Fig 2: Construction and validation of secretory fluorescent protein-tagged albumin(A) Schematic construct design of mNG-Alb (non-secretory negative control), IgkL-mNG (secretory positive control), and Alb-mNG. A three-dimensional (3D) protein structure prediction for Alb-mNG is displayed on the right.(B) Schematic illustration for cell culture testing of the plasmid constructs.(C) Microscopic images of transfected HEK293T cells at 48 h for mNG-Alb, Alb-mNG, and IgKL-mNG. The bottom row is the magnification of the corresponding white squares in the top row. Scale bars, top: 100 µm, bottom: 10 µm.(D) Fluorescence signal ratio of external versus cytosolic signal of microscopic images taken at 24 and 48 h. n = 18.(E) Fluorescence intensity of cell culture medium from 24 to 48 h post-transfection measured via a microplate reader. n = 6.All graphs show mean (line) and individual values; *p < 0.05.

Fig 3: Genetic expression of Alb-mNG is advantageous to fluorescent dextran in long-lasting imaging sessions(A) Experimental approach to compare the genetically expressed Alb-mNG with i.v.-injected Texas Red dextran (70 kDa). Alb-mNG-expressing mice (7–8 weeks) were imaged under ketamine-xylazine anesthesia before and 10, 60, and 120 min after Texas Red dextran injection.(B) Example images at various time points during an imaging session. Alb-mNG signal is present throughout the total imaging session with little attenuation. Texas Red dextran signals diminishes within an hour. Scale bar, 100 µm.(C) Quantification of signal intensity for Alb-mNG and Texas Red dextran for the time course of 120 min (n = 3). Graph shows means ± SEM; post hoc between Alb-mNG and Texas Red, *p < 0.05.(D) Volumetric imaging of brain vasculature covering 450 µm below the pial surface of Alb-mNG-expressing mouse (post-injection 10 weeks).(E) Two-photon images obtained from the same mouse at 3 and 7 weeks of Alb-mNG expression. The zoomed in area (yellow square) depicts neovascularization at 7 weeks (red arrow). Scale bar, 100 µm.(F and G) Two-photon imaging of a capillary at 3 and 7 weeks of Alb-mNG expression at frame rate of 116–220 Hz enables quantification of blood flow velocity by computing the cross-correlogram (right). See histogram on the right side. Pink triangles indicate the flow of an example red blood cell. Scale bar, 10 µm.(H) Experimental setup for functional hyperemia induced by whisker puff. During each session, mice were exposed to two whisker stimulations. Each session started with a 30 s baseline (gray), followed by 5 s of whisker stimulation (red) (50-ms pulses at 10 Hz). After the second stimulation, recording continued for 30 s (gray).(I) Example two-photon images of the same mouse at 7 and 15 weeks expression of Alb-mNG show dilation of artery (marked in red square) compared with vein (marked in blue square) after air puff whisker stimulation. Scale bar 50 µm.(J) Quantification of percentage change of vessel width for artery and vein (n =3 for each vessel type) after two whisker stimulations. Graphs show means ± SEM; Vessel type × time interaction, *p < 0.05.

Fig 4: Premalignant alteration events triggered by AAI exposure in the liver-like tissue at the differentiation endpointA. ALB residues (red) appeared in c-Myc-positive cells (green). B. Lin28B (green) co-expressed with ALB (red) in cytoplasm of hepatocytes.

Fig 5: Comparison between Alb-mNG and Alb-mScarlet macroscopic fluorescent imaging(A) Representative examples of macroscopic imaging with the two fluorescent plasma probes. Scale bars, 50 µm.(B) Signal-to-noise ratio quantification.(C) Shannon’s entropy of macroscopic images.All graphs show means ± SEM and individual values; *p < 0.05. NAlb-mNG = 10, NAlb-mScarlet = 6.