and show that images of beads and biological samples can be reconstructed with a 5-10 fold reduction of light exposure and acquisition time. Our scheme opens a new door towards faster and less damaging 3D fluorescence microscopy.OCIS codes: (110.1758) Computational imaging; (170.2520) Fluorescence microscopy; (170.6900) Three-dimensional microscopy.

Analyzing proteome topology and function by automated multidimensional fluorescence microscopy

2006 ◽  
Vol 24 (10) ◽  
pp. 1270-1278 ◽  
Author(s):  
Walter Schubert ◽  
Bernd Bonnekoh ◽  
Ansgar J Pommer ◽  
Lars Philipsen ◽  
Raik Böckelmann ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
And Function ◽  
Multidimensional Fluorescence
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