Combined Analysis of Polycation/ODN Polyplexes by Analytical Ultracentrifugation and Dynamic Light Scattering Reveals their Size, Refractive Index Increment, Stoichiometry, Porosity, and Molecular Weight

Analytical ultracentrifugation (AUC) and dynamic light scattering (DLS) were combined to characterize polyplexes formed with 10 kDa chitosan or 10 kDa PEI and oligodeoxynucleotides (ODN). Combined analysis revealed that both polyplexes were highly porous (over 80%) and that their weight average hydrodynamic diameters were of 46 and 55 nm for chitosan/ODN and PEI/ODN complexes, respectively. Transformation of the sedimentation coefficient distribution to a size and molecular weight distribution gave an average molecular weight of 19 and 29 MDa for chitosan and PEI polyplexes, respectively. Data from AUC also allowed for the calculation of the actual d<i>n</i>/d<i>c</i> and N/P ratios of each polyplex. Additional data from scanning electron microscopy and static light scattering confirmed the conclusions that were initially derived from AUC and DLS, thus validating that the combination of AUC and DLS is a powerful approach to characterize polyplexes in terms of refractive index increment, size, and molecular weight distributions, as well as porosity

Multispectral detection allows identifying D1 and D2 expressing neurons.

<p>A) Schema of the shutters state and incident signals on the detecting PMTs as the probe passes by a green and a red cell in succession. B) (Left) Micrograph of a GFP fluorescent cell and a microprobe (highlighted with gray contour). Scale bar is 10 µm. (Right) Collected fluorescence in the PMT detector 1 (green dots), and 2 (red dots) as the probe is moved transversally in front of the cell shown in left panel. C) Same result is shown as the probe pass by a tdTomato fluorescent cell. Scale bar is 10 µm. Arrows in (B) and (C) represent the probe displacement in front of the cell. D) Striatal section of a transgenic mouse showing cells expressing fluorescent proteins under the control of D1 (red) or D2 (green) receptor (green) promoters. Some cells co-express both fluorescent proteins (see arrow). E) Average density of the different cell types in the striatum (n = 5 striatal sections). Cells were counted and densities were estimated with the help of the software Image J. F) Histogram of the optically detected cells. Fluorescent cell detections were accompanied by a rise (signal-to-noise >2) and a decay of green and/or red fluorescence as described previously. A total of 29 cells were detected and computed in a histogram according to their fluorescence colour. (G-H) Examples of <i>in vivo</i> simultaneous optical and electrical recordings (inset) as the probe pass by a green (G) or a red (H) cell.</p

Impact of fluorescence collection through the microprobe walls.

<p>A1) Schematics of the coated and uncoated microprobe descents into fluorescent agar. A2) Fluorescence measurements for different penetration depths into fluorescent agar for bare (green) and coated (white) probes (n = 5 probes; 1°<<i>θ</i> <3°)). B) Effect of the coating on the fluorescence DC level when a bare (green) or a coated (black) probe is lowered into cortex and thalamic issue. Arrows show location of fluorescent cells (inset: representation of the probe displacement).</p

Side acceptance of tapered waveguides.

<p>A) 2D schematic representation of ray acceptance within a tapered waveguide (thick black boundaries). Note that for visualization purpose the taper angle was exaggerated in this illustration. B) Critical accepted incidence angle <i>θ</i>_1c as a function of the taper angle <i>θ</i>. Rays with incidence angles ranging from <i>θ</i>_1c to 90° will be accepted in the waveguide (parameters were fixed as follows: <i>R</i> = 100 µm, <i>R</i>_f =5 µm, <i>r</i>_c =60 µm, <i>r</i>_cf = 3 µm, <i>n</i>₀ = 1 (air), <i>n</i>₀ = 1.35 (tissue) <i>n</i>₁ = 1.47 and <i>n</i>₂ = 1.45).</p

Optical and electrical microprobes.

<p>a) Schematic representation of the probe (left) and a metal coated probe adapted to achieve large field recording through the Al coating (middle: 3D representation, right: transverse cut view). Insets are scanning electron microscopy images of the respective electrode tips (scale bars are 2 µm). b) Experimental setup for multispectral detection showing : (1) 543 nm laser (25-LGR-193-249, Melles Griot), (2) 488 nm laser (FCD488 24 mW, JDS Uniphase Corporation), (3) shutters (LS3,Uniblitz), (4) 495 nm dichroic mirror (495DCLP, Chroma Technology Corporation), (5) multiline dichroic mirror (51015bs, Chroma Technology Corporation), (6) 495 nm dichroic mirror (495DCLP, Chroma Technology Corporation), (7) PMT detectors (H6780-20, Hamamatsu) and bandpass filters (ET520/40M and ET005/52M, Chroma Technology Corporation) and (8) objective (UIS-2 Plan-N, NA = 0.25, Olympus Corporation) and the fibre optic launch system (KT110, Thorlabs Inc.).</p

Photoconversion of mEOS.

<p>A–B) Images at different time points of a mEOS2 expressing cell during UV-induced photoconversion. UV illumination with the probe causes an increase in red fluorescence (A) and a decrease in green signal (B). C) Red and green emission of a cell during photo-conversion as a function of time. The region of interest taken into account is shown in (A) (white circle). Note that only two time points were measured for the green signal. Change in background fluorescence around the cell is shown in black.</p

Coated glass microprobes enable dual electrical recordings.

<p>A) Schematic representation of a multimodal microprobe tip. B) Measured resistance as a function of the uninsulated surface. C) Simultaneous recording of field potential oscillations and single unit achieved with the microprobes. Spikes were computed in a time histogram (C1). Inset: overlay of 10 successive spikes (vertical scale bar: 0.1 mV, horizontal scale bar: 1 ms) (C2) according to time of occurrence relative to field maxima (arrows in c1; n = 15) and into an interspike interval histograms (C3).</p