X cite series 120q

Primary hippocampal cultures from P0-1 Tsc1;Rptor;Emx1-Cre mice of different genotypes were plated onto 24 well plates pre-coated with PDL (Corning, Cat # 08774271). On DIV 2, cultures were transduced with AAV1-jRGECO1a (Supplementary Table 5) and maintained for 12 days in Neurobasal media. On DIV 14, neurons were imaged on an AxioObserver.A1 (Zeiss) inverted microscope using a ×10 Zeiss A-Plan objective (Numerical Aperture: 0.25) with wide field fluorescence illumination (X-Cite series 120Q, Lumen Dynamics). Images were taken at 8.91 Hz with a Hamamatsu Orca-er digital camera and Micro-Manager 1.4 software. All imaging conditions including excitation light intensity, camera sensor gain, and exposure time were identical for all calcium imaging experiments. A single field of view (FOV) was imaged from at least 2-3 individual wells per culture (prepped from 1 pup) and approximately 40 neurons were randomly selected and analyzed. Before proceeding to the analysis, we verified that the neurons selected were active at least once during the recording period. At least 3 mice per genotype were examined from at least 3 different litters.

At the end of the treatment, cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. Samples were permeabilized in 2% Triton X-100 for 15 min at room temperature. Actin 488 Ready Probe (Life Technologies) was applied according to manufacturer instructions and incubated on fixed cells at room temperature for 30 min. Nuclei were visualized with DAPI stain for 5 min, or with Hoechst 33342 (Life Technologies) stain for 10 min, incubation at room temperature in the dark in a 1 µg/ml solution. Images were obtained using Axiovert 40 CFL (Zeiss) and a Progres C3 (Jenoptik) camera with an X-Cite series 120Q (Lumen Dynamics) lamp utilizing FITC or DAPI filter (Chroma).

QPI of MCF-7, BT-474, and HeLa cells was performed as described in Yu et al.^{74 (link)}. Fluorescence images were obtained with an EM-CCD C9100 camera (Hamamatsu Photonics) and an X-Cite Series 120 Q (Lumen Dynamics) source. Image collection occurred every 5 min for 12 h at 14–16 imaging locations containing cells plated with sufficient spacing to enable automated image processing and biomass segmentation.

In Gin/Chat-ChR2-EYFP-crossed mice, cholinergic fibers were stimulated by blue light activation of channelrhodopsin (ChR2) (five light pulses, 470 nm, @ 25 Hz) using a DC4100 4- channel LED-driver (Thorlabs, Newton, NJ) or a Fluorescence lamp (X-Cite Series 120q, Lumen Dynamics). In experiments where light stimulation was combined with presynaptic electrical stimulation the first light pulse started 100 ms before the first AP in the presynaptic neuron. The presynaptic stimulation was either with light off or with light on, alternating with 60 s interval. In some experiments we observed feedforward inhibitory responses by blue light, as was reported previously^{12 (link)}. These recordings were excluded from analysis. In layer 5, we sometimes observed feedforward excitatory responses by blue light, which was prevented by reducing the field of illumination.

Ca²⁺ imaging recordings were performed on an Olympus BX51WI upright fluorescence microscope using 10 and 20× Fluor water immersion lenses (Olympus, Center Valley, PA, USA). The tissue was excited at 488 nm, using a X-Cite series 120Q (Lumen Dynamics, Ontario, Canada) and a modified GFP dichroic cube (excitation 488 nm; emission 543 nm; Chroma Technology Corp., Bellows Falls, VT, USA). Movies were captured with an Andor iXon +897 EMCCD camera (normal recording ~2000 frames @ 32.4 Hz) using Andor Solis 4.14 software (Andor Technology, Belfast, UK). Solis (16-bit, tif) files were analyzed on a MacPro desktop computer (Apple Inc., Cupertino, CA, USA) using in-house analysis software (Volumetry G8c, G. W. Hennig).

FISH images were acquired with a Retiga EXi aqua camera (mount on 0.70×) mounted on a DM5500B upright microscope (Leica) and magnified through a 100× oil immersion objective (NA = 1.3). Images of fluorescent probes were excited using an X-Cite Series 120Q light source (Lumen Dynamics) with the appropriate filters. All hardware parts were controlled with Volocity v5.0 software. Data presented in figures are representative fields of images from two biological replicates. For calculating the percentage of mRNA accumulation in the different strains, we manually visualized 150 to 300 cells and counted the ones with visible polyA RNA accumulation in the nucleus.