Figure 1.
Quantum dot uptake in lumbar lymph nodes.
A–D. Right lumbar lymph node, visualized 36 hr after N-9 pretreatment and 24 hr after instillation of 30 µL 1 mM polyarg-streptavidin Qdots. A. GFP spectral window (autofluorescence), false-colored green. B. Qdot spectral window, false-colored red. C. Superposition of A and B. D. Superposition of A and B with B multiplied 5x. While detection sensitivity is low at 2.5x, this lens permits a view of a whole lymph node at once. Many more quantum dots are visible than are shown in B; exposure was set to avoid saturation. E–H. Control left lumbar lymph node (mouse sham-instilled with PBS only). E. GFP spectral window (autofluorescence), false-colored green. F. Qdot spectral window, false-colored red. G. Superposition of E and F. H. Superposition of E and F with F enhanced 5x. The relatively uniform color in H indicates that the background image in F is due to autofluorescence. Imaging times were adjusted to ensure that no pixels were saturated; then all images were adjusted to correspond to uniform exposure in both red channels and the green channel. Background (assessed by averaging 4 off-image fields, one in each quadrant) was subtracted from each image.
Table 1.
Scoring nodes.
Figure 2.
Uptake of quantum dots having three different surface coats into lumbar lymph nodes.
Half of the animals were instilled using 30 µL 1% N-9 in PBS 12 h before instillation of Qdots. Either carboxyl-, streptavidin -, or polyarg-streptavidin Qdots were instilled as described in the Materials and Methods. Lumbar lymph nodes were harvested 36 hr after Qdot instillation, then evaluated by microscopy. Scoring was as described in Table 1. Standard errors are displayed. No standard error is given for carboxyl Qdots instilled without pretreatment, because all scored identically.
Figure 3.
Uptake of mixed quantum dots in lymph nodes.
All mice were pretreated with N-9 by instillation of 30 µL 1% N-9 in PBS 12 h before instillation of Qdots. Qdots were instilled as described in Materials and Methods, except that a total of 15 µl of quantum dots in 30 µL total volume (15 pmoles) were instilled singly, or 15 µLeach mixed to give a total volume of 30 µL(15 pmoles each). Mixtures were prepared so as to eliminate any effects due to Qdot size or emission wavelength; Mixture 1 contained 15 pmoles each 655-nm-emitting streptavidin-polyarg Qdots and 800-nm-emitting PEG Qdots, while the reciprocal Mixture 2 contained 15 pmoles each 655-nm-emitting PEG Qdots and 800-nm-emitting streptavidin-polyarg Qdots. Mice were harvested 36 hr after Qdot instillation, scored as described in Table 1, then integrated brightness was measured as described in Materials and Methods. A, Score; B, Integrated brightness (fluorescence was measured in arbitrary units, “a.u.”). Fluorescence due to 655-nm-emitting Qdots is shown in unfilled columns; fluorescence due to 800-nm-emitting Qdots is shown in filled columns. There are very few artifacts due to miscounts of 800-nm-emitters in nodes from mice exposed to 655 nm Qdots alone, or to miscounts of 655-nm-emitters in nodes from mice exposed to 800 nm Qdots alone (A); this is confirmed by measurements of integrated brightness (B). Standard errors are shown. Note that our scoring method collapses the differences in uptake between lymph nodes as compared to integrated brightness. This is why the standard errors shown in A appear much smaller than those in B. There are no significant differences in uptake between quantum dot having different coats or emission maxima.
Table 2.
Quantification by ICP-MS and Integrated z-Stacks.
Figure 4.
Comparison of Qdot content as assessed by integrated spot brightness with cadmium content by ICP-MS.
Data were from Table 2.
Figure 5.
Uptake into lumbar lymph nodes at indicated times after Qdot instillation.
Animals were pretreated with N-9 and instilled with polyarg-streptavidin Qdots as described in Figure 1. Five animals were analyzed for each time point and Qdot uptake was scored as in Table 1. SEM’s are indicated.
Figure 6.
Qdots in vaginal wall close to cervix.
Top to bottom, increasing magnification of circled areas. Red, Qdots; green, WGA (cell surfaces); blue, nuclei (DAPI). Note penetration of tissue in bottom figure. N-9 pretreatment and instillation were as described in Figure 1. Organs were harvested and flash-frozen without fixation 24 hr after Qdot instillation. Sectioning, staining and microscopy were as described in Materials and Methods.
Figure 7.
Identification of Qdots in draining lymph nodes.
A. CD3 staining (green) and CD4 staining (red) in mouse lymph node. N-9 pretreatment and instillation were as described in Figure 1. Inset shows tiled panel of entire lymph node. B. Region in white circle from A, which contains a Qdot aggregate, false-colored blue, which is not associated with CD4 or CD8 cells (arrow). C. Qdots (red) associated with macrophage (CD11b, green). Blue, DAPI.