Platelet-derived microparticles or exosomes were stained with P4D1 anti-poly-ubiquitin monoclonal antibody and Alexa594-conjugated secondary antibody, incubated in a glass bottomed microwell (MatTek) dish for 30 min, and then imaged at 100X by Total Internal Reflection Microscopy with a 1.46 N.A. objective in a Leica AM TIRF MC System (Leica Microsystems, Wetzlar, Germany) equipped with an ImageEM C9100-13 EMCCD camera (Hamamatsu, Bridgewater, N.J). The 10-mW diode laser was used for excitation and the penetration depth was set to 70 nm.
Am tirf mc system
Manufactured by Leica
Sourced in Germany
The AM TIRF MC system is a modular and configurable microscope platform designed for advanced imaging techniques. It is capable of performing Total Internal Reflection Fluorescence (TIRF) microscopy, which allows for high-contrast visualization of structures and processes near the cell-substrate interface. The core function of this system is to provide a flexible and customizable solution for researchers conducting high-resolution fluorescence imaging experiments.
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Lab products found in correlation
Dmi6000, by Leica (2 mentions)
Af6000 lx, by Leica (2 mentions)
Du 8285 vp camera, by Oxford Instruments (2 mentions)
Alexafluor568 goat anti mouse igg1, by Thermo Fisher Scientific (2 mentions)
Dylight 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
Plc γ py783, by Cell Signaling Technology (2 mentions)
Imageem c9100 13 emccd camera, by Hamamatsu Photonics (1 mentions)
Em ccd camera, by Hamamatsu Photonics (1 mentions)
Application suite x, by Leica (1 mentions)
Dmi8 inverted microscope, by Leica (1 mentions)
Tcs sp8 laser, by Leica (1 mentions)
Ixon du 885 camera, by Oxford Instruments (1 mentions)
Volocity 5, by PerkinElmer (1 mentions)
Tmr phalloidin, by Merck Group (1 mentions)
Arpc3, by Merck Group (1 mentions)
Wave2, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Lat py226, by BD (1 mentions)
16 protocols using am tirf mc system
1
Visualization of Platelet-Derived Microparticles
2
FRET Analysis of APJ Receptor
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The donor plasmid APJ-CFP and receptor plasmid APJ-Venus were co-transfected into CHO cells as the FRET channel. In addition, the donor and acceptor channel are used to eliminate crosstalk into the FRET channel. FRET sample preparations must therefore include references of the donor in the absence of the acceptor (donor only control) and acceptor in the absence of the donor (acceptor only control). APJ-CFP and APJ-Venus were transfected into CHO cells as donor and acceptor channels, respectively, to obtain calibration coefficients to correct for excitation and emission crosstalk. After 12–24 h, FRET signals were detected with a FRET Kit for a Leica AM TIRF MC system (Leica Microsystems).
3
Live-cell TIRF Microscopy Imaging
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A Leica AM TIRF MC system attached to a Leica DMI 6000 inverted epifluorescence microscope was used to image the surface of cells. A 63× oil lens was used. HBS was added to the cells, and the cells were found and focused, and the cell surface plane (mCherry) was found using automated TIRF angles. Frames were taken every 30 s.
4
FRET Analysis of STIM1-Orai1 Interaction
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HEK293 cells were transfected with 3 µg STIM1‐mCherry‐pmax (containing mCherry after position L599 (Alansary et al, 2016 (link)) or STIM1A‐mCherry‐pmax (acceptor) and 1µg Orai1‐GFP‐pmax (donor) 24 h before measurements). Prior to the measurements, TG‐sensitive stores were depleted using 1 µm TG in 0 [Ca2+]o Ringer. For recording fluorescence images, the Leica AM TIRF MC system was used. Images were taken with a 100 × 1.47 oil HCX Plan Apo objective. The GFP‐Donor signal was used to determine the TIRF focal plane. For each cell, three images were taken: I. GFP excited at 488 nm (suppression filter BP 525/50); II. mCherry excitation wavelength at 561 nm (suppression filter BP 600/40); and III. FRET excitation with a 488‐nm laser and suppression filter BP 600/40. To calibrate laser intensities and excitation durations for each day of experiments, single transfected cells were used, only expressing the donor or acceptor construct. Calibrated parameters were constant for all channels and images taken. For image acquisition and analysis, the LAS (Leica Application Suite) FRET module was used. To calculate FRET efficiency according to van Rheenen et al (2004 (link)), all images were corrected for background signal, bleed‐through, and crosstalk factors.
5
Visualizing VEGFR2 in Endothelial Cells
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TIRF microscopy was performed using a Leica AM TIRF MC system mounted on a Leica AF 6000LX workstation with a 63× oil‐immersion objective and a laser penetration depth of 110 nm. ECs were fixed, saturated and permeabilized, and then treated with Ab anti‐VEGFR2 (A3) and appropriate Alexa Fluor secondary Ab.
6
FRET Efficiency Calculation for GPCR Interactions
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To obtain calibration coefficients and eliminate excitation and emission crosstalk, 5-HT1AR-ECFP and OX1R-EYFP were transfected into HEK293 cells as donor and acceptor channels, respectively. After 24 h, FRET signals were detected with a FRET kit using a Leica AM TIRF MC System. FRET efficiency (EA) was calculated as shown in the equation below; A, B, and C correspond to the intensities of the three signals (donor, FRET, and acceptor, respectively), and α, β, γ, and δ are the calibration factors generated by the acceptor-only and donor-only references:
7
TIRF Microscopy of Living Endothelial Cells
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TIRF microscopy on living ECs was performed using a Leica AM TIRF MC system mounted on a Leica AF 6000LX workstation. Cells were plated onto glass-bottom dishes (WillCo-dish; WillCo Wells B.V., Amsterdam, The Netherlands) and placed onto a sample stage within an incubator chamber set to 37 °C, in an atmosphere of 5% CO2, 20% humidity. A Leica HC PL APO 63 × /1.47 NA oil-immersion objective was used, and laser penetration depth was set at 90 nm. Excitation and analysis of fluorescent proteins were performed with a 488 nm (for GFP) and 532 nm (for Cherry) laser. Imaging was recorded on a Hamamatsu EM-CCD camera (C9100-02, Hamamatsu, SZK, Japan).
8
Multimodal Microscopy Techniques
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Confocal microscopy was performed on a Leica TCS SP8 laser-scanning confocal module mounted on a Leica DMi 8 inverted microscope, equipped with a motorized stage, and controlled by the software Leica Application Suite X (ver. 3.5.2.18963). For image acquisition, a HC PL APO CS2 63 × /1.40 oil immersion objective was used. DIC, epifluorescence (EPI), and total internal reflection fluorescence microscopy (TIRFM) of fixed specimens, live time lapse of spreading cells, drug treatments, osmotic shocks, cell stretching (EPI mode only), and cell compression were performed on a Leica AM TIRF MC system. Two different TIRFM-grade objectives were used: HCX PL APO 63 × /1.47NA oil immersion and HCX PL APO 100 × /1.47NA oil immersion. Three different laser lines were used for fluorochrome excitation: 488 nm, 561 nm, and 635 nm. A specific dichroic and emission filter set for each wavelength have been used. The microscope was controlled by Leica Application Suite AF software (ver. 2.6.1.7314), and images were acquired with an Andor iXon DU-8285_VP camera. For live imaging experiments, environmental conditions were maintained by an Okolab temperature and CO2 control system.
9
Vinculin Clusters and Actin Cytoskeleton in PC12 Cells
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PC12 cells were seeded on the different substrates (on Ø24 mm glass cover slips). At the indicated time points (30 min, 1, 4 and 24 h) the cells were fixed and labeled for vinculin and f-actin following the immunofluorescence imaging protocol described above. The images were recorded with a Leica AM TIRF MC system using a Leica HCX PL APO 63X NA 1.47 objective (Leica). To visualize the vinculin clusters, integrated 488 nm laser lines and Andor iXon DU-885 camera (Andor Technology, Belfast, UK) was used. The image recording was done with a laser incident angle of 74° allowing a penetration depth of almost 250 nm. The images were elaborated with ImageJ following a recently described method [96 (link)] to analyze the area and number of clusters per cell. In total, for global statistics 722-3678 clusters from 16–34 cells were analyzed from three independent experiments. The f-actin instead was imaged in epifluorescence mode. Here the cytoskeletal organization of the cells was categorized in three categories: (1) no detectable presence of actin bundles/stress fibers, (2) 1–10 distinct actin bundles/stress fibers and (3) >10 distinct actin bundles/stress fibers.
10
Wound Healing Assay with Amyloid Fibrils
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IGR39 cells were seeded at 80% confluency. After 24 h, the cells were treated with PMEL amyloid fibrils and Verteporfin. After another 12 h a wound was performed manually with a 200 μL pipette tip. Image acquisition was recorded by Leica AM TIRF MC system with 10× objective for 18 h.
IGR37, WM266.4 and BXPC3 cells were seeded at 80% confluency. After 24 h after were treated with DMSO, 3I alone, 3I with PMEL or Aβ40 and in combination with Verteporfin. 12 h after the treatment, a wound was performed manually with a 200 μL pipette tip IGR37 cells were imaged for 48 h, whereas WM266.4 and BXPC3 cells were imaged for 24 h.
IGR37, WM266.4 and BXPC3 cells were seeded at 80% confluency. After 24 h after were treated with DMSO, 3I alone, 3I with PMEL or Aβ40 and in combination with Verteporfin. 12 h after the treatment, a wound was performed manually with a 200 μL pipette tip IGR37 cells were imaged for 48 h, whereas WM266.4 and BXPC3 cells were imaged for 24 h.
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