Chemical biology-based approaches on fluorescent labeling of proteins in live cells

2013 ◽  
Vol 9 (5) ◽  
pp. 862 ◽  
Author(s):  
Deokho Jung ◽  
Kyoungmi Min ◽  
Juyeon Jung ◽  
Wonhee Jang ◽  
Youngeun Kwon
Keyword(s):  
Chemical Biology ◽  
Fluorescent Labeling ◽  
Live Cells

scholarly journals Highly photostable fluorescent labeling of proteins in live cells using exchangeable coiled coils heterodimerization

2020 ◽  
Vol 77 (21) ◽  
pp. 4429-4440 ◽  
Author(s):  
Maxim M. Perfilov ◽  
Nadya G. Gurskaya ◽  
Ekaterina O. Serebrovskaya ◽  
Pavel A. Melnikov ◽  
Sergey L. Kharitonov ◽  
...  
Keyword(s):  
Fluorescent Labeling ◽  
Coiled Coils ◽  
Live Cells

scholarly journals Fringe optimization for structured illumination super-resolution microscope with digital micromirror device

2019 ◽  
Vol 12 (03) ◽  
pp. 1950014 ◽  
Author(s):  
Xibin Yang ◽  
Qian Zhu ◽  
Zhenglong Sun ◽  
Gang Wen ◽  
Xin Jin ◽  
...  
Keyword(s):  
Modulation Depth ◽  
Low Cost ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Live Cells ◽  
Digital Micromirror Device ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Wide Range ◽  
Fringe Patterns

Structured illumination microscopy (SIM) is a promising super-resolution technique for imaging subcellular structures and dynamics due to its compatibility with most commonly used fluorescent labeling methods. Structured illumination can be obtained by either laser interference or projection of fringe patterns. Here, we proposed a fringe projector composed of a compact multi-wavelength LEDs module and a digital micromirror device (DMD) which can be directly attached to most commercial inverted fluorescent microscopes and update it into a SIM system. The effects of the period and duty cycle of fringe patterns on the modulation depth of the structured light field were studied. With the optimized fringe pattern, [Formula: see text] resolution improvement could be obtained with high-end oil objectives. Multicolor imaging and dynamics of subcellular organelles in live cells were also demonstrated. Our method provides a low-cost solution for SIM setup to expand its wide range of applications to most research labs in the field of life science and medicine.

scholarly journals Design of a Multi-Use Photoreactor to Enable Visible Light Photocatalytic Chemical Transformations and Labeling in Live Cells

2020 ◽  
Author(s):  
Noah Bissonnette ◽  
Keun Ah Ryu ◽  
Tamara Reyes-Robles ◽  
Sharon Wilhelm ◽  
Erik Hett ◽  
...  
Keyword(s):  
Visible Light ◽  
Chemical Biology ◽  
Coupling Reaction ◽  
Cross Coupling ◽  
Live Cells ◽  
Chemical Transformations ◽  
Cross Coupling Reaction ◽  
Visible Light Driven ◽  
Visible Light Photocatalytic

<p>Despite the growing utilization of visible light photochemistry in both chemistry and biology, a general low-heat photoreactor for use across these different disciplines does not exist. Herein, we describe the design and utilization of a standardized photoreactor for visible light driven activation and photocatalytic chemical transformations. Using this single benchtop photoreactor, we perform photoredox reactions across multiple visible light wavelengths, a high throughput photocatalytic cross coupling reaction, and <i>in vitro</i> labeling of proteins and live cells. Given the success of this reactor in all tested applications, we envision that this multi-use photoreactor will be widely used in biology, chemical biology, and medicinal chemistry settings.</p>

scholarly journals High-throughput label-free molecular fingerprinting flow cytometry

2019 ◽  
Vol 5 (1) ◽  
pp. eaau0241 ◽  
Author(s):  
Kotaro Hiramatsu ◽  
Takuro Ideguchi ◽  
Yusuke Yonamine ◽  
SangWook Lee ◽  
Yizhi Luo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Fluorescent Labeling ◽  
Live Cells ◽  
Molecular Fingerprinting ◽  
Label Free ◽  
Indispensable Tool

Flow cytometry is an indispensable tool in biology for counting and analyzing single cells in large heterogeneous populations. However, it predominantly relies on fluorescent labeling to differentiate cells and, hence, comes with several fundamental drawbacks. Here, we present a high-throughput Raman flow cytometer on a microfluidic chip that chemically probes single live cells in a label-free manner. It is based on a rapid-scan Fourier-transform coherent anti-Stokes Raman scattering spectrometer as an optical interrogator, enabling us to obtain the broadband molecular vibrational spectrum of every single cell in the fingerprint region (400 to 1600 cm−1) with a record-high throughput of ~2000 events/s. As a practical application of the method not feasible with conventional flow cytometry, we demonstrate high-throughput label-free single-cell analysis of the astaxanthin productivity and photosynthetic dynamics ofHaematococcus lacustris.

scholarly journals Cell Wall Growth and Modulation Dynamics in a Model Unicellular Green Alga—Penium margaritaceum: Live Cell Labeling with Monoclonal Antibodies

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
David S. Domozych ◽  
Hannah Brechka ◽  
Alicia Britton ◽  
Marc Toso
Keyword(s):  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Green Alga ◽  
Fluorescent Labeling ◽  
Land Plants ◽  
Cell Labeling ◽  
Live Cells ◽  
Specific Cell ◽  
Cell Wall Degrading Enzymes ◽  
Cell Wall Polymers

Penium margaritaceum is a unicellular charophycean green alga that possesses cell wall polymers similar to those of land plants. Several wall macromolecules of this alga are recognized by monoclonal antibodies specific for wall polymer epitopes of land plants. Immunofluorescence protocols using these antibodies may be employed to label specific cell wall constituents of live cells. Fluorescent labeling persists for several days, and this attribute allows for tracing of wall epitopes in both long- and short-term studies of cell development. Quantitative analysis of surface area covered by cell wall polymers is also easily performed. We show that significant cell expansion caused by incubation of cells in low levels of osmotically active agents like mannitol, glucose, or sucrose results from the inability of cells to undergo cytokinesis but does not result in significant changes to the amount of new cell wall. We also demonstrate that cells can be maintained for long periods of time in culture medium supplemented with specific cell wall-degrading enzymes where notable changes to wall infrastructure occur. These results demonstrate the great potential value of Penium in elucidating fundamental events during cell wall synthesis and modulation in plant cells.

scholarly journals Dynamic signatures of lipid droplets as new markers to quantify cellular metabolic changes

2020 ◽  
Author(s):  
Chi Zhang ◽  
Stephen A. Boppart
Keyword(s):  
Lipid Droplets ◽  
Distribution Functions ◽  
Fluorescent Labeling ◽  
Metabolic Changes ◽  
Live Cells ◽  
Label Free ◽  
Metabolic States ◽  
Metabolic Properties ◽  
Displacement Speed ◽  
Anti Stokes

AbstractThe metabolic properties of live cells are very susceptible to intra- or extra-cellular perturbations, making their measurements challenging tasks. We show that the dynamics of lipid droplets (LDs) carry information to measure the lipid metabolism of live cells. Coherent anti-Stokes Raman scattering microscopy was used to statistically quantify LD dynamics in living cells in a label-free manner. We introduce dynamic signatures of cells derived from the LD displacement, speed, travel length, and directionality, which allows for the detection of cellular changes induced by stimuli such as fluorescent labeling, temperature change, starvation, and chemical treatment. Histogram fittings of the dynamic signatures using lognormal distribution functions provide quantification of changes in cellular metabolic states. The LD dynamics also enable separation of subpopulations of LDs correlated with different functions. We demonstrate that LD dynamics are new markers to quantify the metabolic changes in live cells.

scholarly journals Fluorophore-Assisted Click Chemistry through Copper(I) Complexation

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 619
Author(s):  
Victor Flon ◽  
Magalie Bénard ◽  
Damien Schapman ◽  
Ludovic Galas ◽  
Pierre-Yves Renard ◽  
...  
Keyword(s):  
Reaction Rate ◽  
Photophysical Properties ◽  
Fluorescent Labeling ◽  
Fluorescence Labeling ◽  
Live Cells ◽  
Proof Of Concept ◽  
Peptide Molecule ◽  
Chemical Strategies ◽  
Complex Copper

The copper-catalyzed alkyne-azide cycloaddition (CuAAC) is one of the most powerful chemical strategies for selective fluorescent labeling of biomolecules in in vitro or biological systems. In order to accelerate the ligation process and ensure efficient formation of conjugates under diluted conditions, external copper(I) ligands or sophisticated copper(I)-chelating azides are used. This latter strategy, however, increases the bulkiness of the triazole linkage, thus perturbing the biological function or dynamic behavior of the conjugates. In a proof-of-concept study, we investigated the use of an extremely compact fluorophore-based copper(I) chelating azide in order to accelerate the CuAAC with concomitant fluorescence labeling; in our strategy, the fluorophore is able to complex copper(I) species while retaining its photophysical properties. It is believed that this unprecedented approach which was applied for the labeling of a short peptide molecule and the fluorescent labeling of live cells, could be extended to other families of nitrogen-based fluorophores in order to tune both the reaction rate and photophysical characteristics.

scholarly journals Exploring structural dynamics of a membrane protein by combining bioorthogonal chemistry and cysteine mutagenesis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Kanchan Gupta ◽  
Gilman ES Toombes ◽  
Kenton J Swartz
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Conformational Changes ◽  
Fluorescent Labeling ◽  
Live Cells ◽  
Bioorthogonal Chemistry ◽  
Kv Channel ◽  
Cysteine Mutagenesis ◽  
Voltage Dependent ◽  
Methionine Residues

The functional mechanisms of membrane proteins are extensively investigated with cysteine mutagenesis. To complement cysteine-based approaches, we engineered a membrane protein with thiol-independent crosslinkable groups using azidohomoalanine (AHA), a non-canonical methionine analogue containing an azide group that can selectively react with cycloalkynes through a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We demonstrate that AHA can be readily incorporated into the Shaker Kv channel in place of methionine residues and modified with azide-reactive alkyne probes in Xenopus oocytes. Using voltage-clamp fluorometry, we show that AHA incorporation permits site-specific fluorescent labeling to track voltage-dependent conformational changes similar to cysteine-based methods. By combining AHA incorporation and cysteine mutagenesis in an orthogonal manner, we were able to site-specifically label the Shaker Kv channel with two different fluorophores simultaneously. Our results identify a facile and straightforward approach for chemical modification of membrane proteins with bioorthogonal chemistry to explore their structure-function relationships in live cells.

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