Abstract:Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER. Keywords: cristae; image analysis; Mitochondria Endoplasmic Reticulum Contacts (MERCs); image processing; ImageJ; TEM analysis; TEM quantification; mitochondria
All Photomicrographs were taken with a Lecia DFC700 color camera (Leica Camera Inc., Allendale, NJ, USA) connected to a Nikon E800 microscope (Nikon Inc., Melville, NY, USA), using the Leica Application Suite software (Version 4.12.0, Leica Camera Inc.). The percentage of dystrophin positive cells was quantified from digitalized dystrophin immunostaining images using the ImageJ software (Version 1.48b, ). The total number of CD8+ cells/filed was quantified from five random microscopic fields (200 X magnification) using the ImageJ software (Version 1.48b).
Cytation 5 combines automated microscopy and conventional microplate detection in a configurable, upgradable platform. The microscopy module offers up to 60x magnification in fluorescence, brightfield, high contrast brightfield, color brightfield, and phase contrast to address many applications and workflows.;The multimode detection modules include filter- and monochromator-based fluorescence detection, luminescence, and UV-Vis absorbance detection. Gen5 software provides complete control over all imaging and data capture, plus powerful image and data analysis.
To quantify GFP expression, images were taken of the area with the strongest GFP fluorescence in each muscle section with a 10X objective at identical settings. The brightness of each image was measured with ImageJ software (W. Rasband, National Institutes of Health, ) on a scale from 0 (darkest) to 255 (brightest) and averaged for each group. To measure nuclear β-gal expression, images of muscle sections showing the area of strongest expression were taken with a 4X objective and nuclei that stained positive with X-Gal were counted using AnalySIS (Soft Imaging System) and ImageJ software. A macro was generated under AnalySIS that performed the following operations sequentially: 1) convert image into gray scale, 2) perform shade correction (corrects for uneven illumination caused by the microscope), 3) set threshold between 0 and 120 (selects the X-Gal-positive areas), 4) perform phase color-coding (shows the selected areas in the image), 5) binarize image, and 6) invert image. The resulting picture is a black and white image that shows the X-Gal-positive areas in black over a white background. This image was then again converted into gray scale and opened in ImageJ. Black dots with a minimum size of 15 pixels (corresponding to β-gal-positive nuclei) were counted with ImageJ and values averaged for each group.
DHD-K12 cells expressing β-gal. DHD-K12 cells were transiently transfected with a plasmid vector expressing LacZ gene. Twenty-four hours after transfection, cells were checked for expression of β-gal through the development of blue colour. Cells expressing β-gal (mark with an arrow-head) ranged between 50% and 60% without significant cell death. The images (20x) was captured using Spot RT software version 3.0 (Diagnostic Instruments, inc) using a conventional inverted microscope.
The Gel Doc system in The james & Lillian Martin Centre is A BioRad ChemiDoc XRS+ system with Image Lab v2.0 software. See guide, below, for details of its use.This instrument has both a uv transilluminator and an XcitaBlue conversion Screen, enabling one to view DNA gels stained with SybrSafe.The attached Laptop will run the machine through Image Lab software, and this includes an automated procedure for identifying lanes and the size of bands. For your convenience, I have added the BIO Line HyperLadder 1kb to the settings, and it has Biorad markers built in. If your ladder is not already present, it is very easy to create it.The log-in is the usual password used for all the other communal lab PCs and the laptop is not yet connected to the internet! Please create a folder on the desktop for your images and back them up yourself!! Happy imaging 2b1af7f3a8