scholarly journals FRAME-tags: genetically encoded fluorescent markers for multiplexed barcoding and time-resolved tracking of live cells

2021 ◽  
Author(s):  
Andrew V. Anzalone ◽  
Miguel Jimenez ◽  
Virginia W. Cornish
Keyword(s):  
Fitness Landscape ◽  
Fluorescent Proteins ◽  
Biological Systems ◽  
Emission Spectra ◽  
Live Cells ◽  
Time Resolved ◽  
Detection Techniques ◽  
Fluorescent Markers ◽  
Spectral Channels

Cellular barcodes offer critical tools for tracking cellular identity in biological systems. Although genetically encoded fluorescent barcodes are ideal for real-time tracking, their scalability is constrained by the broad, overlapping emission spectra characteristic of fluorescent proteins (FPs). Here, we describe a palette of genetically encoded fluorescent barcodes called FRAME- tags, which break this scalability barrier by encoding barcode identity as unique FP expression ratios. FRAME-tags use -1 programmed ribosomal frameshifting RNA motifs to precisely control the translational output of multiple FPs from a single mRNA, leading to extremely narrow and resolvable ratios of the corresponding cellular fluorescence distributions. With this platform, we constructed 20 resolvable FRAME-tags in yeast using just two FPs, and further demonstrated that 100 or more distinguishable FRAME-tags could be made by the addition of a third FP. We used FRAME-tags to map the dynamic fitness landscape of yeast co-cultures, and to characterize the expression pattern of 20 yeast promoters in multiplex across diverse conditions. FRAME-tags offer a valuable new tool for cellular barcoding that enables time- resolved characterization of complex biological systems using widely available fluorescence detection techniques and a minimal number of spectral channels.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

scholarly journals Super-multiplexed fluorescence microscopy via photostability contrast

2018 ◽  
Author(s):  
Antony Orth ◽  
Richik N. Ghosh ◽  
Emma Wilson ◽  
Timothy Doughney ◽  
Hannah Brown ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Emission Spectra ◽  
Biological Research ◽  
Fluorescent Labels ◽  
Wide Range ◽  
Confocal Fluorescence ◽  
High Degree ◽  
Spectral Channels

AbstractMany areas of biological research rely heavily on fluorescence microscopy to observe and quantify the inner workings of the cell. Traditionally, multiple types of cellular structures or biomolecules are visualized simultaneously with spectrally distinct fluorescent labels. A high degree of multiplexing is desirable as it affords the experiment greater information content, speeding up research timelines. Multiplexing can be increased by imaging a larger number of spectral channels, however, the wide emission spectra of most fluorophores limits multiplexing to four or five labels in standard fluorescence microscopes. Further multiplexing requires another dimension of contrast. Here, we show that photostability differences can be used to distinguish between fluorescent labels. By combining photobleaching characteristics with a novel unmixing algorithm, we resolve up to three fluorescent labels in a single spectral channel and unmix fluorescent labels with nearly identical emission spectra. We apply our technique to organic dyes, autofluorescent biomolecules and fluorescent proteins, and show that the latter are particularly well suited to our method as their bleaching is often reversible. Our approach has the potential to triple the multiplexing capabilities of any digital widefield or confocal fluorescence microscope with no additional hardware, making it readily accessible to a wide range of researchers.

Design and Characterization of a Femtosecond Fluorescence Spectrometer Based on Optical Kerr Gating

2005 ◽  
Vol 59 (2) ◽  
pp. 206-220 ◽  
Author(s):  
S. Arzhantsev ◽  
M. Maroncelli
Keyword(s):  
Response Times ◽  
Detection System ◽  
Emission Spectra ◽  
High Repetition Rate ◽  
General Purpose ◽  
Kerr Medium ◽  
Recording Time ◽  
Time Resolved ◽  
Good Signal

Design and characterization of a general-purpose spectrometer for recording time-resolved emission spectra of typical fluorescent species is described. The system is based on a high repetition rate amplified Ti:sapphire system, an optical Kerr shutter for gating the emission, and a polychromator plus charge-coupled device (CCD) detection system. Using 1 mm of liquid benzene as the Kerr medium, and optics designed to provide high polarization quality, emission spectra of dilute solutions of solutes with nanosecond lifetimes can be recorded with good signal-to-noise ratios. The current spectrometer uses excitation wavelengths near 390 nm and provides spectra over the wavelength range 400–650 nm with 4 nm resolution and instrument response times of 450 fs (full width at half-maximum, FWHM). Selected applications are described to demonstrate the utility of this instrument.

scholarly journals Visual Barcodes for Multiplexing Live Microscopy-Based Assays

2020 ◽  
Author(s):  
Tom Kaufman ◽  
Erez Nitzan ◽  
Nir Firestein ◽  
Miriam Ginzberg ◽  
Seshu Iyengar ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Single Cells ◽  
Live Imaging ◽  
Live Cells ◽  
Balance Growth ◽  
Complex Biological System ◽  
Clonal Identity

Abstract While multiplexing samples using DNA barcoding revolutionized the pace of biomedical discovery, multiplexing of live imaging-based applications has been limited by the number of fluorescent proteins that can be deconvoluted using common microscopy equipment. To address this limitation we developed visual barcodes that discriminate the clonal identity of single cells by targeting different fluorescent proteins to specific subcellular locations. We demonstrate that deconvolution of these barcodes is highly accurate and robust to many cellular perturbations. We then used visual barcodes to generate ‘Signalome’ cell-lines by multiplexing live reporters to monitor the simultaneous activity in 12 branches of signaling, in live cells, at single cell resolution, over time. Using the ‘Signalome’ we identified two distinct clusters of signaling pathways that balance growth and proliferation, emphasizing the importance of growth homeostasis as a central organizing principle in cancer signaling. The ability to multiplex samples in live imaging applications, both in vitro and in vivo may allow better high-content characterization of complex biological system

scholarly journals Characterization of melanoidin formed from glucose with alanine  by Fluorescence spectroscopy and redox chemistry in vitro cells: تشخيص الميلانويدين المتشكل من الكلوكوز مع الألنين بمطياف الفلوره ودراسة تأثيره التأكسدي على خلايا CHO-K1 المختبرية

2020 ◽  
Vol 4 (1) ◽  
Author(s):  
Ghassan Faisal Mohsin, Abdulridha Ati Jaafar
Keyword(s):  
Fluorescence Spectroscopy ◽  
Chinese Hamster ◽  
Emission Spectra ◽  
Green Fluorescence Protein ◽  
Time Resolved ◽  
Yellow Fluorescence Protein ◽  
Fluorescence Protein ◽  
Dialysis Time

Possible identify and describe a polymer formed from Glc with Ala by Fluorescence spectroscopy. Melanoidins before dialysis was higher than those of melanoidin after dialysis. Time‐resolved fluorescence has been applied in the characterization of melanoidin and was observed. Melanoidins show a decrease in the absorption after the addition of hydrogen peroxide when the concentrations of H2O2 from 100 - 200 μl are used. However, the data suggests that the melanoidin did not penetrate into the cells (CHO-K1) after 1 and 2 hours, but it can be observed after 24 hours. Abbreviation: TRES, time-resolved emission spectra ; Glc, Glucose ; Ala, Alanine ; YFP, Yellow fluorescence protein ; GFP, green fluorescence protein ; CHO, Chinese hamster ovary

scholarly journals Fluorescent Markers: Proteins and Nanocrystals

2021 ◽  
Author(s):  
Anielle Christine Almeida Silva ◽  
Jerusa Maria de Oliveira ◽  
Kelen Talita Romão da Silva ◽  
Francisco Rubens Alves dos Santos ◽  
João Paulo Santos de Carvalho ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Biological Systems ◽  
Biological Molecules ◽  
Fluorescent Reporter ◽  
Fluorescent Markers ◽  
Drosophila Model ◽  
Reporter Proteins ◽  
Red Luminescence

This book chapter will comment on fluorescent reporter proteins and nanocrystals’ applicability as fluorescent markers. Fluorescent reporter proteins in the Drosophila model system offer a degree of specificity that allows monitoring cellular and biochemical phenomena in vivo, such as autophagy, mitophagy, and changes in the redox state of cells. Titanium dioxide (TiO2) nanocrystals (NCs) have several biological applications and emit in the ultraviolet, with doping of europium ions can be visualized in the red luminescence. Therefore, it is possible to monitor nanocrystals in biological systems using different emission channels. CdSe/CdS magic-sized quantum dots (MSQDs) show high luminescence stability in biological systems and can be bioconjugated with biological molecules. Therefore, this chapter will show exciting results of the group using fluorescent proteins and nanocrystals in biological systems.

Characterization of a Motile Cellular Domain Arising During the Amoebo-Flagellate Transformation of Physarum Polycephalum

1980 ◽  
Vol 38 ◽  
pp. 552-553
Author(s):  
K.I. Pagh ◽  
M.R. Adelman
Keyword(s):  
Cell Shape ◽  
Physarum Polycephalum ◽  
Slime Mold ◽  
Live Cells ◽  
Cellular Domain

Unicellular amoebae of the slime mold Physarum polycephalum undergo marked changes in cell shape and motility during their conversion into flagellate swimming cells (l). To understand the processes underlying motile activities expressed during the amoebo-flagellate transformation, we have undertaken detailed investigations of the organization, formation and functions of subcellular structures or domains of the cell which are hypothesized to play a role in movement. One focus of our studies is on a structure, termed the “ridge” which appears as a flattened extension of the periphery along the length of transforming cells (Fig. 1). Observations of live cells using Nomarski optics reveal two types of movement in this region:propagation of undulations along the length of the ridge and formation and retraction of filopodial projections from its edge. The differing activities appear to be associated with two characteristic morphologies, illustrated in Fig. 1.

Characterization of Amethysts from Sukamara, Central Kalimantan, Using Laser-Induced Breakdown Spectroscopy (LIBS)

2020 ◽  
Vol 1 (2) ◽  
pp. 5-8
Author(s):  
Komang Gde Suastika, Heri Suyanto, Gunarjo, Sadiana, Darmaji
Keyword(s):  
Emission Intensity ◽  
Laser Energy ◽  
Emission Spectra ◽  
Atomic Emission ◽  
Laser Induced Breakdown Spectroscopy ◽  
Chemical Formula ◽  
Central Kalimantan ◽  
Breakdown Spectroscopy ◽  
Laser Induced Breakdown

Abstract - Laser-Induced Breakdown Spectroscopy (LIBS) is one method of atomic emission spectroscopy using laser ablation as an energy source. This method is used to characterize the type of amethysts that originally come from Sukamara, Central Kalimantan. The result of amethyst characterization can be used as a reference for claiming the natural wealth of the amethyst. The amethyst samples are directly taken from the amethyst mining field in the District Gem Amethyst and consist of four color variations: white, black, yellow, and purple. These samples were analyzed by LIBS, using laser energy of 120 mJ, delay time detection of 2 μs and accumulation of 3, with and without cleaning. The purpose of this study is to determine emission spectra characteristics, contained elements, and physical characteristics of each amethyst sample. The spectra show that the amethyst samples contain some elements such as Al, Ca, K, Fe, Gd, Ba, Si, Be, H, O, N, Cl and Pu with various emission intensities. The value of emission intensity corresponds to concentration of element in the sample. Hence, the characteristics of the amethysts are based on their concentration value. The element with the highest concentration in all samples is Si, which is related to the chemical formula of SiO2. The element with the lowest concentration in all samples is Ca that is found in black and yellow amethysts. The emission intensity of Fe element can distinguish between white, purple, and yellow amethyst. If Fe emission intensity is very low, it indicates yellow sample. Thus, we may conclude that LIBS is a method that can be used to characterize the amethyst samples.Key words: amethyst, impurity, laser-induced, breakdown spectroscopy, characteristic, gemstones

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