Hi Leila Hambaba . When you are talking about:

"best fluorescent molecules used in microscopy to tag a specific protein with great precision and high affinity"

you are talking about labeled monoclonal antibodies.

Thank you Dr Steingrimur Stefansson for your answer, but i'm asking about the best molecules that can be used as fluorescent dyes like Fluorescein isothiocyanate. For exemple, a molecule that have an important excited lifetime.

Best regards,

Pr. Hambaba

Hi Leila Hambaba

I used FITC to see HIV antigens in infected cells. It was very distinct. I do not have experience in using comparative fluors.

Hello Pr Salequl Islam,

It's really interesting, thank you.

Best regards,

Pr. Hambaba

One method is to introduce into the cell a gene encoding a fusion between the molecule of interest and a fluorescent protein. Most of these are variants of green fluorescent protein from the jellyfish Aequorea Victoria or red florescence protein from from Discosoma, although by now, variants covering the whole visible spectrum have been developed ( http://www.olympusconfocal.com/applications/fpcolorpalette.html , http://klingon.uab.cat/dynamics/research_photo.html ). These are proteins that, upon folding, autocatalytically convert three amino acids within their sequence into a fluorophor. They do this within the living cell, allowing to follow the localisation of the fused protein. To label cell surface receptors, you can chemically couple organic fluorophors to the ligands for these receptors, allowing for example to use fluorescence activated cell sorting to collect cells what express high levels of functional, properly folded receptor. This has e.g. been used to identify GPCR mutants with increased stability for crystallisation.

Most fluorophors do not by themselves bind with high specificity to particular proteins, but you need to either chemically couple or genetically fuse them to a recognition module or molecule of interest, and then make sure the construct can get to to the target within the cell, either by penetrating the membrane (hydrophobic small molecules), by using specific transport mechanism, being expressed within the cell, or through working with fixed cells permeabilised by detergents or organic solvents.

This site contains a long list of frequently uses fluorophors: https://www.leica-microsystems.com/science-lab/fluorescent-dyes/

Thank you Pr Annemarie Honegger for these informative, interesting and clear explications.

Best regards,

Pr. Hambaba