FLIM was recorded on a Zeiss LSM-780 NLO confocal/multiphoton microscopy system comprising an inverted Axio Observer Z1 microscope, an X-Cite 120PC Q mercury arc light source (Excelitas Technologies) for cell selection, a motorized stage for automated scanning, an IR Chameleon Vision-II ultrafast Ti:sapphire laser for multiphoton excitation (Coherent), a Zeiss 40 × 1.3 NA oil immersion Planapo objective, an environmental chamber (PeCon GmbH, Germany) that envelops the microscope stage to control temperature and CO2 level, and a 3-channel FLIM system based on three HPM-100–40 GaAsP-based hybrid detectors and 3 SPC-150 TCSPC boards (Becker & Hickl). The SPC-150 boards are synchronized with the 2-photon excitation laser and the Zeiss LSM-780 NLO scan head signal. Ex 740 nm; Em450/50 nm.
Lsm 780 nlo
Manufactured by Zeiss
Sourced in Germany, United States
The LSM 780 NLO is a confocal laser scanning microscope system designed for multiphoton imaging. It is capable of high-resolution, non-linear optical imaging, providing researchers with advanced tools for visualizing and analyzing biological samples.
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Lab products found in correlation
Zen software, by Zeiss (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Axio observer z1, by Zeiss (6 mentions)
Axio observer z1 microscope, by Zeiss (5 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (4 mentions)
Axio observer a1, by Zeiss (3 mentions)
Alexa fluor 647 dextran conjugate, by Thermo Fisher Scientific (2 mentions)
Axio observer z1 microscope, by Excelitas (2 mentions)
X cite 120pc q mercury arc, by Excelitas (2 mentions)
Spc 150 tcspc boards, by Becker & Hickl (2 mentions)
Spc 150 boards, by Zeiss (2 mentions)
Environmental chamber, by Pecon (2 mentions)
Pngase f, by New England Biolabs (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Zen 2, by Zeiss (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Oct compound, by Sakura Finetek (2 mentions)
C apochromat 1.2 na w objectives, by Zeiss (2 mentions)
Lsm 510 duoscan, by Zeiss (2 mentions)
183 protocols using lsm 780 nlo
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Multiphoton Microscopy Imaging Setup
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Multiphoton Microscopy Imaging Setup
3
Imaging mitochondria and actin cytoskeleton
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Cells were inoculated into 24-well plates with coverslips overnight. After treatment with 80 μM IR-783, cells were stained with 100 nM MitoTracker Red CMXRos (Beyotime Institute of Biotechnology) for 30 min at 37°C in the dark, then washed three times with culture medium for 5 min each time. Subsequently, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS for three times, and observed using a laser-scanning confocal microscope (LSM780NLO; Carl Zeiss AG) at x63 magnification, the representative images were randomly selected from five fields of view. For staining of the actin cytoskeleton, cells were treated with 80 μM IR-783 for 24 h at 37°C, then cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. F-actin was incubated with Alexa Fluor® 488 Phalloidin (CST, 8878S) for 30 min at 37°C in the dark; cells were counterstained with DAPI for 5 min at room temperature (cat. no. C1002; Beyotime Institute of Biotechnology). Cell images were collected using a laser-scanning confocal microscope (LSM780NLO; Carl Zeiss AG) at x63 magnification, the representative images were randomly selected from five fields of view.
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Immunofluorescence Imaging of VLCAD in PPGs
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The 24 PPGs were fixed in 4% paraformaldehyde for 30 min and then permeabilized with 0.1% Triton® X-100 for 30 min. The PPGs were incubated overnight at 4°C with 10 μg/mL of anti-acyl-CoA dehydrogenase primary mouse monoclonal antibody (Very long-chain acyl-CoA dehydrogenase, VLCAD, monoclonal antibody, Molecular Probes). Additionally, Cy5-conjugated goat anti-mouse IgG (H+L) (Molecular Probes) diluted 1:1,000 for 1 h was used as secondary antibody. A solution of 10% goat serum was used as a blocking agent at all stages. The cell nucleus was counterstained with DAPI.
The markings (VLCAD and nucleus) in the PPGs were documented in full assembly by making use of a confocal laser scanning microscope (Zeiss LSM780-NLO). Fluorescent images were obtained using lasers with 405 nm and 633 nm excitation wavelengths. The optical sections were acquired in suitable Z-axis sectioning step sizes (0.63 μm and 0.67 μm). Different modules of the software Zeiss LSM780-NLO, as well as the software ImageJ, were used for the confocal analysis, including maximum projection.
In order to verify possible fluorescence emissions, PPG incubated only with Cy5 secondary antibody was used as the negative control.
The markings (VLCAD and nucleus) in the PPGs were documented in full assembly by making use of a confocal laser scanning microscope (Zeiss LSM780-NLO). Fluorescent images were obtained using lasers with 405 nm and 633 nm excitation wavelengths. The optical sections were acquired in suitable Z-axis sectioning step sizes (0.63 μm and 0.67 μm). Different modules of the software Zeiss LSM780-NLO, as well as the software ImageJ, were used for the confocal analysis, including maximum projection.
In order to verify possible fluorescence emissions, PPG incubated only with Cy5 secondary antibody was used as the negative control.
5
Intracellular Localization of SPG-ODN 1826
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Intracellular localization of SPG-ODN 1826 was analyzed through confocal laser scanning microscopy (CLSM, Zeiss, LSM 780NLO, Oberkochen, Germany) as previously described [27 (link)]. In brief, 1.5 × 104 J774A.1 cells were transferred onto a single chamber cell culture slide (SP Lifesciences) and were allowed to adhere. The J774A.1 cells were stimulated using LPS as stated above. Post-stimulation, the cells were exposed to SPG-ODN 1826 nanovehicles for 6 h. Subsequently, the cells were stained using FM 4-64 following the manufacturer’s instructions, and fixed using 4% paraformaldehyde. The slides were finally analyzed using a Carl Zeiss microscope (Zeiss, LSM 780NLO, Oberkochen, Germany).
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Visualizing Isolated EpCAM-Negative CTCs
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Visualization of isolated subsets of stem EpCAM-negative CTCs was carried out on confocal microscope LSM 780 NLO (Carl Zeiss, On Cohen, Germany). In order to do that cell suspension after sorting was dried on glass with poly-l -lysin coating, fixated with cold methanol and incubated with 3% BSA in 1× PBS with 0.02% Tween 20 (Amresco, Dallas, TX, USA) during 45 min for preventing non-specific antibody binding. Further the primary antibodies rabbit anti-EpCAM (polyclonal, 1:2000, Abcam, Cambridge, UK) and goatanti-CK7 (polyclonal, 1:50, Santa Cruz Biotechnology, Dallas, TX, USA) in 1% BSA were added and incubated in dark during 30 min. Then were washed with PBS and after that the cocktail of second conjugated antibodies Anti-Rabbit IgG H&L (Cy3) (Abcam, Cambridge, UK) and Anti-Goat IgG H&L (AlexaFluor 647) (Abcam, Cambridge, UK) was added. Sections were covered by Mounting Medium with DAPI (Dako, Carpinteria, CA, USA) and analyzed with the laser scanning microscope LSM 780 NLO (Carl Zeiss, Oberkochen, Germany) with magnification ×630.
7
Islet Imaging and Morphological Analysis
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Single islet images were obtained using an upright laser scanning confocal microscope (LSM 780 NLO, Zeiss) with a Plan-Apochromat 20x/1$0 water-immersion objective (Zeiss) using a stack separation of 1.5 mm. For the assessment of 2D islet morphology, 10-20 islets per patient were imaged from at least 3 different slices and analysis was performed manually on three z-planes per islet at a distance of 15 mm using Fiji (Schindelin et al., 2012) . Islet cell composition was expressed as the average of the three planes analyzed. For imaging of entire tissue slices, tile-scans were performed with 2-4 stained slices per patient mounted under a poly-L-lysine coated 22x22mm glass coverslip submerged in PBS (pH 7.4) using an upright laser scanning confocal microscope (LSM 780 NLO, Zeiss), with a Plan-Apochromat 20x/1.0 water-immersion objective (Zeiss) and an automated stage (Zeiss). Single tiles were imaged at a resolution of 0.75x0.75mm, a tile overlap of 15%, and stack separation of 2.5mm. The bounding grid was set around the slice such that the entire slice was included in the tile-scan, and the stack boundaries were set to include the entire slice.
8
Visualizing Polyphosphate in Sponge Tissues
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The sponge tissue (approximately 0.3 cm3) was washed for 10 min in each of a series of solutions: 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol, 50% xylene/ethanol (vol/vol), and 100% xylene and embedded in paraffin. Next, 10 μm thick paraffin sections were cut, placed on microscope slides, dewaxed in xylene and ethanol respectively, and washed three times in MilliQ water. The sections were stained with 20 μM DAPI solution for 2 min, washed with MilliQ water three times and visualized under a Zeiss LSM780NLO (Carl Zeiss AG, 73447, Oberkochen, Germany) inverted confocal microscope equipped with a 20 × Axio Observer Z1 automatic inverted fluorescence/lens. Sample sections were visualized by excitation with a 405-nm laser light source with emission signals separated by a NFT515 filter into two channels. Emission wavelength from 420–515 nm resulting from DAPI bound to nucleotides was collected in Channel 1. The emission wavelength above 530 nm, including the peak of 550 nm resulting from DAPI bound to polyP, was collected in Channel 2. The polyP standard curve was shown in Figure S3 .
9
Quantifying Cellular Oxidative Stress
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To analyse oxidative stress, cells were incubated with the indicated drugs for 2 h before CellROX Green reagent (Molecular Probes, ThermoFisher) was added to the medium at a final concentration of 5 μM for 30 min at 37 °C. Cells were then washed three times with PBS and placed under confinement for 2 h. Cells were finally fixed for 20 min in 4% paraformaldehyde and washed twice in PBS. CellROX Green fluorescence was then measured (Ex. 488/Em. 520) by confocal microscopy using identical settings (Zeiss LSM 780 NLO, Zeiss, Germany). Experiments were performed in triplicate.
10
Quantification of Advanced Glycation End-Products
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Several measurements were performed on transverse slices of skin biopsies. Immunohistochemical staining was conducted with the anti-AGE antibodies anti-CML (ab30917, Abcam, Cambridge, UK) and anti-Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine ([MG-H1], STA-011, cell biolabs, inc., NL) using goat anti-mouse IgG alkaline phosphatase (AP) conjugate ([Go-a-Mo-AP], D0486, Dako, Glostrup, DK) as chromogenic reporter. Histological localization of CML and MG-H1 was evaluated semi-quantitatively by two independent investigators (I.M.A and G.F.H.D). Hematoxylin and eosin (HE) stains were added to be able to identify individual cells.
Invasively assessed intrinsic fluorescence was measured by confocal microscopy using the ZEISS LSM 780 NLO (Zeiss, GER) and quantified by ImageJ. We used single (405nm and 440nm) and 2-photon (750nm, equivalent to 375nm) excitation, which corresponds to the excitation area of the AGE Reader.
In the second skin biopsy, including epidermis and dermis, the concentrations of CML, MG-H1 and pentosidine were assessed by ultra-performance liquid chromatography tandem mass spectrometry measurements (UPLC-MS/MS) and high-performance liquid chromatography (HPLC), respectively [20 , 21 (link), 22 (link)]. (Supplementary material).
Invasively assessed intrinsic fluorescence was measured by confocal microscopy using the ZEISS LSM 780 NLO (Zeiss, GER) and quantified by ImageJ. We used single (405nm and 440nm) and 2-photon (750nm, equivalent to 375nm) excitation, which corresponds to the excitation area of the AGE Reader.
In the second skin biopsy, including epidermis and dermis, the concentrations of CML, MG-H1 and pentosidine were assessed by ultra-performance liquid chromatography tandem mass spectrometry measurements (UPLC-MS/MS) and high-performance liquid chromatography (HPLC), respectively [20 , 21 (link), 22 (link)]. (Supplementary material).
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