Two-photon imaging was performed either with an upright Bio-Rad microscope or an inverted ZEISS LSM510 META/NLO (capturing equivalent T cell behavior). Briefly, with the Bio-Rad setup, GFP and DyLight594 were excited simultaneously with a laser tuned to a wavelength of 800 nm and detected with PMTs after separation of the emitted fluorescent light with a suitable filter cube (dichroic mirror: 575 nm; bandpass filters: 510/40 nm and 670/45 nm). Movie sequences were captured with the LaserSharp2000 software. For the ZEISS LSM510 META/NLO imaging set-up, a Chameleon Ti:Sapphire laser tuned to 800 nm (Coherent) and suitable filter cube (dichroic mirror: 560 nm; bandpass filters: 500–550 nm and 575–640 nm) were used for specific detection of GFP and DyLight594. Movies were captured with the ZEN user interface (Zeiss). In both systems, time-lapse movies were composed of a series of image volumes (depth: 84 to 156 µm; step size: 6 or 12 µm) separated by 90” (movie duration >2 h) or 45” (movie duration < 2 h).
Lsm510 meta nlo
Manufactured by Zeiss
Sourced in Germany
The LSM510 META NLO is a high-performance confocal microscope system designed for advanced imaging applications. It features a multi-track imaging capability, allowing for the simultaneous acquisition of multiple fluorescence signals. The system utilizes a tunable multi-photon laser source for non-linear optical (NLO) imaging, providing enhanced penetration depth and reduced photodamage. The LSM510 META NLO is a versatile tool for researchers working in fields such as developmental biology, neuroscience, and tissue engineering.
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Lab products found in correlation
Mouse anti gfap, by Abcam (5 mentions)
Mouse anti gfap, by Merck Group (5 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (4 mentions)
Lsm 510 meta, by Zeiss (3 mentions)
Cy3 conjugated goat anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Cy3 conjugated goat anti mouse igg, by Jackson ImmunoResearch (3 mentions)
Fitc conjugated goat anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Wiseven woc high clean air oven, by Daihan Scientific (3 mentions)
Lsm image browser software, by Zeiss (3 mentions)
Mouse anti glial fibrillary acidic protein, by Merck Group (3 mentions)
Vectashield, by Vector Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Goat anti iba1, by Abcam (2 mentions)
Smi 31, by Merck Group (2 mentions)
Anti psd 95 6g6, by Abcam (2 mentions)
Anti emx1 sc 28220, by Santa Cruz Biotechnology (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Fitc conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Cy3 conjugated anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
93 protocols using lsm510 meta nlo
1
Two-Photon Imaging of T Cell Behavior
2
Imaging Metabolic Dynamics in Yeast Cells
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Fluorescence images of ACDAN were obtained on an inverted multiphoton excitation fluorescence microscope (Zeiss LSM 510 META NLO, Carl Zeiss, Jena, Germany) equipped with a Ti:Sa MaiTai XF-W2S laser (Broadband Mai Tai with 10 W Millennia pump laser with a tuneable excitation range of 710–980 nm, Spectra Physics, Mountain View, CA). The fluorescence signal was collected through either a 63X water objective, NA 1.2 or a 63X oil objective, NA 1.4. For two-photon excitation, the signal was split to two detectors (H7422 PMT, Hammamatsu, Denmark) by a 460 nm long-pass dichroic mirror and then filtered by a 520 ± 17.5 nm, 462 ± 24 nm or a 438 ± 12 nm bandpass filter (all from AHF Analysen Technik AG, Tübingen, Germany). Acquisition of the two-photon excitation fluorescence image was performed by adding 300 μl of ACDAN-labelled resting yeast cells at a density of 10% by weight (suspended in 100 mM potassium phosphate buffer, pH 6.8) to an 8-well plastic chamber (Lab-tek Brand Products, Naperville, IL). The chamber containing the sample was placed in the microscope, and the images were acquired a few minutes later to allow the cells to sink to the bottom of the well. For measurement of NADH signal, an additional 30 mM glucose was added to the buffer. Image analysis was done using either a MATLAB script or ImageJ.
3
Time-lapse Imaging of Chick Spinal Cord
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Detailed materials and methods are provided in Supplementary Materials and Methods. In ovo electroporation, immunohistochemistry, and in situ hybridizations were performed as described 38 (link). For time-lapse imaging, spinal cord transversal slices (400 μm) were dissected from E3 chick embryos using a Leica vibratome. Video imaging of the in situ dividing cells was performed with a Zeiss LSM510 META NLO (Carl Zeiss, Germany) 2-photon laser scanning microscope. Stacks of ∼50 μm were acquired every 3–5 min for 4–6 h. Ethical permission N200/11 was approved by the Stockholm regional ethics committee for animal research.
4
Imaging Salivary Particle Internalization
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Calcein-labeled salivary particles were observed and photographed by DM6 B upright microscope (Leica Microsystems). For salivary particle internalization study, calcein-stained salivary particles resuspended in DMEM were added to the culture of THP-1 cells or GEC stained with CellMask Orange at volumetric ratio of 1:10. Internalization of calcein-labeled salivary particles by cells in culture was monitored by a multiphoton laser-scanning microscope (Zeiss LSM 510 META NLO; Carl Zeiss) while maintained at 37 °C with a heating unit (XL S and TempModule S; Carl Zeiss).
5
Fluorescence and SEM Imaging of Cells
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Mowiol 4/88 (Sigma Aldrich, the Netherlands) was applied to the sections and a cover glass was put on Mowiol to fix the sections overnight. A confocal microscope (Zeiss LSM510 META NLO, Zeiss Nederland, the Netherlands) was used to obtain all the fluorescence images. For the invasion study, a 20× objective was used. For the assessment of the morphology of the cells, a 63× oil immersion lens and confocal mode of the microscope was used. A plane interval of 3 μm was set.
All samples were sputter coated with 5 nm of gold prior to scanning electron microscopy. A scanning electron microscope (Quanta 600F ESEM, Fei, the Netherlands) with high vacuum settings and working distance of 10 mm was used.
All samples were sputter coated with 5 nm of gold prior to scanning electron microscopy. A scanning electron microscope (Quanta 600F ESEM, Fei, the Netherlands) with high vacuum settings and working distance of 10 mm was used.
6
Quantifying Oligodendrocyte Morphology
7
Microscopy Analysis of Oocyte Morphology
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Slides were examined with the Axiovert 25 inverted microscope (Zeiss, Thornwood, NY) using Texas Red (red) and FITC (green) fluorescent filters with excitation and emission wavelengths of 470 and 525 nm, and 596 and 613 nm, respectively. Confocal images were obtained utilizing a Zeiss LSM 510 META NLO (Zeiss, Germany) microscope as previously described [27 (link)]. Three independent observers blinded to the assigned treatment groups performed the categorization of oocytes based on MT and CH status. Observers used comprehensive evaluation of the individual optical sections and the 3-D reconstructed images.
8
Live Imaging of Optic Nerves
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Live imaging of optic nerves was performed using an up-right confocal laser scanning microscope (Zeiss LSM 510 META/NLO, Zeiss, Oberkochen, Germany) equipped with an Argon laser and a 63x objective (Zeiss 63x IR-Achroplan 0.9 W). The objective was immersed into the aCSF superfusing the optic nerve. Theoretical optical sections of 1.7 μm over a total scanned area of 66.7 μm x 66.7 μm (512 × 512 pixels) of the optic nerve were obtained every 10.4 s in three channels, referred as CFP (excitation 458 nm; emission 470–500 nm), FRET (Ex 458 nm; Em long pass 530 nm) and YFP (Ex 514 nm; Em long pass 530 nm).
9
Bacterial Viability Assay Protocol
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To determine the viability of the treated cells, 1 mL of samples was centrifuged at 18,200× g for 5 min. Cell pellets were washed with 1 mL of 0.85% NaCl and stained using the BacLight™ Bacterial Viability Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The samples were incubated at room temperature in the dark for 15 min and 8 µL of the stained bacterial suspension were placed between a glass slide and a 170 µm thick coverslip. Samples were observed under a Zeiss LSM 510 META (Zeiss LSM 510 META NLO, Carl Zeiss Ltd., Jena, Germany) confocal microscope [30 ].
10
Characterizing Carbonaceous Particles in Urine
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The carbonaceous particles in the urine samples were analyzed and images collected using a Zeiss LSM510 META NLO (Carl Zeiss, Jena, Germany) mounted on an Axiovert 200 M equipped with a two-photon femtosecond pulsed laser (MaiTai DeepSee, Spectra-Physics, USA). A detailed description of the set-up and imaging protocol can be found in the online supplement.
From optimization measurements we found that 120 images obtained from 10-frame time lapses at three different positions in four different aliquots of one urine sample are necessary to gain highly reproducible results (<5 % coefficient of variation of three repeated measurements for 20 individuals, data not shown). Urine samples were aliquoted at 200 µL/well in Ibidi µ-slide 8 well plates (Ibidi GmbH, Germany). All images were taken 300 µm above the bottom glass.
To count the number of black carbon particles in the time frames of each urine sample, a peakfind algorithm in Matlab (Matlab 2010, MathWorks, The Netherlands) was used. A detailed description of the function of the customized Matlab routine can be found in online supplement.
From optimization measurements we found that 120 images obtained from 10-frame time lapses at three different positions in four different aliquots of one urine sample are necessary to gain highly reproducible results (<5 % coefficient of variation of three repeated measurements for 20 individuals, data not shown). Urine samples were aliquoted at 200 µL/well in Ibidi µ-slide 8 well plates (Ibidi GmbH, Germany). All images were taken 300 µm above the bottom glass.
To count the number of black carbon particles in the time frames of each urine sample, a peakfind algorithm in Matlab (Matlab 2010, MathWorks, The Netherlands) was used. A detailed description of the function of the customized Matlab routine can be found in online supplement.
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