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passive diffusion, exhibiting lateral diffusion sometimes followed by membrane flip-

flop. Furthermore, while the octaarginine behavior shares some common features with

protein uptake (endocytotic) processes, in that a subpopulation exhibits very low

diffusion coefficients, it also exhibits dissimilar diffusional properties in that another

subpopulation exhibits much larger diffusion coefficients. From our observations, we

conclude that the mode by which octaarginine penetrates the cell membrane appears to

either be a multi-mechanism uptake process or a mechanism different from passive

diffusion and endocytosis. These results have relevance to the understanding the

mechanism and optimization of cellular uptake of guanidinium-rich transporters

conjugated to small molecules, drugs and probes (MW ca. <3000).

Experimental 3.2

Fluorescent Conjugates 3.2.1

Arg8-DCDHF-V, Arg4-DCDHF-V, DCDHF-V-12 (further abbreviated as D-

V-12), and Transferrin-Alexa594 are the fluorescent conjugates used for this single-

molecule study. Arg8-DCDHF-V is the primary octaarginine molecular probe under

scrutiny; Arg4-DCDHF-V is the negative control, as Arg4 has been shown to be

insufficient for cellular uptake; D-V-12 is a fluorescent lipid analog, used for

mimicking passive lipid diffusion; Transferrin-Alexa594 is known to enter the cellular

interior via receptor- and clathrin-mediated endocytosis. The preparation of the

DCDHF-V maleimide, its Arg8 and Arg4- conjugates, and the lipid analog D-V-12 are

detailed in the supplementary online material of the published manuscript.49 The

synthesis of DCDHF-V has been previously reported.41, 46, 50 The transferrin-Alexa594

conjugate was purchased from Molecular Probes.

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Figure 3-3 A. Structures of DCDHF-V labeled octaarginine (Arg8-D-V) and

tetraarginine (Arg4-D-V) B. Structure of DCDHF-V labeled lipid analog (D-V-12).
Reprinted with permission from Ref.49. Copyright 2011 American Chemical Society.

Epi-illumination for single-molecule imaging 3.2.2

Both white-light transmission images of the cells and epi-fluorescence images

of single molecules were acquired using an inverted microscope (IX71, Olympus,

Center Valley, PA). Bright field white light illumination from a condenser lens

allowed the direct visualization of the edges of the cells. The fluorescence imaging of

the cells was performed with wide-field epi-illumination in an area of ~ 15 µm  15

µm (see Figure 3-4). For more details on the specific requirements and

recommendations for instrumentation, please see Chapter 2.

He-Ne laser illumination at 594 nm provided an intensity of ~ 0.14 kW/cm

the sample focal plane. The resulting epi-fluorescence was collected with a 100

magnification, 1.3 NA, oil-immersion objective (UPlanApo, Olympus, Center Valley,

PA) and imaged through a 620 nm long-pass filter and a 610 nm dichroic mirror

(Omega Optical Inc., Brattleboro, VT) on an EMCCD-camera (IXonDV887, Andor,

South Windsor, CT). The pumping beam intensity (~ 140 W/cm
) was selected to

achieve acceptable signal-to-background ratio (~4-6) while extending the time before

photobleaching of the fluorophores to obtain the longest single-molecule trajectories

possible. The total number of detected photons from a single molecule was determined

by integrating all detected signal counts from a single spot and then computing the

total detected photons taking into account the known detector quantum efficiency and

measured multiplication gain.

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