scholarly journals Imaging and manipulation of intra-cellular structures in live cells using multiphoton excitation

2008 ◽  
Vol 36 (Supplement) ◽  
pp. S8-S9
Author(s):  
W. Watanabe ◽  
S. Matsunaga ◽  
T. Higashi ◽  
K. Fukui ◽  
K. Itoh
Keyword(s):  
Cellular Structures ◽  
Live Cells ◽  
Multiphoton Excitation

scholarly journals Mapping Protein Numbers in Living Cells

2021 ◽  
Author(s):  
Derek J Thirstrup ◽  
Jie Yao ◽  
Jamie Sherman ◽  
Irina A Mueller ◽  
Winfried Wiegraebe
Keyword(s):  
Single Cell ◽  
Laser Scanning ◽  
Light Sheet ◽  
Living Cells ◽  
Cellular Protein ◽  
Cellular Structures ◽  
Live Cells ◽  
Total Proteins ◽  
Cell Gene Expression ◽  
Induced Pluripotent

We introduce a new, robust method to map the numbers of proteins in living cells. The method can be applied to laser scanning, spinning disk, and lattice light-sheet microscopes in a robust, reproducible, and scalable fashion. The method uses calibrated EGFP solutions that are imaged with the appropriate microscope modality to create a calibration curve that is then applied to convert the fluorescence intensities from 3D microscope images into molecule numbers. We applied this method to human induced pluripotent stem cells in which proteins representing key cellular structures were endogenously tagged with mEGFP. We used the ratio of mEGFP-tagged proteins to total proteins to create 3D maps of live cells showing the density of total proteins measured in molecules per µm3. The method opens the door to new quantitative single cell analyses of cellular protein numbers in the context of single cell gene expression, associations with cellular complexes, and changes in cellular behaviors. The method is capable of quantifying protein numbers, over three orders of magnitude, in the cytoplasm or within various cellular structures while offering the unique advantages of each microscopy modality.

5-HT spatial distribution imaging with multiphoton excitation of 5-HT correlative visible fluorescence in live cells

2002 ◽  
Author(s):  
Zhihong Zhang ◽  
Shaoqun Zeng ◽  
Yafeng Liu ◽  
Wei Zhou ◽  
Tongsheng Chen ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
Live Cells ◽  
Multiphoton Excitation

scholarly journals BICD2, dynactin, and LIS1 cooperate in regulating dynein recruitment to cellular structures

2012 ◽  
Vol 23 (21) ◽  
pp. 4226-4241 ◽  
Author(s):  
Daniël Splinter ◽  
David S. Razafsky ◽  
Max A. Schlager ◽  
Andrea Serra-Marques ◽  
Ilya Grigoriev ◽  
...  
Keyword(s):  
Cytoplasmic Dynein ◽  
Cellular Structures ◽  
Live Cells ◽  
Concerted Action ◽  
Membrane Tethering ◽  
Directed Motility ◽  
Cellular Motor ◽  
Dynactin Complex

Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we focus on dynein–dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N–dynein–dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end–directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors.

scholarly journals Hyperspectral Counting of Multiplexed Nanoparticle Emitters in Single Cells and Organelles

2021 ◽  
Author(s):  
Prakrit V Jena ◽  
Mitchell A Gravely ◽  
Christian Cupo ◽  
Mohammad Moein Safaee ◽  
Daniel Roxbury ◽  
...  
Keyword(s):  
Carbon Nanotube ◽  
Single Cell ◽  
Single Cells ◽  
Cellular Structures ◽  
Live Cells ◽  
Tissue Cell ◽  
Significant Heterogeneity ◽  
The Subject ◽  
Non Gaussian ◽  
Accurate Quantification

Nanomaterials are the subject of a range of biomedical, commercial, and environmental investigations involving measurements in living cells and tissues. Accurate quantification of nanomaterials, at the tissue, cell, and organelle levels, is often difficult, however, in part due to their inhomogeneity. Here, we propose a method that uses the diverse optical properties of a nanomaterial preparation in order to improve quantification at the single-cell and organelle level. We developed 'hyperspectral counting', which employs diffraction-limited imaging via hyperspectral microscopy of a diverse set of nanomaterial emitters, to estimate nanomaterial counts in live cells and sub-cellular structures. A mathematical model was developed, and Monte Carlo simulations were employed, to improve the accuracy of these estimates, enabling quantification with single-cell and single-endosome resolution. We applied this nanometrology technique to identify an upper-limit of the rate of uptake into cells -approximately 3,000 particles endocytosed within 30 minutes. In contrast, conventional ROI counting results in a 230% undercount. The method identified significant heterogeneity and a broad non-Gaussian distribution of carbon nanotube uptake within cells. For example, while a particular cell contained an average of 1 nanotube per endosome, the heterogenous distribution resulted in over 7 nanotubes localizing within some endosomes, substantially changing the accounting of subcellular nanoparticle concentration distributions. This work presents a method to quantify cellular and subcellular concentrations of a heterogeneous carbon nanotube reference material, with implications for nanotoxicology, drug/gene delivery, and nanosensor fields.

scholarly journals Robust integrated intracellular organization of the human iPS cell: where, how much, and how variable?

2020 ◽  
Author(s):  
Matheus P. Viana ◽  
Jianxu Chen ◽  
Theo A. Knijnenburg ◽  
Ritvik Vasan ◽  
Calysta Yan ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Integrated Approach ◽  
Cellular Structures ◽  
Live Cells ◽  
Shape Variation ◽  
Ips Cell ◽  
Intracellular Organization ◽  
Wide Range ◽  
Cell Organization ◽  
Induced Pluripotent

SummaryDespite the intimate link between cell organization and function, the principles underlying intracellular organization and the relation between organization, gene expression and phenotype are not well understood. We address this by creating a benchmark for mean cell organization and the natural range of cell-to-cell variation. This benchmark can be used for comparison to other normal or abnormal cell states. To do this, we developed a reproducible microscope imaging pipeline to generate a high quality dataset of 3D, high-resolution images of over 200,000 live cells from 25 isogenic human induced pluripotent stem cell (hiPSC) lines from the Allen Cell Collection. Each line contains one fluorescently tagged protein, created via endogenous CRISPR/Cas9 gene editing, representing a key cellular structure or organelle. We used these images to develop a new multi-part generalizable analysis approach of the locations, amounts, and variation of the 25 cellular structures. Taking an integrated approach, we found that both the extent to which a structure’s individual location varied (“stereotypy”) and the extent to which the structure localized relative to all the other cellular structures (“concordance”) were robust to a wide range of cell shape variation, from flatter to taller, smaller to larger, or less to more polarized cells. We also found that these cellular structures varied greatly in how their volumes scaled with cell and nuclear size. These analyses create a data-driven set of quantitative rules for the locations, amounts, and variation of 25 cellular structures within the hiPSC as a normal baseline for cell organization.

scholarly journals Response of Cytoskeleton of Murine Osteoblast Cultures to Two-step Freezing

2005 ◽  
Vol 37 (12) ◽  
pp. 814-818 ◽  
Author(s):  
Bao-Lin Liu ◽  
John Mcgrath
Keyword(s):  
Actin Filaments ◽  
Damage Mechanism ◽  
Cellular Structures ◽  
Live Cells ◽  
Freezing Process ◽  
Fluorescence Staining ◽  
Specific Fluorescence ◽  
The Status

Abstract Understanding the ultrastructural response of cells to the freezing process is important for designing cryopreservation strategies for cells and tissues. The cellular structures of attached cells are targets of cryopreservation-induced damage. Specific fluorescence staining was used to assess the status of the actin filaments (F-actin) of murine osteoblasts attached to hydroxyapatite discs and plastic coverslips for a two-step freezing process. The F-actin of dead cells was depolymerized and distorted in the freezing process, whereas that of live cells had little change. The results suggest that the cytoskeleton may support the robustness of cells during cryopreservation. The present study helps to investigate the damage mechanism of attached cells during the freezing process.

Preparatory Procedures for Electron Microscopic Analysis at the Molecular Level of Resolution

1978 ◽  
Vol 36 (3) ◽  
pp. 516-520
Author(s):  
F.J. Sjostrand
Keyword(s):  
Electron Microscope ◽  
Molecular Level ◽  
Microscopic Analysis ◽  
Resolving Power ◽  
Cellular Structures ◽  
Electron Microscopic ◽  
Systematic Analysis ◽  
Technical Developments ◽  
Thin Sectioning ◽  
Obvious Reason

In the 1940's and 1950's electron microscopy conferences were attended with everybody interested in learning about the latest technical developments for one very obvious reason. There was the electron microscope with its outstanding performance but nobody could make very much use of it because we were lacking proper techniques to prepare biological specimens. The development of the thin sectioning technique with its perfectioning in 1952 changed the situation and systematic analysis of the structure of cells could now be pursued. Since then electron microscopists have in general become satisfied with the level of resolution at which cellular structures can be analyzed when applying this technique. There has been little interest in trying to push the limit of resolution closer to that determined by the resolving power of the electron microscope.

Some aspects of the defect structure in an austenitic stainless steel

1978 ◽  
Vol 36 (1) ◽  
pp. 314-315
Author(s):  
R. Gonzalez ◽  
L. Bru
Keyword(s):  
Stainless Steel ◽  
Austenitic Stainless Steel ◽  
Cellular Structures ◽  
Total Strain Amplitude ◽  
Total Deformation ◽  
Density Of Dislocations ◽  
Planar Arrays ◽  
Stacking Fault Tetrahedra ◽  
Distribution Of Dislocations ◽  
Very High

The analysis of stacking fault tetrahedra (SFT) in fatigued metals (1,2) is somewhat complicated, due partly to their relatively low density, but principally to the presence of a very high density of dislocations which hides them. In order to overcome this second difficulty, we have used in this work an austenitic stainless steel that deforms in a planar mode and, as expected, examination of the substructure revealed planar arrays of dislocation dipoles rather than the cellular structures which appear both in single and polycrystals of cyclically deformed copper and silver. This more uniform distribution of dislocations allows a better identification of the SFT.The samples were fatigue deformed at the constant total strain amplitude Δε = 0.025 for 5 cycles at three temperatures: 85, 293 and 773 K. One of the samples was tensile strained with a total deformation of 3.5%.

4-dimensional, high-resolution imaging of living cells

1992 ◽  
Vol 50 (1) ◽  
pp. 416-417
Author(s):  
Shinya Inoué
Keyword(s):  
Light Microscope ◽  
Living Cells ◽  
Live Cells ◽  
Video Tape ◽  
Active Living ◽  
High Resolution Imaging ◽  
3 Dimensional ◽  
Dynamic Architecture ◽  
Resolution Imaging ◽  
Disk Memory

This paper reports progress of our effort to rapidly capture, and display in time-lapsed mode, the 3-dimensional dynamic architecture of active living cells and developing embryos at the highest resolution of the light microscope. Our approach entails: (A) real-time video tape recording of through-focal, ultrathin optical sections of live cells at the highest resolution of the light microscope; (B) repeat of A at time-lapsed intervals; (C) once each time-lapsed interval, an image at home focus is recorded onto Optical Disk Memory Recorder (OMDR); (D) periods of interest are selected using the OMDR and video tape records; (E) selected stacks of optical sections are converted into plane projections representing different view angles (±4 degrees for stereo view, additional angles when revolving stereos are desired); (F) analysis using A - D.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

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