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Bioluminescent Substrates

Bioluminescent substrates are commonly utilized for non-invasive monitoring of biological processes, for example in bioluminescence imaging (BLI) in both in vivo and in vitro settings. Luciferase substrates emit light when they become oxidized, which can be detected using fluorescence microscopy.

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Cat. No. Product Name / Activity
7753 BL660-NO
Nitric oxide activity-based sensing NIR bioluminescent probe
5427 D-Luciferin sodium salt
Firefly luciferase substrate; cell permeable.
6555 TokeOni
NIR-emission luciferin analog; ideal for deep tissue applications; orally bioavailable and brain penetrant

Luminescence is the spontaneous emission of light caused by chemical reactions, electrical energy and subatomic motion. There are many types of luminescence, including chemiluminescence and photoluminescence. Bioluminescence is a type of chemiluminescence, and refers to the production of light or photons in living organisms.

Bioluminescence occurs naturally in multiple organisms, such as fungi and bacteria, as well as a whole host of marine animals and insects. One of the most well-known examples of bioluminescence in nature is the spectacle produced by the firefly.

The main sources of natural luciferase for biological applications are:
  • Firefly luciferase, which requires the substrate D-luciferin.
  • Renilla luciferase (derived from the sea pansy), which requires its substrate coelenterazine; coelenterazine has poor bioavailability because P-glycoprotein/MDR1 pumps it out of mammalian cells.
  • Bacterial luciferase (e.g. Vibrio fischeri), which requires the substrate luciferin.

The reaction between the oxidative enzyme luciferase and a light-emitting substrate luciferin, produces bioluminescence. Luciferases and their substrates are widely used in fluorescence microscopy, and in biotechnology as reporter genes. Techniques such as bioluminescence imaging (BLI) and bioluminescence tomography (BLT) also utilize bioluminescence for non-invasive in vivo studies. DNA encoding the luciferase enzyme is incorporated into viral vectors for transfection in cell lines and animal models, or a transgenic animal expressing luciferase is created through genetic engineering. Generally, luciferase is expressed under the control of a promoter of interest, which is specific to the investigation being carried out.

Firefly luciferase (Fluc) and D-luciferin

The Firefly luciferase (Fluc) and D-luciferin BLI system has long been utilized in standard lab techniques such as monitoring tumor growth and intracellular signaling activity in vitro and in vivo. While this system is very useful for many applications, it is less suitable for experiments requiring detection in deep tissue, because of the relatively short emission wavelength produced by D-luciferin: emission maximum (λmax) of 562 nm. This wavelength is very similar to those of melanin (λmax = ~600 nm) and hemoglobin (λmax = 415-577 nm), making signal detection of D-luciferin emanating deep from with tissue challenging to detect.

Deep Tissue Bioluminescent Imaging: TokeOni-AkaLuc

A major breakthrough in bioluminescent imaging came from engineering luciferases and substrates for near infrared (NIR) wavelength emission. Directed evolution on Fluc resulted in the optimized 28 amino acid AkaLuc. This engineered luciferase produced a significantly brighter bioluminescence, as well as exhibiting lower toxicity than Fluc, allowing high concentrations of AkaLuc to accumulate inside the cell.

Engineered AkaLuc substrate TokeOni (Akalumine-HCl), with an emission spectrum in the NIR range (λmax = 677 nm), generates exceptional signal sensitivity from deep within tissue, exhibits good brain permeability and is orally bioavailable. Compared to the TokeOni-Fluc system, the TokeOni-AkaLuc system demonstrated a 40-fold brighter bioluminescence signal in vitro, and 100- to 1000-fold brighter in vivo bioluminescence. The improved properties of the TokeOni- AkaLuc pairing has opened up a new range of applications including monitoring neuronal activity-dependent gene expression, tracking tumor metastasis and immune cell migration, as well as proving a useful tool for assessing the efficiency of gene delivery and gene editing technologies.

Bioluminescent Substrate Emission Spectras
Bioluminescent Substrate Emission Spectras

Figure 1: Emission spectras of bioluminescent substrates, D-luciferin, CycLuc 1 and TokeOni span the color spectrum, making them suitable for different applications. TokeOni is well suited to deep tissue in vivo applications because of it's NIR emission wavelength.

Literature for Bioluminescent Substrates

Tocris offers the following scientific literature for Bioluminescent Substrates to showcase our products. We invite you to request* your copy today!

*Please note that Tocris will only send literature to established scientific business / institute addresses.

Fluorescent Dyes and Probes Research Product Guide

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