UPDATE: SUSHMITHA GET A POSTER AWARD !!!
Highly sensitive voltage-sensitive dyes emitting in the near infrared (NIR-VSD) in the scope of the EMIM 2015 in Tubingen.
Friday 20 March 2015
Time: 09:30h - 11:10h
Poster #: 57
Poster Walk Name/ID: Optical Imaging, Mass Spectrometry, Microscopy - Technology & Methods (PW 4)
Sushmitha Raja1, Rokhaya Faye2, Philippe Pasdois2, Richard D Walton2, Olivier Bernus2, Nasire Mahmudi3, ChistopheLanneau3, Nicolas Redon3, Gihad Dargazanli3, Alan Urban1
1Optogenetics and Brain Imaging team, Centre de Psychiatrie et Neurosciences, INSERM U894, Hôpital Sainte-Anne, Paris, France.
2INSERM U1045 -LIRYC L'Institut de Rythmologie et Modélisation Cardiaque, Bordeaux, France
3SANOFI Research and Development, Exploratory unit, Chilly-Mazarin, France.
Whether for basic research or drug discovery, precise measurement of voltage changes at the cell membrane is essential for understanding function, pathology, and potential therapeutic effects in electrically active cells.
Voltage-sensitive fluorescent dyes provide bright, membrane-localized signal but they suffer from poor signal to noise, secondary side effects and difficulties to achieve single-cell resolution. Moreover, capturing transient, millisecond events such as action potentials requires an indicator that responds very quickly to voltage changes.
We developed novel near-infrared (NIR) VSDs based on 3 different heterocyclic fluorophores that offers advantages of high sensitivity, deep photon penetration, reduced light scattering and minimal autofluorescence from living tissues, rendering them valuable for noninvasive in vivo imaging of cardiac and neuronal activity.
The properties of these dyes were first assessed in vitro using patch clamp and high-speed fluorescence imaging (1 KHz).
We evaluated voltage sensitivities of these NIR-VSDs in both HEK cells and primary neuronal cultures and demonstrated that sensitivity curves for all dyes are well fit by a log-normal function with nonlinearity at the spectral edge. Moreover, we observed that NIR-VSD are fast and sensitive enough to resolve single action potentials without averaging.
Then, NIR-VSDs were used ex vivo in Langendorff perfused rat hearts. Spectral properties were determined in these conditions and we observed optimal peak emission wavelengths for all dyes were found between 700 nm and 750 nm. These results are in good agreement with values observed in vitro.
Finally, SNR and kinetics were measured in epifluorescence imaging experiments. We showed that NIR-VSDs have sensitivity ranging from 6.5 % to 20 %, compared to 5.5% for the reference di-4-ANEPPS dye. Large Stoke shift up to 250 nm were observed. Moreover, NIR-VSDs show reduced internalization and photo-bleaching compared to di-4-ANEPPS as confirmed by the enhanced stability of the signal during optical recordings.
These properties suggest that NIR-VSD voltage sensitivity could extend the capabilities of modernelectrophysiological techniques for probing brain and heart function, and allow for the investigation of previously inaccessible research studies.
Keywords: voltage sensitive dye, near-infrared , in vivo imaging, fluorescent dyes.