Flying membrane samples were analyzed using a Leica TCS SP2 laser scanning confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) with a 405 nm diode laser for excitation and a 10x/0.4 objective. Lambda scans were performed using a 20 nm spectral window shifted continuously from 400 nm to 750 nm through the spectrum in 40 steps, thus yielding an 8.25 nm step. The obtained lambda stacks were analyzed with Leica LCS (Leica Microsystems GmbH) software and FIJI51 (link). The calibration curve of riboflavin solutions (100, 80, and 50 μg ml−1) was analyzed in a focused drop on a cover slip under the same conditions as those of the lambda scans of the flying membranes. Linear curve fittings were computed in FIJI through the highest spectral points, yielding the linear regression equation y = 2.80777x + 4.68651, r2 > 0.999. Spectral unmixing of the natural background and fungal lesion spot spectrum were done in LCS software. The spectrally unmixed channel of the fungal lesion spot was then compared to the obtained calibration curve and the maximum and mean riboflavin concentrations were estimated. Three-dimensional lambda stacks were used for 3D reconstruction.
Tcs sp2 laser
Manufactured by Leica
Sourced in Germany
The TCS SP2 is a laser-scanning confocal microscope produced by Leica. It is designed to capture high-resolution, three-dimensional images of samples. The core function of the TCS SP2 is to provide researchers with a tool for advanced imaging and analysis of biological and materials science specimens.
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Lab products found in correlation
Vectashield, by Vector Laboratories (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Az100, by Nikon (1 mentions)
Axioskop 2 microscope, by Zeiss (1 mentions)
Dc300f camera, by Leica (1 mentions)
Dmire2 microscope, by Leica (1 mentions)
Dmire2, by Leica (1 mentions)
Dfc350fx, by Leica (1 mentions)
Axioskop2, by Leica (1 mentions)
10 protocols using tcs sp2 laser
1
Quantitative Spectral Analysis of Fungal Lesions
2
Quantifying Cellular Autophagy via Confocal Microscopy
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The cellular autophagy level was further detected by examining using confocal fluorescence microscopy. Briefly, PANC-1 and BxPC-3 cells were seeded into eight-well chamber slide. The cells were treated with ALS at 0.1 μM, 1 μM, and 5 μM for 24 hours. After the ALS treatment, the cells were washed with 1× assay buffer given in Cyto-ID® Autophagy detection kit, followed by incubation with 100 μL of microscopy dual detection reagent for 30 minutes at 37°C in the dark. After the incubation, the cells were washed with 1× assay buffer to remove detection reagent and then the cells were examined using a Leica TCS SP2 laser scanning confocal microscopy (Leica Microsystems, Wetzlar, Germany) using a standard FITC filter set for imaging the autophagic signal at wavelengths of 405/488 nm.
3
Confocal Microscopy of Fixed Samples
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Confocal imaging was performed at the Bordeaux Imaging Center at University of BordeauxII using a Leica TCS SP2 laser scanning confocal microscope with excitation lines from a 488 nm and 543 nm lasers in the sequential mode. Samples for confocal microscopy were fixed in 4% paraformaldehyde with 4% sucrose in PBS and mounted in Vectashield (Vectorlabs, USA).
4
Visualizing Chromosome Dynamics in Live Cells
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The stained HeLa cells were scanned using a Leica TCS SP2 laser scanning confocal system same as described before [6 ].
The live images of the histone 2B-GFP in HeLa cells or MCF-7 cells stained with 0.2 μM SiR-DNA to illustrate the chromosome morphologies were recorded using a Nikon A1R fully automated high-speed confocal imaging system with a time interval of 5 min over 24 h.
ImageJ, Photoshop, and NIS-elements were used for quantification, editing of the fluorescent intensities of the complex or live imaging processing where appropriate.
The live images of the histone 2B-GFP in HeLa cells or MCF-7 cells stained with 0.2 μM SiR-DNA to illustrate the chromosome morphologies were recorded using a Nikon A1R fully automated high-speed confocal imaging system with a time interval of 5 min over 24 h.
ImageJ, Photoshop, and NIS-elements were used for quantification, editing of the fluorescent intensities of the complex or live imaging processing where appropriate.
5
Fluorescence Monitoring of GFP in P. xanthii and A. nidulans
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To monitor fluorescence associated with GFP, P. xanthii colonies growing onto cotyledons were inspected in a confocal laser-scanning microscope (Leica) equipped with a TCS SP2 Laser (Ar/Kr, Gre/Ne, He/Ne; traditional phase contrast). GFP fluorescence was excited with a 488 nm laser line and detected at 515–530 nm. The analysis of putative transformants of A. nidulans for GFP fluorescence was conducted in an epifluorescence stereomicroscope AZ-100 (Nikon) with 485 and 530 nm filters.
6
Visualizing Nostoc Filament Fluorescence
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Fluorescence of Nostoc sp. PCC 7120 filaments carrying plasmid pELV75 growing on top of solidified nitrogen-free medium, was analyzed by confocal microscopy and quantified as described [32 (link)] using a Leica HCX PLAN-APO 63× 1.4 NA oil immersion objective attached to a Leica TCS SP2 laser-scanning confocal microscope. Samples were excited at 488 nm by an argon ion laser and the fluorescent emission was monitored by collection across windows of 500–538 nm (GFP) and 630–700 nm (cyanobacterial autofluorescence). Filaments were stained with Alcian blue and visualized at the microscope as described [33 (link)].
7
Localization and Expression of RIP140 in Kupffer Cells
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To identify the location and expression level of RIP140 in KCs under different circumstances, an immunofiuorescence assay was performed. After exposure to the 100 ng/ml LPS stimuli for 6 h, RIP140 observation was performed with a Leica TCS SP2 laser scanning confocal microscope, using excitation spectral laser lines at 405 nm and 594 nm, as described elsewhere [16 (link)]. DAPI was used to show the position of KC nuclei, which appeared as blue dye cell nuclei. RIP140 expression showed a distinct nuclear pattern in green.
8
Fluorescence Microscopy of A. tumefaciens-Infected T. borchii
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Sections (3 × 3 mm) of either control, untransformed T. borchii mycelia or mycelia co-cultivated with A. tumefaciens for 3 days were washed 5 times with distilled water and cefotaxime (400 μM) prior to microscopic analysis. An Axioskop 2 microscope (Carl Zeiss International, Oberkochen, Germany) equipped with a 100X oil immersion objective (Plan Neofluar), a Zeiss filter set (green excitation filter 450–490 HB) and a Zeiss AttoArc2 HB-100 W mercury lamp were used for fluorescence microscopy analysis. Images were captured with a Microcolor camera (RGB-MS-C; CRI, Boston, MA) and processed with Diffraction Micro CCD software and Adobe Photoshop (Adobe System, San Jose, CA). Typical exposure times were 30–60 ms for the SGFP and Nomarski images. Confocal laser scanning microscopy was performed with a Leica microscope equipped with a TCS SP2 Laser (Ar/Kr, Gre/Ne, He/Ne; standard phase contrast plus Normaski contrast). SGFP was excited with a 488-nm laser line and detected at 515-530 nm.
9
Confocal Microscopy for Immunostaining
10
Confocal Imaging of Brain Sections
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Confocal images of brain sections were acquired with a Leica TCS SP2 laser scanning microscope using 40x 1,25NA and 63x 1,4NA lenses. Epifluorescent and time lapse images were obtained in a Zeiss Axioskop 2 and Leica DMIRE2, equipped with a DFC350Fx camera. Adobe Photoshop (CS and Elements 15, Adobe Systems) and ImageJ (Fiji 1.51, NIH) were used for digital processing of images (linear adjustments of brightness and contrast). Statistical analysis was performed with R [42 ].
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