Light microscopy Flashcards
What is the smallest measurement visible to the naked eye?
0.2mm
What are the 2 steps to which a classic compound microscope magnifies?
- Objective lens produces enlarged image of object in ‘real’ image plane
- Real image magnified by ocular lens on eyepiece = produces virtual image
Define magnification
Ratio of size of image to size of object
How do you calculate the total magnification?
Power of objective x power of eyepiece
Define resolution
Clarity of image, illumination & quality of the optics
Define contrast
Contrast between lightest & darkest areas of the sample
What is the numerical aperture of a lens?
The size of the cone of light coming from each point on the specimen
Who 1st named cells?
Robert Hooke
What did Antony van Leeuwenhoek do?
improved production of lenses & observes cell types e.g. RBCs
What did Zeiss do?
Made lenses with resolution at the theoretical limits
What do all microscopes have?
Light source Stage Objectives Lens Eyepiece
What does the objective lens do?
Collect a cone of light rays to create an image
What does the condenser lens do?
Focuses cone of light rays onto each point of specimen
What are the 5 types of light microscopy?
> Bright field > Phase contrast > DIC = differential interference contrast > Fluorescence > Confocal
What do bright field microscopes produce?
Relatively poor images compared w/ Phase contrast & DIC
What do phase contrast microscopes produce?
High contrast images
- can see organelles clearly
How are phase contrast images produced?
Diffracted & un-diffracted rays give rise to change in brightness
What do DIC microscopes produce?
Images w/ depth
How does a DIC microscope work?
Uses polarised light
- -> through beam splitter prism
- -> through specimen
- -> through-combining prism
How are the different colours on DIC images produced?
1 beam diffracted by specimen
–> recombined by prism
–> interfere
= generates contrast from black to white
Beams not diffracted
–> don’t interfere when recombined
=pale grey
Briefly, how can cellular components, cell types or cell viability be detected?
- add dyes
- chromogenic enzyme substrates
- detect whether dyes have been included or excluded
Which dyes interact w/ phenolic compounds?
Histo- or cyto-chemical dyes
How can enzyme substrates be used in microscopy?
Specific chemical bind to enzymes and give rise to coloured products that can reveal sites of activity
e.g. proteases
How can living or dead cells be identified?
Dead = dye not retained
(damaged vacuolar membrane)
Alive = vacuoles take up dye
What is fluorescence?
Property of absorbing light of a particular wavelength & then emitting light of a diff colour & wavelength
What is the difference between the path light takes in bright field vs epifluorescence microscopy?
Epi:
Light comes from side
Goes through filter
Monochromatic green light reflects off mirror onto specimen
Bright field:
Single beam of light from below the specimen
What are the autofluorescence examples?
Lignin
Avenacin
anti-fungal compound in epidermis of oat roots
How do confocal microscopes work?
They focus on 1 plane of a 3D specimen at a time
What can confocal microscopes be used for?
> Looking at co-localisation
Intra-cellular studies
On thick specimens
3D studies
