Ccd camera

A pressure-driven microfluidic
pump (Fluigent MFCZ-EZ, France) was connected to the PDMS based microfluidic
device and used to fill the dead-end channel with the desired solution.
After filling the dead-end channel, an air bubble is passed through
to empty the main channel while leaving the original solution inside
the dead-end channel. Meanwhile, a particle suspension was sonicated
for at least 5 min in ElmaSonic P (Elma Schmidbauer GmbH, Singen,
Germany). Afterward, the particle suspension is passed through the
main channel by a syringe pump (Harvard Apparatus, PHD-Ultra, Massachusetts,
United States) using a 250 μL glass syringe (Hamilton, 1725RN
Syringe, Nevada, United States). To minimize the particle–particle
interactions and be able to track individual particles, the particle
concentration was set to 0.01% w/v for PS-carboxylate and 0.05% w/v
for PS–PEG. An inverted microscope (Zeiss Avio Observer. Z1,
Carl-Zeiss, Jena, Germany) was employed with a 20×f/0.4 objective
(depth of field is 5.8 μm, Zeiss LD Plan-Neofluar, Carl-Zeiss)
and a 20HE (Carl-Zeiss, Jena, Germany) filter. The particle movement
in the dead-end channel was captured by a CCD camera (Hamamatsu, Japan)
with 1376 × 1040 pixels mounted in the inverted microscope. The
images are sequentially captured for 6 min at 10 frames per second
(fps).

For immunostaining, LGs were dissected from matured third instar larvae and fixed in 3.7% paraformaldehyde for 15 min. After repeated washing, the fixed samples were incubated with primary antibody at 4°C for overnight. The following anti-Mmp1 antibodies (#3A6B4, #3B8D12, and #5H7B11) were mixed and used (1:100 for each; DSHB, IA, USA). After extensive washing, specimens were incubated with Alexa 594 secondary antibody (1:400; Molecular Probe, USA). The LG specimens were observed under a fluorescence microscope (Olympus, Tokyo, Japan, model: IX81), outfitted with excitation, emission filter wheels (Olympus). The fluorescence signals were collected using a 10x dry objective lens. Specimens were illuminated with UV filtered and shuttered light using the appropriate filter wheel combinations through a GFP filter cube. GFP fluorescence images were captured with a CCD camera (Hamamatsu Photonics, Shizuoka, Japan). Image acquisition was controlled through the Metamorph software version 7.6 (Molecular Devices, Sunnyvale, CA, USA) and processed with Adobe Photoshop CS. The basement membrane of the LG cells was observed under a confocal microscope from the surface to the inside of the tissue (Fv10i, Olympus, Tokyo, Japan) by altering the focus along the z-axis. The confocal images obtained were then processed by the Fv10i software and Adobe photoshop CS (Adobe KK, Tokyo, Japan).

Brain section images were acquired by using automated slide scanning and analysis software (MetaMorph) in a high-capacity computer coupled with a fluorescent BX61 Olympus microscope and a high-sensitive Hamamatsu CCD camera. Under a 10× objective, we were able to obtain images of sufficient resolution for all subsequent computer-based analyses. Image stitching, overlaying, cell counting, and further imaging analysis were completed by using MetaMorph imaging and analysis tools. In addition, we imaged labeled cells in selected sections with a confocal microscope (LSM 700/780, Carl Zeiss Microscopy) coupled with z-stack and tile scanning features under a 20× objective lens. Image stitching, overlaying, maximum projections, and export were performed by using the ZEN software analysis tools.
Quantitative examinations across the series of sections were conducted for complete and unbiased analyses of rabies-mediated, direct synaptic connections to targeted Cre-defined cell types by using either MetaMorph or Adobe Photoshop software (CS4 extended version, Adobe Systems). For mapping rabies-labeled presynaptic neurons (expressing mCherry only), digital images of brain sections were examined to identify and mark the locations of mCherry-expressing cell bodies. These labeled cells were assigned to specific anatomic structures for regional input quantification.

HT1080 cells containing HAC/dGFP cells (6500 cells/cm²) were seeded in a six-well tissue culture plate and cultivated in the presence of 10 µg/mL blasticidin for 210 h using Cell-IQ high-content in vivo imaging system equipped with 20× LUCPlanFLN Olympus Objective and Hamamatsu CCD camera. The growth curve was performed by time-lapse cell population analysis, recognizing each cell by its peculiar image using a phase-contrast microscopy using computer vision as well as fluorescent signal analysis to identify GFP-positive cells. The growth curve was generated automatically using Cell-IQ Analyzer software after the image library was performed, and each cell was marked with a specific dot marker plotted on the image mask for an operator's visual control (Supplemental Fig. S15).

For Ca²⁺ imaging, iAstro lines grown onto 24 mm round coverslips were loaded with Fura-2/AM (Life Technologies, Milan, Italy, Cat. No. F1201) in the presence of 0.005% Pluronic F-127 (Life Technologies, Cat. No. P6867) and 10 μM sulfinpyrazone (Sigma, Cat. No. S9509) in KRB solution (125 mM NaCl, 5 mM KCl, 1 mM Na₃PO₄, 1 mM MgSO₄, 5.5 mM glucose, 20 mM HEPES, pH 7.4) supplemented with 2 mM CaCl₂. After loading and 30 min of de-esterification, the coverslips were mounted in an acquisition chamber on the stage of a Leica epifluorescence microscope equipped with a S Fluor 40×/1.3 objective. Cells were alternatively excited at 340/380 nm by the monochromator Polichrome V (Till Photonics, Munich, Germany) and the fluorescent signal was collected by a CCD camera (Hamamatsu, Japan) through bandpass 510 nm filter; the experiments were controlled and images analyzed with MetaFluor (Molecular Devices, Sunnyvale, CA, USA) software. The cells were stimulated by 20 μM ATP. To quantify the difference in the amplitude of Ca²⁺ transients, the ratio values were normalized according to the formula (ΔF)/F₀ (referred to as norm. Fura ratio).