Images were acquired with a LSM780 confocal microscope (Zeiss) using a 40 × 1.4 NA oil objective (Plan Apochromat 40 x/1.4 oil DIC M27) and a pinhole setting of 90 μm. All lasers were used at 2 % power. Images were acquired in 8 bit mode as Z-stacks with 1024 × 1024 pixels (or 2048 × 2048 for better visualization) xy resolution through the entire thickness of the cell with optical slice thickness set for two times oversampling to allow 3D reconstruction, pixel dwell times of 0.39 to 0.79 μs and the detector gain in each channel adjusted to cover the full dynamic range but to avoid saturated pixels. Imaging conditions were held constant within an experiment.
Plan apochromat 40x 1.4 oil dic m27
Manufactured by Zeiss
The Plan-Apochromat 40x/1.4 Oil DIC M27 is a high-magnification, high-numerical aperture objective lens designed for use in microscopy. It features plan-apochromatic optical correction and a numerical aperture of 1.4, providing high-resolution, high-contrast imaging. The lens is compatible with differential interference contrast (DIC) microscopy techniques and has a M27 thread mount.
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Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Lsm 710, by Zeiss (2 mentions)
Fm4 64 dye, by Thermo Fisher Scientific (1 mentions)
Lsm 710 axioobserver confocal microscope, by Zeiss (1 mentions)
Pentr d topo vector, by Thermo Fisher Scientific (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Gatan orius 200, by Ametek (1 mentions)
Led laser, by Leica (1 mentions)
Hcx irapo l 25x 0.95w, by Leica (1 mentions)
Cm 120, by Ametek (1 mentions)
X cite 120led, by Excelitas (1 mentions)
3 protocols using plan apochromat 40x 1.4 oil dic m27
1
Confocal Microscopy Imaging Protocol
2
Transient Expression of MWL-1 and MWL-2 in Populus
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Genes encoding MWL-1 and MWL-2 were amplified from Arabidopsis Col-0 cDNA using gene-specific forward primers and modified reverse primers where the stop codon was removed (S1 Table ). The PCR products were cloned into the pENTR™/D-TOPO® vector (Invitrogen™), sequenced verified, and thereafter recombined into the transient expression vector pSAT-DEST-GFP-N1B (CD3-1654, TAIR) downstream a double 35S CaMV promoter and in-frame with a C-terminal Green Florescent Protein (GFP). Protoplast isolation and PEG-calcium transfection was carried out in Populus alba × tremula, P717 protoplasts using the method described by Guo et al. (2012) [13 (link)]. FM® 4–64 dye (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide; ThermoFisher Scientific) was used as a positive membrane marker. Florescence was detected 24-hours post-transfection using a Zeiss LSM 710 AxioObserver confocal microscope with the Plan-Apochromat 40x/1.4 Oil DIC M27. Excitation wavelength for GFP and FM® 4–64 was 488 and 515 nm, respectively, while emission was detected at 495–540 and 640 nm, respectively. Images were processed with Zen 2 lite blue edition (Zeiss).
3
Multimodal Microscopy of Plant Pollen
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Maize pollen development analyzes were performed on epifluorecence microscope Imager M2 Axio (Zeiss®), harboring a lamp LED X-Cite® 120LED (Excelitas®). Objectives X20 and X40 were used (Plan-ApoChomat, Zeiss). Confocal imaging of maize pollen marked with both mCitrine and mCherry fluorescent reporters was done on a Leica SP8 up-right confocal microscope, with a water immersion objective (HCX IRAPO L 25x/0.95 W). Fluorophores were excited using Led laser (Leica Microsystems, Wetzlar, Germany) emitting at wavelengths of 514 nm for mCitrine and 552 nm for mCherry. Images were collected at 521-550 nm for mCitrine, 610-650 nm for mCherry. Arabidopsis root cells and pollen grain observations for both Arabidopsis and maize were done on an inverted Zeiss LSM710 confocal microscope mounted on AxioImager Z2. Samples were observed using a X40 oil objective (Plan-Apochromat 40x/1.4 Oil DIC M27, Zeiss). Dual-colour images were acquired by sequential line switching, allowing the separation of channels by both excitation and emission. Depending on the fluorophores observed, different wavelengths of excitation and band pass filters were used: mCitrine, YFP, VENUS: 514 nm / 520-580 nm and DAPI: 405 nm / 410-480 nm (excitation / band pass). Immunogold labelling imaging was done with a TEM (transmission electron microscopy) Philips CM120 at 120 kV using a CCD camera Gatan Orius 200.
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