Determination of the fluorescence lifetime of novel red fluorescent proteins in fixed cells

Session

Poster Session

Authors

Ralph Palmisano (1)

Affiliations

1. OICE FAU

Keywords

Multi-Photon

Fluorescence Life-Time

Novel Red Fluorescent Proteins

FLIM

Abstract text

In biological and medical research, the use of fluorescent proteins (FPs) to study chemical processes has become a standard method. This makes it possible to gain insights into cells at the molecular level and to follow real-time processes such as the spread of cancer cells and the behavior of nerve cells in the brain. The most widely used and best known FP is the green fluorescent protein (GFP), which was first isolated from the jellyfish Aequorea victoria in 1962 by Osamu Shimomura. This led to the discovery and further development of new fluorescent proteins, as a result of which a large number of different FPs with different spectral properties are available today. The labeling and microscopic visualization of cell proteins is limited to the number of FPs already available. Often, different biomarkers can only be labeled with FPs whose emission spectra strongly overlap. However, this prevents a spectral separation of the FPs used and thus the different labeled epitopes cannot be separated.

This work therefore investigated a property of fluorescent substances that can circumvent this limitation. The fluorescence lifetime (FLT) is a property that is independent of the wavelength and fluorescence intensity. Fluorescent proteins with the same or very similar emission wavelength can have different FLTs, which would allow their microscopic separation. Therefore, the FLT of nine red fluorescent proteins (RFPs) with similar spectral properties was determined in this work. The FLT can be measured using pulsed excitation lasers and special detectors that can register single photons, so-called fluorescence lifetime imaging microscopy (FLIM) detectors. The FLT for the RFPs mCherry, mCherry2, mCarmine, mKate2, TagRFP, mScarlet, mScarlet-I and mScarlet-H was determined comparatively in fixed HeLa cells. The nine expression vectors of the different RFPs were all tagged with a localization sequence for actin and were transiently expressed in HeLa cells. The FLT of the nine RFPs differed slightly from each other. RFP variants with an identical genetic origin showed fewer FLT differences.

References