Transformed P. tricornutum cells were screened for GFP fluorescence using a Leica TCS SP2 confocal laser scanning microscope with an HCX PL APO 40×/1.25–0.75 Oil CS objective. Excitation of eGFP and chlorophyll fluorescence occurred at 488 nm with a 65 mW Argon laser, whereas fluorescence emission was detected at a bandwidth of 500–520 nm for eGFP and 625–720 nm for chlorophyll (plastid autofluorescence), respectively.
Hcx pl apo 40 1.25 0 75 oil cs objective
Manufactured by Leica
Sourced in Germany
The HCX PL APO 40×/1.25–0.75 Oil CS objective is a high-performance objective lens designed for microscopy applications. It features a numerical aperture range of 1.25 to 0.75 and a magnification of 40x. The lens is optimized for use with oil immersion.
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4 protocols using hcx pl apo 40 1.25 0 75 oil cs objective
1
Confocal Microscopy of GFP-Expressing P. tricornutum
2
In vivo Localization of eGFP Fusion Proteins
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To analyze the in vivo localization of each eGFP fusion protein, gene expression was induced by incubating the cells in f/2 medium containing 0.9 nM NaNO3 instead of 1.5 nM NH4Cl for 24 h in sterile reaction tubes under the conditions described above. Localization of GFP fusion proteins was performed using a Leica TCS SP2 confocal laser scanning microscope (Leica, Wetzlar, Germany) with an HCXPL APO40/1.25-0.75 Oil CS objective. Excitation of eGFP and plastid autofluorescence was performed at 488 nm using a 65-mW argon laser. For eGFP, emission was detected at a bandwidth of 500–520 nm and autofluorescence at 625–720 nm.
3
Visualizing eGFP Fusion Protein Localization
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For in vivo localization the expression of the eGFP fusion proteins was induced with 0.9 mM of NaNO3 for 2 days. The localization was visualized with a confocal laser scanning microscope Leica TCS SP2 using a HCX PL APO 40×/1.25 − 0.75 Oil CS objective. eGFP and chlorophyll fluorescence was excited at 488 nm. eGFP fluorescence was detected at a bandwidth of 500–520 nm and the chlorophyll (plastid autofluorescence) at a bandwidth of 625–720 nm, respectively. Pictures were processed with Fiji ImageJ with usage of BioFormats Importer plug-in (Linkert et al. 2010 (link); Schindelin et al. 2012 (link); Schneider et al. 2012 (link); Rueden et al. 2017 (link)).
4
Visualizing Protein Interactions in Plant Cells
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Reconstituted sYFP in BiFC assays was monitored in epidermal cells of N. benthamiana-infiltrated tissue at 72 h post-infiltration using a Leica TCS SL confocal microscope with an HCX PL APO ×40/1.25–0.75 oil CS objective. sYFP fluorescence was recorded by excitation with 488 nm argon laser line with emission being collected through a bandpass filter from 505 to 550 nm. For mRFP visualization, excitation was performed by means of a 543-nm green-neon laser line, and fluorescence emission was collected at 610 to 630 nm.
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