Confocal fluorescence anisotropy (CFA) microscopy of AS-C4-FlAsH aggregates in SH-SY5Y cells.

<p>(A) CFA image (<i>I_f,</i>) and (B) associated fluorescence anisotropy (<i>r</i>) image. (C) 2D-histogram <i>I_f</i> vs. <i>r</i>. The histogram values were sectioned using threshold values for <i>I_f</i> and <i>r</i>, and the resulting (<i>I_f</i>, <i>r</i>) pairs were backmapped on a pixel-by-pixel basis onto the fluorescence intensity image (red pixels). Four distinct groups were defined: (i) pixels with high <i>I_f</i> and low <i>r</i> (top left), (ii) with high <i>I</i>_f and <i>r</i> (top right), (iii) low <i>I</i>_f and <i>r</i> (bottom left), and (iv) low <i>I</i>_f and high <i>r</i> (bottom right). The colored scale bar represents frequency (number of pixels). The insets in the images in groups (i) and (ii) show an example of an aggregate that displays high <i>I</i>_f and both low and high <i>r</i>, reflecting different dynamics of AS-C4 within its structure.</p

FRAP microscopy of fluorescently labeled AS-C4 aggregates in SH-SY5Y cells.

<p>(A) Cells were transiently transfected to express the biarsenical-binding AS-C4 version of AS, and ReAsH labeling allowed the identification of aggregated and non-aggregated regions with the protein, as opposed to non-transfected control samples that only exhibit a low background staining signal (the transmission image was included for a proper observation of the location of the cell). For a typical FRAP experiment performed on one cell, the regions of interest (<i>ROI</i>s, diameter ∼4 µm) before (B) and immediately after (C) photobleaching, are shown (colored circles): <i>ROI</i> with aggregates (yellow), <i>ROI</i> with no apparent aggregated AS-C4 (green), scanning-photobleaching control <i>ROI</i> (blue), and background <i>ROI</i> (white). (D) Normalized fluorescence recovery for the corresponding ROIs and their fits according to a simple monoexponential model. Fluorescence intensity values before (<i>I_i</i>) and after (<i>I₀</i>) photobleaching, and at the end of the experiment (<i>I_∞</i>), are shown. The green arrow indicates the region of fast recovery for non-aggregated protein. The inset images correspond to the <i>ROI</i> with aggregated protein before photobleaching (<i>t</i> = 0 s), immediately after photobleaching (<i>t</i> = 17 s) and post-bleaching at <i>t</i> = 60, 90 and 120 s. Data shown as means ± standard errors (sample set <i>n</i> = 5).</p

Modulation of a Photoswitchable Dual-Color Quantum Dot containing a Photochromic FRET Acceptor and an Internal Standard

Photoswitchable semiconductor nanoparticles, quantum dots (QDs), couple the advantages of conventional QDs with the ability to reversibly modulate the QD emission, thereby improving signal detection by rejection of background signals. Using a simple coating methodology with polymers incorporating a diheteroarylethene photochromic FRET acceptor as well as a spectrally distinct organic fluorophore, photoswitchable QDs were prepared that are small, biocompatible, and feature ratiometric dual emission. With programmed irradiation, the fluorescence intensity ratio can be modified by up to ∼100%

Magnetic field induced activation of EGFR.

<p>Confocal image analysis-left column after 60 sec magnetization or right column without magnetiztion. (a,b) MS- Alexa488-biocytin, green; (a′, b′) EGFR activation (pY-EGFR1068) red; (a′′,b′′) overlay of red, green and DAPI DNA staining, blue, images; and DIC image (bottom). Scale bar is 10 µm. (c) Two-dimensional colocalization histogram for the same confocal sections a and a′ after background subtraction. SEM images of A431 cells reacted with MS after (d,d′) and without (e,e′) application of magnetic field. Scale bars, 1 µm for (d & e) and 500 nm for (d′ & e′), respectively.</p

Shc activation as a result of magnetic activation of EGFR.

<p>Confocal immunofluorescence images of cells incubated for 15 min at 37°C after 0 sec (a) or after 30 sec (b) or 180 sec (c) magnetic field activation. Image columns left to right: MS Alexa-488 biocytin (green); indirect immunofluorescence of MAb against pY-317 Shc protein and GARIG-CY5 (red); overlay of the first 2 columns; two-dimensional colocalization histograms of MS and pY317-Shc fluorescence signals after deconvolution of 50 optical sections using SVI Huygens software.</p

Effect of magnetization time on the level of EGFR phosphorylation.

<p>(a, a′, a′′) The time axis shown vertically. Confocal images of MS 488Alexa biocytin signal (green, left panels) and of anti-pY-EGFR 1068 and GARIG-Cy5 (rainbow intensity scale, middle panels) on A431 cells as a function of the applied magnetic field for time intervals of 30, 60 and 180 s. Overlay images are depicted with green/red LUTs, Alexa488-biocytin/GARIG-Cy5 respectively (right panels). Scale bar, 10 µm. (b) Fluorescence intensity ratio of pY-EGFR to MS signals as a function of magnetization. (c) Mean pixel intensity of the pY-EGFR signal from 5 images for each time point as a function of MS magnetization time. (e) Western blot analysis of A431 cell extracts for pY-EGFR 1148. Lane 1, sample obtained from A431 cells incubated with MS in the absence of a magnetic field. Lanes 2–4, pY-EGFR signals for 30, 60 or 180 s of MS magnetization. Lane 5 and 6, pY-EGFR signals after treatment with 30 nM (saturating) or 100 pM EGF. Lane 7, extract of untreated cells. (e) Signal intensities of the positive pY-EGFR lanes in (d) relative to the lane from 30 nM EGF treated cells.</p

Schematic depiction of the composition and use targeted SPION magnetic switches for ligand-free activation of EGFR in a cell membrane.

<p>Magnetic switches (MS) consisting of SPION covalently coupled to streptavidin further reacted with biotinylated anti-EGFR MAb, in most cases, and biocytin-fluorophore.</p

Mitochondrial membrane potential (MMP) and energy balance.

<p><b>A) Fluorescence microscopy</b> of MMP in live NPCs from patient (SNCA-Tri) and control (Ctrl) loaded with 100 nM TMRM in normal growth medium (HG), medium plus 20 µM Rotenone (HG+R) or with 1 µM of the ionophore CCCP (HG+CCCP) as negative control (Scale bar: 10 µm). <b>B)</b><b>Plate reader based high throughput screen (HTS) of MMP</b> in live NPCs loaded with 20 µM JC-10 for 45 min. Cells were also treated with medium w/o glucose (NG). Shown are log ratios of reduced (Ex./Em. 540 nm/590 nm) to oxidized JC-10 (Ex./Em. 488 nm/520 nm) normalized to Hoechst 33342 (Log Norm. JC-10 Ratio) after 60 min. (n = 8, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD for HG+R: 202/29/194 (xE04), *<i>p</i>≤0.05; for NG: 92/30/118 (xE03) **<i>p</i>≤0.006). <b>C) Plate reader based HTS for MMP loss</b> in live NPCs prepared and analyzed as under B). Fluorescence measurements were acquired as under B) every 5 min for 10 cycles and loss of MMP with time graphed as ΔRFU/min. (n = 8, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: −0.02/−0.06/−0.01, *p≤0.05; HG+R: −0.17/−0.70/−0.22 ***p<0.001, NG: −0.08/−0.33/−0.04, *p≤0.05). <b>D)</b><b>Luminescence plate reader based HTS</b><b>of ATP levels</b> in Ctrl, SNCA-Tri and SNCA-Tri KD NPCs under the above growth conditions (HG, HG+R, NG) assayed by a coupled luciferin/luciferase assay. Depicted are ATP contents in cells treated with 20 µM rotenone (R) for 18 hrs. (n = 8, mean ± SD nMATP/ug protein in: Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 1.66/0.75/1.37, **<i>p</i> = 0.003; NG: 0.69/0.45/0.51, *<i>p</i> = 0.04). <b>E and F) Mitochondrial metabolic activity</b> studied by Seahorse XF24 analysis. <b>E)</b> Oxygen Consumption Rate (OCR) and <b>F)</b> Extracellular Acidification Rate (ECAR). Shown are relative OCR compared to basal values as a function of the sequential addition of mitochondrial inhibitors Oligomycin (1 µM), CCCP (1.5 µM) and Rotenone (Rot, 5 µM) + Antimycin A (Ant, 1 µM). Significant changes compared to basal OCR rates (*p<0.05) and differences between lines treated with and without 6-OHDA (250 µM) for 1 hr are indicated by # (#p<0.05, mean ± SEM, n≥17; from five independent experiments).</p

Apoptosis sensitivity and caspase activation.

<p><b>A) Caspase 3 activity</b> in cell lysates from adherent NPCs either left untreated or treated with 20 µM rotenone (R) for 18 hrs and then exposed to 1 uM staurosporine for 120 min before analysis. HTS analysis for caspase 3 activity from cell lysates was by activation of the fluorescent caspase substrate 7-amino-4-methylcoumarin (AMC) (Ex./Em. 340/440 nm) (n = 9, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD, HG: 33/69/42, HG+R: 42/129/87, NG: 55/138/85, *p≤0.050, **p≤0.0035; from three independent experiments). <b>B)</b><b>Kinetics of caspase 3/7</b><b>activity</b> in permeabilized NPCs pretreated as described under B) and assayed 15 min after staurosporine treatment. Changes in caspase 3 activity are depicted as ΔµM AMC fluorescence/min + mg cellular protein (detected by Bradford protein assay) (n = 9, mean ± SEM).</p

Mitochondrial integrity, MPT opening, and apoptosis.

<p><b>A) Mitochondrial calcein loading</b> by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl₂ were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 µM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean ± SD, Ctrl/SNCA-Tri: 3.4/4.9, *<i>p</i> = 0.039). <b>B)</b><b>MPT-induced mitochondrial calcein loss</b> in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl₂ were assayed 1 hr. after treatment with 4 µM staurosporine under NG conditions. MPT opening results in entry of CoCl₂ into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 µm). <b>Inserts:</b> Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 µm). <b>C) HCI automated fluorescence microscopy analysis</b> of MPT in NPCs treated with 4 µM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean ± SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, *<i>p</i>≤0.01). <b>D)</b><b>Kinetic evaluation of MPT opening</b> and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS analysis. NPCs treated and prepared as under B) were loaded with 4 µM stauropsporine and changes in calcein signal normalized to cell number and mitochondrial content (Δ Norm. RFU) were recorded every 1 min for 20 min (n = 8, mean ± SD, Ctrl/SNCA-Tri, HG: −0.06/−0.12, HG+R: −0.17/−0.28, HG+Iono: −0.03/−0.04, *<i>p</i>≤0.01). <b>E)</b><b>Cytochrome c immuno-cytochemistry</b> in Ctrl and SNCA-tri NPCs challenged with 200 µM paraquat (PQ) 15 min. before fixation. Shown are permeabilized cells probed with cytochrome c antibody, detected by an Alexa-488 nm labeled secondary antibody (green). Cells were counter stained with Hoechst 33342 (blue) (Scale bar: 100 µm, insert: 10 µm). <b>F)</b><b>Immunoblot analysis of cytochrome c levels</b> in sub-cellular fractions containing either cellular organelles (containing bound cytochrome c) or cytosolic proteins (with soluble cytochrome c) from NPC cell lysates (Ctrl and SNCA-Tri) treated with paraquat (PQ) as under E). Cytochrome c (14 kDa) and GAPDH (40 kDa) specific antibodies were detected by a secondary IR-dye conjugate.</p