Dm6000 b upright light microscope

Manufactured by Leica

The DM6000 B is an upright light microscope designed by Leica. It is a versatile instrument that can be used for a variety of microscopy techniques, including bright-field, phase-contrast, and differential interference contrast (DIC) imaging. The DM6000 B features a motorized focusing system and a high-resolution optical system to provide clear and detailed images of specimens.

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2 protocols using dm6000 b upright light microscope

Quantitative Analysis of Grafted Cell Density

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Fluorescent images were captured using a Zeiss Meta laser scanning confocal upright microscope. To provide macroscopic illustrations of GFP immunoreactivity, montages of single darkfield images captured using a ×20 objective were constructed using a Leica DM6000 B upright light microscope equipped with a motorized stage. The sections and GFP+ fiber patterns were reconstructed by tracing over the montaged images using Canvas software (ACD systems). Morphological profiles of biocytin filled cells were generated by accurately tracing over collapsed z‐stacks of the streptavidin‐555 labeled dendrites using Adobe photoshop software.
Graft volumes were estimated through extrapolation of the area of GFP measured in every 12th section according to Cavalieri's principle 26. The cellular densities of the grafts were estimated through counting of DAPI‐labeled nuclei in three separate ×40 (225µm²) fields of view for each graft in five animals. The proportion of NeuN and TBr1 expressing neurons in three of the grafts was estimated by inspection of every DAPI cell in three separate ×20 (425µm²) fields of view in each of three grafts.

Niclis J.C., Turner C., Durnall J., McDougal S., Kauhausen J.A., Leaw B., Dottori M., Parish C.L, & Thompson L.H. (2017). Long‐Distance Axonal Growth and Protracted Functional Maturation of Neurons Derived from Human Induced Pluripotent Stem Cells After Intracerebral Transplantation. Stem Cells Translational Medicine, 6(6), 1547-1556.

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Quantifying Neuronal and Glial Morphometry

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Images were captured using a Leica DM6000 B upright light microscope equipped with a motorised stage to capture whole coronal sections labelled for NeuN. Cortical and striatal area were measured at rostro-caudal levels corresponding to approximately 2.20, 1.30, 0.48, −0.30 and −1.30 mm relative to bregma. The volume of each structure was extrapolated from the cumulative area according to the method of Cavalieri [55 (link)]. Quantification of NeuN+ neurons or Iba1+ microglia were performed using a fixed field of view (20× objective) and thresholding of the photomicrographs in ImageJ to produced binarized images. For neurons, total NeuN numbers were counted via the automated function in ImageJ. For microglia, where many of the cells were overlapping, optical density was calculated as the fraction of the field of view occupied by positive signal for Iba1. Darkfield imaging was used to visualise and quantify white matter area, including the corpus callosum (CC) measured as an area and periventricular white matter bundles (PWMB) measured as the fractional contribution of white matter to the fixed field of view (20× objective).

Kagan B.J., Ermine C.M., Frausin S., Parish C.L., Nithianantharajah J, & Thompson L.H. (2021). Focal Ischemic Injury to the Early Neonatal Rat Brain Models Cognitive and Motor Deficits with Associated Histopathological Outcomes Relevant to Human Neonatal Brain Injury. International Journal of Molecular Sciences, 22(9), 4740.

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