Advanced Microscopy Methods

How are specimens for light microscopy fixed?

Fixed with chemicals that cross-link most proteins/DNA

How does Cross-Linking work in Light Microscopy?

Amino groups on adjacent molecules cross linked, stabilising protein-protein interactions - stabilising sample

How are tissues sectioned for light microscopy?

Tissue embedded in paraffin wax + cut w/ microtome

What happens after tissues are sectioned in light microscopy?

Sections mounted on slides to be stained

What does H&E Stain stand for?

Haematoxylin-Eosin

What does the H&E Stain do?

Stains nuclei and cytoplasm

What is the benefit of H&E Stains?

Shows tissue’s basic morphology in different colours

Why do Pathologists need H&E stains?

Diagnosis

How are H&E stains used in protein detection?

• Antibody used to detect a protein through Immunohistochemistry (IHC)

• H&E visualises cells where protein is detected

Specificity of Brightfield and Optical Microscopy

Limited as not molecular

Purpose of Molecular Tagging

Reveals disease progression

Purpose of Fluorescence Microscopy

Reveals locations of specific molecules w/in cells by labelling w/ dyes or antibodies

How do Fluorescent Substances work?

Absorb light of 1 wL then emit at a longer one

What is Immunofluorescence?

Fluorescent dye instead of coloured

How are antibodies used in Immunofluorescence

Specific to target antigen & fluorescent molecule revels where it is

What does Immunofluorescence do for Cellular Molecules?

Permits localisation of specific cellular molecules

What do Fluorochromes do?

Glow against dark background

What is the benefit of Fluorochromes?

Cells may be fixed/living

Why is fluorescence used?

Selective labelling, high contrast, good 3D

What are the important parameters of microscopy?

Magnification, Resolution, Contrast

What is Magnification?

Ratio of object’s image size to real

What is Resolution?

Measure of clarity of image (minimum distance of 2 distinguishable points)

What is Contrast?

Visible differences in brightness between parts of sample

What is Super Resolution Microscopy?

Allows 1 to go below diffraction limit of conventional resolution

What is Light-Sheet Microscopy?

Allows gentle, long-term timelapse imaging - keeps sample happy

What is the benefit of Confocal Microscopy?

Sharper images of 3D tissues/cells

What are the 4 main considerations for live imaging?

Sample health, spatial resolution, temporal resolution, signal:nose ratio

What is the Pyramid of Frustration

The 4 main considerations for live imaging

What is the main principle of the Pyramid of Fusion?

No single parameter can be optimised w/o compromising others