Luciferin - firefly luciferase bioluminescence in vivo imaging is quickly becoming a standard procedure. D-luciferin [(S)-2-(6′-hydroxy-2′-benzothiazolyl)thiazoline-4-carboxylic acid] is the substrate of the North American firefly Photinus Pyralis luciferase and Clickbeetle Red and Clickbeetle Green luciferase. Luciferin is a low molecular weight (318.41 g/mole) organic compound that consists of a benzothiazole moiety attached to a thiazole carboxylic acid moiety. Luciferin is a small molecule which freely diffuses across membranes. When luciferin is injected in vivo it is not metabolized but excreted via the kidneys. Due to its small size, luciferin makes a poor antigen and immune responses to luciferin are unlikely. Luciferin is able to pass the blood brain barrier, the blood placenta barrier and the blood testis barrier, toxicity appears to be low.

 

Bacterial luciferase uses the substrate bacterial luciferin which is a different molecule from D-luciferin. The plasmid encoding for bacterial luciferase also encodes for the substrate which is then synthesized by the cell, eliminating the neeed for exogenous substrate administration.

Renilla and Gaussia luciferase uses the substrate coelenterazine, again different from D-luciferin.

 

Firefly or clickbeetle luciferase catalyzes the oxidization of D-luciferin to oxyluciferin in the presence of ATP, Mg²⁺, and oxygen with production of a yellow-green light, shifting to red light in vivo at 37^oC. Since ATP is required as a co-factor, Firefly luciferase can be used as an indicator of the presence of energy or “life” and function as a life-death stain.

Dissolve 1g of D-luciferin potassium or sodium salt in 33.3ml sterile water to make a 30 mg/ml * stock solution.

Mix gently, syringe filter (0.2 um), aliquot, purge with nitrogen or argon (inert gas prevents oxidation) and freeze under at -80°C for future use (up to 1 year), protect from light.

Thaw and keep on ice, protect from light. If the luciferin does not dissolve, briefly bring to temperature in a waterbath and gently mix.  Dilute stock at 1:200 in complete tissue culture medium to 150ug/ml . incubate 5 min prior to imaging.

 

Upon IP injection, the D-luciferin is slowly absorbed into circulation, perfuses tissue and is excreted by the kidneys. Since D-luciferin is not metabolized, a simple pharmacokinetics of inflow and outflow isconcentration driven. Initially inflow is greater than outflow, then inflow equals outlfow (this is the plateau phase in the light emission time curve), then outflow exceeds inflow. It is important to image/quantify the signal during the plateau phase. With an IP injection, the signal lasts 2 hrs. The plateau phase usually occurs at 15 min post luciferin injection and lasts for 15-20 min. It is important to establish a time kinetics curve in a pilot study since every model is different. If the plateau phase is short lived, it is recommended to measure peak values. Take a couple of measurement surrounding the expected timepoint of peak post luciferin injection and use the peak value.

Intravenous kinetics:

since the substrate is delivered as a bolus as opposed to slow absorption (IP injection) the luciferase light signal will peak at 2 min post injection and disappear by 30 min. The peak upon IV injection is also several fold higher.

See Burgos et al. Biotechniques. 2003 Jun;34(6):1184-8.

Uptake kinetics and biodistribution of 14C-D-luciferin--a radiolabeled substrate for the firefly luciferase catalyzed bioluminescence reaction: impact on bioluminescence based reporter gene imaging. Berger et al., Eur J Nucl Med Mol Imaging. 2008 Dec;35(12):2275-85

 

Cell uptake and tissue distribution of radioiodine labelled D-luciferin: implications for luciferase based gene imaging. Lee et al., Nucl Med Commun. 2003 Sep;24(9):1003-9