DEV2011 Lecture Notes - Lecture 3: Confocal Microscopy, X-Ray Microtomography, Electron Microscope
25 May 2018
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Lecture 3 – Imaging
Macro-scale Imaging
• Ultarsound
o Originally used to detect flaws during ship building
o Standard tool for tracking development of babies
▪ Detects early signs of serious developmental abnormality →
immediate support/medical attention for child
• Micro-CT
• Optical Projection Tomography (OPT)
o Construct 3D mouse embryo models
o Method: clear tissue → passing visible light through embryo →
rendered semi-transparent by chemical treatment → capture projected
image
o Images record how much light passes through specimen
▪ Darker regions → indicate thicker or darker tissues
o Can visualise unstained biological specimen in 3D
▪ Detecting patterns of colorimetric or fluorescent staining
• Stain different tissues different colours
Micro Imaging Technologies
• Light microscopy
o Transmitted light microscopy
o Resolution – ability to distinguish two very small and closely spaced
objects as separate entities
▪ Limited by defraction and diffusion of normal light
o Diffraction limit of light limits resolution to around 200 nm
o Magnification range: ~40x to 1000x
o Light contrasts
▪ Bright field, cross polarised, dark field, phase contrast
• Confocal microscopy
o See within nucleus/cell
o Takes slice of image through a sample
o Very high contrast and resolution in X, Y, Z planes
▪ Looking on one focal plane
o Focuses image
o Different fluorescent stains used to visualise different compartments
and components of the cells
• Electron microscopy
o Resolution much higher
▪ Much shorter wavelengths (100,000x)
▪ Resolution up to 50 pm can be achieved, so structure of much
smaller objects
Histochemical Stains of Cells
• Haematoxylin – stains nucleus
• Eosin – stains cytoplasm
• Alcian blue – connective tissues
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