Comprehensive BioImaging Study of the Red Permanent Marker Ink: Re-purposing for Cells Imaging Including Cytoplasmic Membrane Visualization and Comparison with Rhodamine 6G, Deep Red Cell Mask, and DiBAC

In this paper, we aim to present a new intravital cells visualization method, which is based on use of a dye called ABDS ("A Beautiful dye for staining"), which can be prepared using a marker pen and is useful for eukaryotic cell research. Using a wide range of instruments, including optical measurements, microscopy studies and wet biology techniques, we have shown that ABDS is close by properties to Rhodamine 6G dye (R6G), which is well known as endoplasmic reticulum stainer. However, by the careful examination of the ABDS and R6G images (ABDS/R6G), we have proved for the first time that these dyes also stain the cytoplasmic membranes. The significant contrast between ABDS/R6G signal from cell membrane and endoplasmic reticulum allows them to be distinguished in the fluorescence photographs. Other important properties of ABDS are its availability, simplicity in manufacturing, safety for living cells in vitro, and bright stable fluorescence, which in contrast to commercial dye like DiBAC allows us to study cells in space and time with high detalization. The paper includes a method for preparing ABDS, a data set with its characteristics, comparison with other commercial dyes, as well as examples of ABDS usage in cells research. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/717455v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@f1ceacorg.highwire.dtl.DTLVardef@137abd2org.highwire.dtl.DTLVardef@1f19efcorg.highwire.dtl.DTLVardef@1fcbc9e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA protocol for high-resolution vital staining of the cells using an inexpensive dye based on permanent marker ink is proposed. C_LIO_LIThe absorption, emission and Raman spectra of the proposed dye are presented, and a direct comparison with commercial dyes Rhodamine 6G, DiBAC and Deep Red Cell Mask dye is made. C_LIO_LIThe main characteristics of the proposed dye are low toxicity, long-term fluorescence, and the ability to separately stain the endoplasmic reticulum and cytoplasmic membrane. C_LIO_LIThe ability of the Rhodamine 6G dye to stain cell membranes also has been proved. C_LI