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Sp8 falcon microscope

Manufactured by Leica
Sourced in Germany

The Leica SP8 Falcon is a high-performance confocal microscope that enables advanced fluorescence imaging. It features a supercontin uum white light laser, offering a wide range of excitation wavelengths. The SP8 Falcon provides high-speed scanning and superior optical performance for a variety of applications.

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8 protocols using sp8 falcon microscope

1

Immunofluorescence Staining of Transfected Cells

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The transfected cells were cultured on coverslips for 48 h, fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100 for 20 min at room temperature, blocked with 10% FBS in PBS for 1h at room temperature, probed with primary antibody at 4 °C overnight, and incubated in the dark with fluorescence-conjugated secondary antibody and Hoechst for 30 min at room temperature. Confocal fluorescence microscopy was performed on a Leica SP8 FALCON microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica TCS SP8 X scanner.
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2

Imaging of Fragile Organoids

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Organoids generated by the aggregation of 30,000 UB PalmGFP cell line cells and 50,000 mTmG MM cells were live imaged with Leica SP8 FALCON microscope (Leica Microsystems, Germany). Due to the fragility of organoids, they were subjected to live imaging at day 2 of generation.
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3

Time-lapse Imaging of Ciliary Dynamics

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3D z-stacks covering entire cilia were collected at 5 min intervals using an 40x oil immersion Plan-Apochromat objective (Leica, NA = 1.3) on a Leica SP8 Falcon microscope at 2x the Nyquist limit and 1 Airy disc. Donor sGFP fluorescence was excited by a pulsed (40 MHz) white light laser tuned at 488 nm, and emitted photons between 490 nm and 550 nm were collected with line repetition = 8.
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4

Fluorescence Correlation Spectroscopy in U-2 OS Cells

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FCS was performed on a Leica SP8 FALCON microscope (as described above) using a HC PL APO CS2 ×40/1.10 water objective with a motorized correction collar. FCS traces (30 s) were measured in U-2 OS cells expressing either HaloTag7 or HaloTag9 in the cytosol (no induction). The cells were labeled with MaP618-CA (150 nM, 2 h) and washed twice for 1 min. Excitation and emission collection was performed as indicated in Supplementary Table 19. Five traces per cell and a total of 36 cells from three biological replicates were measured. Data analysis (correlation and fitting) was performed using the LAS X software (Leica Microsystems) fitting a free three-dimensional diffusion model including a triplet component56 . The diffusion amplitude (G(0)) as well as the mean photon counts (PC) over the 30 s trace were used to calculate the molecular brightness (mB, equation (9)). mB=G0×PC
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5

Synaptic Density Quantification in Neuronal Cultures

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Synaptic density and synapse composition was assayed in 22 DIV neuronal cell cultures. Cultures were fixed in cold 4% PFA with 4% sucrose for 20 min at RT. Primary antibodies were incubated overnight at 4 °C (Supplementary data 9). Secondary antibodies (Invitrogen) were incubated for 3 h at RT. Hippocampal primary culture: pyramidal cells were selected based on their morphology and confocal images were acquired on a Leica SP8 Falcon microscope using 63X (NA 1.4) with a zoom power of 3 and analyzed with Fiji software. After deconvolution (huygens professional), images were subsequently thresholded, and subsequent analyses were performed by an investigator blind to cell culture treatment107 .
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6

Time-lapse Imaging of Ciliary Dynamics

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3D z-stacks covering entire cilia were collected at 5 min intervals using an 40x oil immersion Plan-Apochromat objective (Leica, NA = 1.3) on a Leica SP8 Falcon microscope at 2x the Nyquist limit and 1 Airy disc. Donor sGFP fluorescence was excited by a pulsed (40 MHz) white light laser tuned at 488 nm, and emitted photons between 490 nm and 550 nm were collected with line repetition = 8.
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7

Fluorescence Correlation Spectroscopy Analysis

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FCS trajectories were obtained using a Leica SP8 Falcon microscope equipped with a Leica APO 86x/N.A. = 1.20 water-immersion objective with the motCORR automated correction collar, as previously described (Gandin et al., 2021 (link)). Mouse embryonic stem cells (mESCs) in which Halo and SNAPf tag were inserted into the EIF4E1 and EIF4G1 alleles, respectively, were differentiated into fibroblasts (Gandin et al., 2021 (link)). Twenty-four hours later, cells were labelled with 100 nM JF585-Halo tag ligand for 10 min and 500 nM JF64-6SNAPf tag ligand for 45 min. A set of two measurements (10 s each) were performed in the cytoplasm and in the nucleus for the indicated conditions in at least 5 cells per condition. FCS and FCCS values were exported and averaged using GraphPad Prism v7.
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8

Whole Lung Imaging Using Confocal Microscopy

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The whole cleared lungs have been imaged using a custom-made FluoClearBABB compatible 20× objective on an upright confocal SP8 Falcon microscope (Leica) using the Lightning module. A list of antibodies is included in Supplementary Table 8.
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