Sp5 inverted confocal scanning microscope

Manufactured by Leica

The Leica SP5 inverted confocal scanning microscope is a laboratory equipment designed for high-resolution imaging of biological samples. The core function of the SP5 is to provide optical sectioning and three-dimensional reconstruction of specimens through the use of confocal scanning technology.

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2 protocols using sp5 inverted confocal scanning microscope

Confocal Imaging of Insect Leg Neuroanatomy

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Specimens previously preserved and cleared in methyl salicylate (as above) were returned to 100% ethanol for 2 × 30 min washes, and then transferred to 0.05% eosin-Y in 100% ethanol for 45 min. They were de-stained in two washes of 100% ethanol (50 and 30 min) and transferred to 100% methyl salicylate. Z-stacks of 2 μm thick optical sections were obtained with a Leica SP5 inverted confocal scanning microscope, using a 10x objective with the laser excitation at λ = 514 nm and emission at λ = 540–650 nm. Stacked images were successfully obtained from fore- and midlegs and were processed with Google Picasa to produce videos to allow scanning and analysis of internal tibial structure from posterior to anterior sides. The eosin provided general staining of background soft tissue as well as strong staining of neuronal somata and dendrites. To reconstruct 3D models of the confocal scans, the Z-stacks were imported and rendered in AVISO (described above) and Drishti (Drishti v 2.6.1,The Australian National University, Canberra, Australia) using a 2D transfer function.

Strauß J., Lomas K, & Field L.H. (2017). The complex tibial organ of the New Zealand ground weta: sensory adaptations for vibrational signal detection. Scientific Reports, 7, 2031.

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Confocal Imaging of Bacteria-Neutrophil Interactions

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A Leica SP5 inverted confocal scanning microscope was utilized for all in vitro imaging. GFP-tagged bacteria and neutrophils were excited with the 488 nm and 561 nm laser lines, respectively. A LiveCell (Pathology Devices, CA, USA) environmental chamber system was utilized to maintain 5% CO₂, 20% O₂, 50% humidity, and 37°C for sample incubation duri ng imaging. Image stacks with 1-μm z-slices were recorded sequentially at 1–2 min intervals over a 4-h time course using a 20X objective lens. At least two fields of view from each chamber of the dish were generally imaged per experiment. Each well was then imaged using a 7 × 7 stitched tile scan image with a 10x objective to quantify total amount of bacteria and count neutrophils remaining on the surface.

Pettygrove B.A., Kratofil R.M., Alhede M., Jensen P.Ø., Newton M., Qvortrup K., Pallister K.B., Bjarnsholt T., Kubes P., Voyich J.M, & Stewart P.S. (2021). Delayed Neutrophil Recruitment Allows Nascent Staphylococcus aureus Biofilm Formation and Immune Evasion. Biomaterials, 275, 120775.

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