THE NEW STATE-OF-THE-ART SOLUTION FOR FRET & RATIOMETRIC IMAGING THAT COMBINES PERFECTLY WITH MULTIDIMENSIONAL MICROSCOPY ACQUISITIONS
Inscoper liveRATIO is a comprehensive image acquisition and processing solution developed for ratiometric and FRET imaging. It consists of an electronic unit for triggering and synchronizing the microscope signals, and complete image acquisition and processing software for operating all devices and functions of the microscope.
The solution is compatible with all microscope equipment that is commonly involved for ratiometric imaging: fluorescence illumination source, filter wheel, one or more camera detectors, etc.
Ratiometric imaging of lymphocytes labelled with Fura-2.
Microscopy system equipped with Inscoper liveRATIO.
FOR RATIOMETRIC IMAGING
The liveRATIO solution is a variation of the Inscoper imaging solution for fluorescence microscopy that is specialized for ratiometric imaging and its multiple applications.
The software allows biologists to monitor the evolution of the fluorescence ratios in real time and to replay them afterwards, with an acquisition frame rate at the maximum of the microscope’s capabilities thanks to the elimination of all software latency through our proprietary technology.
Ratiometric imaging can easily be combined within multidimensional acquisition sequences in the same software environment and even with microfluidics.
USER CASE @ GRENOBLE, FRANCE
FRET & multi purpose shared system
LATENCY-FREE AUTOMATION
REAL TIME IMAGING
Perfect synchronization of the microscope trigger signals and the camera exposure time as allowed by INSCOPER technology is a key advantage for imaging fast biological events such as calcium flux or protein-protein
interactions.
The solution also allows biology users to interact in real time with the sample for analysis purposes. The convenient management of the image background and the LUTs for colorization help the output of directly usable images.
SUPPORT SERVICE
Inscoper liveRATIO user interface. Acquisition in progress
RATIOMETRIC IMAGING FOR A LARGE PANEL OF BIOLOGICAL APPLICATIONS
In microscopy, ratiometric imaging is based on the calculation of a ratio between (two or more) fluorescence intensities. This technique is used for sensitive measurements of living cells to prevent artifacts due to bleaching, modification of the laser intensity, or variation of the fluorescent probe concentration. To obtain this ratio, we most often use either two excitation lasers (dual excitation indicator) or two detection ranges (dual emission indicators). More illumination modalities could be combined with this live imaging approach.
Inscoper liveRATIO is a multi-application solution developed for ratiometric imaging. It offers to users a large panel of in vitro applications such as FRET (Förster Resonance Energy Transfer) for protein-protein interaction, calcium imaging, ions concentration measurement (Mg2+, K+, Na+, Cl–,…) and intracellular pH characterization.
Biology applications
T cells activation monitored by ratiometric imaging with the Inscoper liveRATIO
(A) Representative images of γδ T cells labeled with Fura-2 with DMSO or phospho-antigen (PA) stimulation. Surfaces plots are extracted from each dashed square. Scale bar = 100μm.
(B) Kymograph representing the evolution of the fluorescence intensity as a function of time. The black arrowhead represents the addition of DMSO or PA in the medium.
(C) Calcium fluctuation induced by DMSO or phospho-antigen on γδ T lymphocytes labeled with Fura-2. The black arrowhead represents the addition of DMSO or PA in the medium. Data are expressed according to the mean ± SEM.
CUSTOMIZABLE SOLUTION TO SUITE WITH YOUR EXPERIMENTAL DESIGN
The liveRATIO solution provides all the tools needed for ratiometric imaging. First, biologists can select ROI (regions of interest) to analyze their sample using the user-friendly software interface. Users also benefit from the performance of the Inscoper technology for optimized control, which guarantees the highest possible acquisition rate.
Experiments with Inscoper products strictly synchronize the illumination time with the exposure time, thus limiting the phototoxicity development in living samples. It even enables imaging the evolution of the fastest biological phenomena. Image visualization can also be improved using a dedicated LUT for ratiometric imaging and the background subtraction feature while the sequence is still running.
LIVE MONITORING OF FLUORESCENCE INTENSITY MODIFICATION
Real time analyses of all ROI allow researchers to visualize the evolution of their experiment. Each acquisition leads to the generation of .tif images and a .csv file containing all raw data of the fluorescence intensity changes for each ROI. A performance file is also generated to archive the acquisition sequence and the chain of commands. Such a file can be used for metrology applications for example.
The Inscoper liveRATIO is a modern and versatile solution to perform ratiometric imaging experiments on living samples with high spatiotemporal resolution.
Live calcium imaging to monitor T cells activation
using Inscoper liveRATIO
Application note