Whole-body survey of nerves in mice (n = 3) 4 h after injection with 450 nmoles of FAM-NP41. (a,b) Brachial plexus. Reflectance image (a) showing left brachial plexus (arrow). Smaller branches (~50–100 µm) are easily seen (arrowheads) with fluorescence labeling (b) but not in reflectance image. (c,d) Sciatic nerve. Reflectance image (c) showing right sciatic nerve (arrow). Many more small branches (~50–100 µm) are seen (arrowheads) with fluorescence imaging (b) compared to reflectance. (e,f) Phrenic nerve. Reflectance image (e) showing left phrenic nerve (arrow) descending from the mediastinum to innervate the diaphragm. Note that the nerve is seen as a single linear fluorescent structure (arrow, f) compared to the bundle of nerve and connective tissue seen with reflectance (arrow, e). Arrowhead points to an intercostal nerve which is easily seen with fluorescence but not with reflectance. (g,h) Dorsal cutaneous nerves. Reflectance image (g) showing the dorsal musculature. Fluorescence imaging highlights the dorsal intercostal nerves (arrows, h) that are not easily seen with reflectance. (i,j) Facial nerve. Main facial nerve branches (arrows) are easily seen with both reflectance (i) and fluorescence (j). However, a small branch of nerve arborization (arrowhead) leading to the upper face can be distinguished from surrounding tissue with fluorescence but not with reflectance. Insert shows arborizations (~50 µm diameter) of the lower division of the facial nerve that can be easily seen with fluorescence labeling. (k,l) Dorsal view of skull base. Reflectance image (k) showing left facial nerve (large arrow) wrapping around the ear, trigeminal nerves (small arrows), optic nerves (large arrowhead) and optic chiasm (small arrowhead). Fluorescence image (l) shows fluorescence labeling of the facial and trigeminal nerves (peripheral nervous system) but not the optic nerves and chiasm (central nervous system). Scale bar (a–l), 5 mm; inserts (i and j), 1 mm.

Cy5-NP41 (acetyl-SHSNTQTLAKAPEHTGC-(Cy5)-amide) labeling of sciatic nerve in Thy1-YFP transgenic mice. (a) Low-power brightfield view of left exposed sciatic nerve. Inset shows magnified view of central boxed region. (b) Same nerve as in a with YFP fluorescence (pseudocolored yellow) superimposed on the brightfield image, showing transgenic expression of YFP in axons. (c) Same nerve as in a and b viewed with Cy5 fluorescence (pseudocolored cyan for maximal contrast during live surgery) superimposed on the brightfield image, showing nerve labeling with Cy5-NP41. Arrows in b and c point to thin buried nerve branches that are better revealed by the long-wavelength Cy5 fluorescence than by brightfield reflectance or shorter-wavelength YFP fluorescence. There is some nonspecific labeling of skin (asterisk) and cut edges of muscle (arrowhead) by Cy5-NP41. Fortunately, such nonspecific labeling hardly ever has the filamentous appearance of nerves, so an experienced surgeon can usually distinguish nonspecific from specific targets. (d) Low magnification longitudinal section showing myelin within the sciatic nerve using differential interference contrast (DIC), pseudocolored blue. (e) Same nerve as in d showing axoplasmic YFP pseudocolored green (arrows). (f) Same nerve as in d and e, showing Cy5-NP41 labeling (pseudocolored red) of epineurium (arrows) and endoneurium (arrowheads). (g) Composite image of d, e and f showing that NP41 labeling does not colocalize with either myelin or axoplasm. (h–k) Cross-sectional images corresponding to panels e–g.

Time course and dose response of FAM-NP41 binding to nerve and nonnerve tissue. (a–d) Nerve fluorescence peaked at around 10 min after administration (a), then declined (half-life ~50 min) to a plateau sustained between 3 and 6 h (see inset with expanded intensity scale, Student’s t-test, two–tailed, P = 0.002 at 2 h, 0.0007 at 3 h, 0.0007 at 4 h, 0.003 at 5 h, 0.006 at 6 h). In contrast, muscle fluorescence was highest immediately after intravenous administration of the peptide, then declined steadily with a half-life of ~20 min (a). Serum half-life was calculated at ~10 min (b). Useful contrast between nerve and surrounding muscle developed by 2 h and lasts several hours (c). Nerve-to–surrounding tissue contrast ratio increased with increasing amount of peptide injected, concentration from 15 to 5,000 nmoles per mouse injected (n = 2) (d), because surrounding nonnerve tissue fluorescence seems more saturable than nerve binding with increasing peptide concentration (inset).

NP41 can highlight buried nerve branches invisible by standard illumination. (a–c) Right facial nerve and its arborizations in a thy1-YFP mouse treated with Cy5-NP41, viewed by (a) white light reflectance, (b) Cy5 fluorescence (pseudocolored cyan) overlaid on reflectance and (c) YFP fluorescence (pseudocolored yellow), also overlaid on reflectance. The short arrow marks a nerve branch visible by all three imaging modes. The arrowheads point to branches that are difficult to differentiate from muscle fascia in reflectance, but clearly distinguishable in both fluorescence images. The long arrow indicates a deeply buried branch visible only by Cy5-NP41 due to the better penetration of far-red wavelengths. (d–f) Left sciatic nerve (arrow) and its arborization in a mouse with a syngeneic 8119 mammary tumor graft^17,18, viewed by (d) white light reflectance, (e) FAM fluorescence 2 h after intravenous injection of NP41 (150 nmoles) (pseudocolored cyan, overlaid on reflectance) and (f) Cy5 fluorescence (pseudocolored green, overlaid on reflectance) from conjugates of activatable cell-penetrating peptides and dendrimers (ACPPDs). The large arrowheads in d and e point to a nerve branch buried under tumor, visible only by FAM fluorescence. Small arrowheads in f denote tumor. See Supplementary Video 1.