# A System to Optically Determine the Absolute Membrane Potential in Human iPSCD Cardiac Myocytes

> **NIH NIH R43** · CYTOCYBERNETICS, INC. · 2020 · $250,000

## Abstract

Optical assays are a powerful tool in cellular electrophysiology. However, currently-available approaches have not
reached their full potential. The major limitation of existing optical systems is that they are unable to determine the
absolute voltage in the cell. Current commercial approaches report only qualitative relative changes in voltage, &
there is no information on absolute resting potential, diastolic potential, or action potential amplitude. Prior research
attempts to develop absolute voltage reports have been unsuccessful. In addition, currently available voltage-
sensitive dyes (VSDs) offer a very limited experimental duration (typically < 30 min) dyes due to: 1) high washout &
internalization rates, which removes them from the electrically active cell membrane; 2) high photo-toxicity, which
reduces the possible exposure time for measurements; & 3) acute dye toxicity, which limits membrane loading &
illumination, resulting in small signals & low signal to noise ratio.
 This proposal overcomes these 2 major obstacles to develop & optimize our novel system which combines our
VSDs & our unique & robust optical & analytical system which determines absolute membrane potential. Our
integrated quantitative Optical Electrophysiology (qOEP) system consists of our patented long lasting VSDs,
optimized experimental protocols, optical detection system, & analytical software. Our VSDs, which operate in the
red/NIR spectral range, have reduced acute chemical & photo-toxicity, increased sensitivity, & slower washout/
internalization rate. This gives them the ability to be used in experiments up to 4 hours. This dramatic improvement
revolutionizes the types of experiment which can be performed. Specifically, slower internalization rate gives the
experimenter time to calibrate the VSD, so that the measured light intensity can be directly correlated with
transmembrane potential. The spectral properties & stability of this new generation of VSDs has been combined
with advances in electronics & circuitry that increase signal sensitivity & allow for qOEP. Dye performance & signal
processing are species & organ/cell type-specific. These systems have high degrees of cellular heterogeneity &
connective tissue relative to cultured cells. To develop a consistent system we will optimize our qOEP system
specifically for work with electrically syncytial preparations of induced pluripotent stem cell derived (IPSCD) cardiac
myocytes. The goal is to optimize a cell system (stem cell derived cardiac myocytes) & the dyes to make an
integrated optical system that makes qOEP available to almost any lab. This transformation will be similar to the
way that the advent of molecular biology kits made complex molecular biological techniques accessible to all. The
long term commercial opportunity is in cardiac safety screening to determine the arrhythmogenic potential of new drug
candidates in stem cell derived cardiac myocytes. Our novel system has the potential to have sign...

## Key facts

- **NIH application ID:** 10081467
- **Project number:** 1R43HL150989-01A1
- **Recipient organization:** CYTOCYBERNETICS, INC.
- **Principal Investigator:** Anthony John Costantino
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $250,000
- **Award type:** 1
- **Project period:** 2020-07-09 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10081467

## Citation

> US National Institutes of Health, RePORTER application 10081467, A System to Optically Determine the Absolute Membrane Potential in Human iPSCD Cardiac Myocytes (1R43HL150989-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10081467. Licensed CC0.

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