Published May 13, 2024 | Version v2
Software Open

Ratioscope

Creators

  • 1. ROR icon French National Centre for Scientific Research
  • 2. ROR icon Institut de Génomique Fonctionnelle

Contributors

  • 1. ROR icon University of Castilla-La Mancha
  • 2. Sorbonne Université Campus Pierre et Marie Curie
  • 3. ROR icon Sorbonne University

Description

Genetically-encoded biosensors are heavily used in biology to monitor biological processes in real time within living cells. Many of such biosensors use two different emission wavelengths that change in opposite directions: the biological signal is measured as a change in the ratio of these two emissions. This so-called ratiometric quantification is standard and applies to many situations.
Ratioscope, which runs in the Igor Pro environment, offers two packages:
- Ratio analysis of fluorescence images: image corrections, display in pseudocolor, measurements, kymograph, analysis of periodic activity, movies...
- Fusion module: statistics on series of experiments, to compare different experimental conditions; generate editor-ready graphs and plots.

Technical info (English)

Image reading:
It reads new images as they are acquired by the imaging system, to provide a real-time feedback of what happens during the experiment. 
It can also read time stacks with different storage schemes. Besides pairs of images acquired alternately, it can also handle images acquired with an image splitter (2 emission wavelength projected side by side on the camera chip). In addition to the two ratioed channels it can process additional channels such as transmitted light, other fluorescence channels... It reads regular tif files.

Instrument-dependent image corrections:

  • rotation/mirror
  • normalization to exposure duration
  • shading correction
  • subtraction of background image or value 
  • camera / photomultiplier gain 

User-defined image corrections:

  • automatic correction of preparation movements
  • automatic registration of the two emission channels

Ratio calculation:

  • ratio is calculated for every pixel
  • interactive drawing of regions of interest
  • ratio measurement weighted by pixel intensity for better reliability
  • intensity-modulated pseudocolor 

More...

  • kymograph representations (intensity/ratio over time along user-defined lines)
  • analysis of periodic changes (changes in beating heart)

Statistics: the "Fusion" module handles measurements from several different experiments. It automatically updates plots and statistics as more experiments are added.

Some examples of applications:

  • dynamics of cAMP, cGMP, calcium signals in various neuronal types in brain slice preparations
  • ratiometric imaging of calcium, voltage, ATP, cAMP… with ratiometric biosensors in the beating heart of zebrafish larvae, which corrects most of the motion artifacts

Files

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Additional details

Related works

Is cited by
Publication: 10.1111/bph.15664 (DOI)
Publication: 10.7150/thno.64734 (DOI)
Publication: 10.1126/sciadv.aba3992 (DOI)
Publication: 10.1101/2023.10.07.560741 (DOI)
Is derived from
Publication: 2354507 (PMID)

Software

Programming language
IGOR Pro
Development Status
Active