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The sample chamber between the illumination objective and detection objective 1 is drawn in more detail on the right side of the schematic showing the position of the scattering layer and of the glass capillary containing the sample.
The high NA of the illumination objective resulted in a strong convergence of the focused beam and a corresponding short working distance of the illumination objective.
As illumination objective we used a plan apochromat 10×, NA 0.28 long working distance objective lens (Mitutoyo, Japan).
A special long working distance illumination objective (10×; NA = 0.28; working distance, 33.5 mm; Mitutoyo, Japan) met all construction conditions.
The light sheet had to be created within the working distance of 280 µm of the detection objective and was produced by a dedicated illumination objective lens with a high NA in order to achieve a thin light sheet with an axial extension of only 3 µm full-width-at-half-maximum (FWHM).
The laser is incident vertically onto a resonant mirror (RM) which is conjugated to the image plane of the illumination objective (Nikon CFI Plan Fluor 10 ×W).
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Light sheets were generated with cylindrical lenses (f=50 mm) and projected with telescopes and the illumination objectives onto the focal plane of the detection lens.
Inflorescences were further dissected under an Olympus dissecting scope, and photographs were taken using a ×3.8 Ultrapak epi-illumination objective (Posluszny et al. 1980; Charlton et al. 1989) on a Leitz Orthoplan microscope equipped with a Nikon Digital Sight 5M digital camera, as described by Bartlett et al. (2008).
The number of BrdU + cells in the RMS of this optimal section was quantified under brightfield illumination (20× objective; Zeiss 200 M Axiovert inverted microscope equipped with Axiovision 4.6 software).
First, the microvasculature of the spinal cord was observed within the green light epi-illumination (×4 objective) by intra-carotid injection of the pre-warmed fluorescent plasma marker TRITC-conjugated Dextran (1%, MW = 155,000; Sigma-Aldrich, Switzerland) in 0.9% isotonic NaCl.
Using the blue light epi-illumination (10× objective), 4 × 10 Cell Tracker™ green-labeled activated OT-I CD8+ T cells were slowly infused in 3 aliquots of 100 μL and were directly observed within the spinal cord microcirculation where they initiated contact with the inflamed spinal cord white matter endothelium.
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