Infrared fluorescent proteins created by structure-based engineering of a bacteriophytochrome. (A) 14 residues surrounding the biliverdin (violet) in DrCBD (PDB ID: 1ztu) (14) were divided into 7 groups (shown in different colors) and targeted for mutagenesis. (B) Normalized excitation (blue) and emission (red) spectra of IFP1.4.

Imaging of GFP, mKate, and IFP1.1 in living mice. (A) Liver fluorescence of living mice injected with Ad5I (top row) and Ad5K (bottom row), imaged in the IFP excitation/emission channel before and after BV administration, mKate channel before and after BV, and GFP channel (14). Images through the mKate channel have been 5X brightened in software to render them visible, so the relative gains of the IFP channel, mKate channel, and GFP channel were 1, 5, and 1 respectively. The labeling above the images indicates the fluorescence channel. Arrows point to the liver. Note that the GFP images are dominated by autofluorescence, rendering the livers invisible. (B) Time course of averaged and normalized Ad5I liver fluorescence before and after BV injection. (C) Images of IFP-expressing mouse showing spectrally deconvoluted liver fluorescence (left, red) and its overlay (right) with autofluorescence (grey).

Imaging of IFP1.4 and IFP1.4-PH^AKT1 in HEK293A cells. (A) Fluorescence image of IFP1.4 taken with Cy5.5 filter set (665 ± 22.5 nm excitation, 725 ± 25 nm emission). (B) Confocal laser-scanning microscopy of IFP1.4-PH^AKT1 before and after insulin stimulation (excitation by 635 nm laser, emission by 650 nm long pass filter).

Analysis of mKate and IFP1.1 visibility and expression levels in livers of mice infected with Ad5I and Ad5K. (A) IFP/mKate fluorescence images before dissection (skin on), after removal of skin (skin off), and after removal of overlying peritoneum and ribcage (liver exposed). mKate images were 2.5X brightened relative to IFP images. Note that Ad5I infected mouse was imaged after 250 nmole IV injection of BV. See bright field images in Fig. S9. (B) Fluorescence intensity (signal over noise ratio) analysis of livers from (A). Average of 80 pixels in the horizontal x axis divided by the average of the 30 most rostral horizontal lines from each of the images starting below the liver (i.e. signal/noise ratio), moving rostrally for 300 lines. (C)Frozen sections were imaged to show IFP, mKate and GFP expression using a fluorescence stereomicroscope (Lumar, Zeiss) (14), displayed with relative intensity gains of 10, 5, and 1 respectively. Scale bar is 200 µm.