Algorithms for the selection of fluorescent reporters
Preprint posted on 16 May 2020 https://www.biorxiv.org/content/10.1101/2020.05.15.098186v1
Article now published in Communications Biology at http://dx.doi.org/10.1038/s42003-020-01599-5
Various fields in biology extensively use fluorescent probes to visualize a plethora of phenomena including cell-cell interactions, protein dynamics, immune responses, and signaling among others. Fluorophores fall into various categories, but they all share general properties, including excitation and emission spectra for example. One important challenge has been to maximize the number of different signals that can be distinguished in a single measurement, to maximize the number of probes that can be used simultaneously. The challenge arises from the fact that many fluorescent probes emit light spectra that overlap with one another, making it difficult to separate signals from different probes (a problem known as spectral spill-over, or bleed-through). While it is possible to correct for spectral spillover using algebraic operations for spectral compensation, if compensation is incorrectly performed, it can lead to incorrect biological measurements and/or incorrect conclusions. Choosing the right set of fluorophores and detectors is key for the experimental setup. In their work, Vaidyanathan, Appeleton et al generated algorithms and implemented them in an open-source software tool, to allow users to design an n-colour panel of fluorophores optimized for maximal signal and minimal bleed-through (1).
Key findings and developments
The authors designed an open-source web-application and command-line tool that allows users to design an n-colour panel for a specific measurement instrument: http://fpselection.org. The solution can be constructed from a library of fluorophores for a fluorescence measurement instrument to find the optimal panel configurations, as well as allowing the user to upload values such as emission and excitation spectra, autofluorescence of samples used, and brightness of fluorophores. Two properties were chosen to optimize the n-colour panel: a) the amount of signal measured by a detector for the fluorophore it is supposed to detect, and b) the amount of bleed-through from all other fluorophores in that detector. The authors highlight that obtaining a valid panel is not trivial, and that on one hand the probability of obtaining a valid panel using the maximum number of fluorophores is relatively low, while a valid panel may not measure fluorescence efficiently. The authors found a logarithmic approach for fluorophore selection: FP selection uses the search algorithm to find a valid panel where the amount of signal is each detector is maximized and bleed-through is minimized. The recommended algorithm uses simulated annealing to quickly and reliably find an optimal result. The authors tested the efficiency of the search algorithm by comparing it against a list of valid panels for 3 different flow cytometers with unique sets of lasers and detectors. They noticed that the run-time for simulated annealing was constant and each run took less than 1 second to complete. Moreover, they found that simulated annealing performs well as a heuristic, and returned an optimal solution in most runs.
The authors then went on to compare the computational predictions against experimental measurements, considering two metrics: a) the number of panels where, for all detectors in the panel, the fluorophores with normalized values equaling 1 matched, and b) the number of panels where for all detectors in the panel, the difference between each normalized predictions and measurement value must be within 0.05, 0.10, and 0,20 of one another. Altogether, they demonstrate that computational predictions of signal and bleed-through reliably match experimental observations.
What I like about this preprint
I like that the paper provides a new tool for optimal selection of fluorophores for different experimental setups. Working with fluorescence-based setups myself, I know that fluorophore choice can be very time-consuming and limiting. I find that a tool which would allow me to create an optimal panel reliably, would be hugely time-saving and perhaps result in less experimental problems/error with bleed-through.
- Experimentally, what are important limitations of your tool that users should be aware of?
- Does your tool allow for correction of autofluorescence during the generation of an optimal panel?
- Is this compatible with live imaging, whereby perhaps large averaging or scan speed might not be possible?
- Is it possible to account for fluorescence decrease upon treatments of cells, such as fixation, permeabilitzation or mounting, for the generation of optimal panels?
- Vaidyanathan P, Appleton E, et al . Algorithms for the selection of fluorescent reporters, bioRxiv, 2020.
Posted on: 30 June 2020 , updated on: 7 July 2020
doi: https://doi.org/10.1242/prelights.22430Read preprint
Also in the cell biology category:
Interspecies blastocyst complementation generates functional rat cell-derived forebrain tissues in mice
Coxiella burnetii actively blocks IL-17-induced oxidative stress in macrophages
Complete male-to-female sex reversal in XY mice lacking the miR-17∼92 cluster
preListscell biology category:in the
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
|List by||Helen Zenner|
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
|List by||Martin Estermann|
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
|List by||Sergio Menchero et al.|
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
|List by||Nadja Hümpfer et al.|
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
|List by||Osvaldo Contreras|
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
|List by||Alex Eve|
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
|List by||Ana Dorrego-Rivas|
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
|List by||Ana Dorrego-Rivas|
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
|List by||Dey Lab, Samantha Seah|
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
|List by||Yamini Ravichandran|
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
|List by||Maiko Kitaoka et al.|
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
|List by||Paul Gerald L. Sanchez and Stefano Vianello|
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
|List by||Hiral Shah|
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
|List by||Madhuja Samaddar et al.|
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
|List by||Dey Lab|
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
|List by||Sandra Malmgren Hill|
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
|List by||Rob Hynds|
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
|List by||Pablo Ranea Robles|
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
|List by||Dey Lab|
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
|List by||Sandra Franco Iborra|
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
|List by||Joseph Jose Thottacherry|
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
|List by||Dey Lab, Amanda Haage|