Chapter 2 Microscopes Flashcards
Advantage of light microscope
Disadvantage
- easily avalanche cheap, used in field, observes living organisms and dead, prepared specimens
- not best resolution
- or ,angifivation
How a compound light microscope works!
Two lenses , objective lens placed near specimen and eyepiece lens you look through
Objective lens is magnficed more by eyepiece which is normally x 10
Illumination provided by light undenwarh which goes through the image to magnrify, so thin sample needed
Resolution is the a measure of how small two poijts can be distinguished as separate entities, closers distance between two points that are seen as separate injected per
Magnification is a ratio of how much the image is compared to actual length of doeciemn
Why must we stain in cells?
IMAGES tend to have low contrast as most cells do not absorb a lot of light , and cytosol is transparent
- so stains increase contrast and allow different components to take up different amounts if stains, which thus allows you to identify species compjemtstoo
What to do before staining
Place sample on slide and aird ray
Hear did
Different ways of koh Tim get
Dry konnte I for just a solid thing
Wet mount for aquatic
Squash for samples
Smear for like blood
Resolution bad in my irises?
Due to diffraction if big wavelentgh of light, resolution is limited. Something with smaller wavelentgh like electrons will give higher resolution
Differential staining
Distinguish between two types of organisms or organelles that would otherwise be hard to identity
Gram staining an example, grap negative bacteria are thin bacteria walls, they don’t retain the crystal violte initial stains fterbeign washed with alachol and so the safaranin redye when counter stained . Whereas gram sportive are blue
This useful for seeing bacteria , as some resctto antibiotics mich different , like oenicnilm t d
What stains are postohe wmd what ensgtivr
So Crystal violated and methylene blue positive .
Pmigrosina nd Congo red negative as attracted to negative bacteria thing
Actually attrscted to negatively charged cytosol
What else is a differential staining technique
Acid fast
Magnification bs resolution
Magnification is a ratio between the image size and the size of the actual object, whereas resolution is the smallest distance that can be distinguished between two points as separate entities
This is linitte by the wavelength of your object, as the wavelength of light caudesdiffrsftion so need something with smaller so that is electron , light overlapped will happen because ti spreads and reflects from structured, and this overlap means detail is lost
Basically if your structures aew less than half the wavelength of light apart it babble to be resolved
So as a result to increases resolution a smaller wavelength msut be used , which diffracts still but the fact that the infivusl beams are much closer together than before they overlap means they can be seen without a blur
So basically smaller the resolution requires smaller wavelengths so infivusl beams can be closer together before overlapping sfterndiffracriojn
Thus electron microbe hiyash
Why call I rate?
Need to calibrate because true magnrufwruojnornthe different kensee can vary slightly from microcode to kcrospe so need to callibrste first
Use a stage micrometer and a gratifle. See how many eye liece units is = to some divisions smd make a scale , and the whole thing is one mm
Increasing mag but nitnreosituojn
Isnjitnint toofn
Electron microscopes
Higher magnification and resoltionna selection of wavelength much less used lmao
Good because we now know things which we would otherwise never know
Bad bevahse
- very expensive
- hard to prepare, a carefully controlled environment must be needed and setup long
- BEAMS CAN DAMAGE THE SAMPLE
- preparation process very complex
- and ARTEFAVTS OFTEN MADE (this is a made made introduction into the sample which appears in the image and shouldn’t be there
Two types of electron microbe
Transmission electron microscope
- sends a beam of electrons through u specimen and focussed to produce a picture, has highest khanrifiwrjom and the best resolution
- THIN SPECIMENR NEEDED AS A RESUKT
Scanning electron microspheres
- across the specimen, and reflected electrons oclledted
- produced a 3D pircture
- less resolution , but still good magnification
- gives good info about
- THIN SPECIKENT NOT NEEDED
Light vs em
Light is - not expensive - portable - sample easy to do - specimen alive - can be done in a lab - VACUUM NOT REQUIRED - natural colour sample seen and staining -
Em
- better reidltionnandmag, 3D can be produced
- has to be dead
,- hard to produce , sample , set up, expensive etcl
-VACCUJ NEEEDED
- dead
- black and whiten
-aretefwfrs often made and beam of electrons often damages beam
