We then explored the fluorescent properties of the in vitro translated peptide containing 4CNW. Translated reaction mixtures (10 µL) were run in a Tricine-SDS PAGE gel and imaged with a trans-UV 302 nm excitation light source. In-gel fluorescence was observed for the translation sample with 4CNW, but not with tryptophan (Figure ) confirming that 4CNW is a highly sensitive fluorophore that can be detected in low quantities, such as those present in a 10 µL in vitro translation mixture. In-gel visualisation of AzAla (λmax= 276 nm) was not attempted due to a suitable excitation light source being unavailable. Finally, we assessed the potential of using either 4CNW or AzAla for biological imaging of peptides. Gai and co-workers have previously demonstrated 4CNW as a satisfactory fluorophore for imaging by coupling to an antimicrobial peptide. However, to the best of our knowledge, the use of AzAla in fluorescence microscopy has not yet been explored. To evaluate their suitability for tracking peptide penetration into cells, 4CNW/AzAla were appended onto the Nterminus of a well-characterized cell penetrating peptide, TAT-NLS P3 (tryptophan was included as a negative control). Human bone osteosarcoma epithelial cells (U2OS) were incubated with peptides 4CNW-P3, AzAla-P3, W-P3 for 20 minutes at a concentration of 50 µM in OPTI-MEM. Cells were then washed once with OPTI-MEM and imaged on a Nikon Ti Eclipse inverted microscope with fluorescence excitation at 340 nm. An ET460/50m single bandpass emission filter was used to image 4CNW-P3 and a ET395/25x single bandpass emission filter was used to image AzAla-P3. Our images show considerable intracellular fluorescence suggesting 4CNW and AzAla are suitable fluorophores for evaluating cell permeability of peptides when coupled with fluorescence microscopy (Figure ). We noted significant levels of cytotoxicity for all three TAT-NLS peptides over longer incubation periods (e.g. 2 h), as measured by the release of lactate dehydrogenase (Figure ). We envisage the cytotoxicity may be attributed to the combination of the TAT-NLS sequence and the additional N-terminal appendage, such that the evaluation of cell viability must be considered when combining these fluorescent amino acids with novel peptide sequences.