Chapter 1: Microscopy Flashcards
What is used to produce ultra-thin slices of specimen to observe under a microscope? what are these clices called?
A microtome.
Sections.
What was the theory of spontaneous generation? Why was it proposed and believed?
That life could be produced from non living matter. Proposed from the existence of life in seemingly sterile conditions.
How was spontaneous generation disproved?
Louis Pasteur’s swan-neck experiment, demonstrated how bacteria only grew in sterile conditions after being exposed to air (which contained bacteria)
how do light microscopes work?
light from a mirror is reflected through a specimen into the objective lens, which magnifies it. hen it is further magnified by the eyepiece lens
advantage of compound microscopes over simple ones?
reduced chromatic aberration/distortion
advantages of light microscopes?
- can observe living specimens
- no harsh chemicals involved
- easy to set up
- inexpensive
- can see in colour or if using a stain
- vacuum not required
- sample preparation doesn’t lead to distortion
disadvantages of light microscopes?
- low magnification
- low resolution
- cannot see ribosomes
magnification/resolution of LM
M = 2000 R = 200 nm
how does a laser scanning confocal microscope work?
a single spot of laser light of high energy illuminates chemical dyes on a specimen, causing them to fluoresce.
what is fluorescence?
absorption and re-radiation of light to a longer wavelength and lower energy / thus fluoresce
advantages of LSCM
- high depth selectivity and so can veiw whole cells
- can see living specimens
can track molecules - high secolution than LM
disadvantages of LSCM?
- more expensive
- more complex
how can we get 3D images from LSCM?
by using light from more than one focal point
why is a laser used instead of light in LSCM?
higher intensities, improves illumination, and higher energy, so light re-emmitted is fluorescent
define the term relsolution (microscopy)
the ability of a microscope to distinguish between two points on a specimen / the smallest interval measurable by a microscope
define magnification
how much an image can be magnified from the original
uses of LSCM?
- used to observe fungal filaments with in the cornea
- new drugs
- virtual biopsies
how does a TEM work?
- beam of electrons fired from cathode and focused using magnets
- ransmitted through a specimen and focused
- form image
advantages of TEM?
- can see details inside cells
disadvantages of TEM?
- general EM dis
magnification/resolution fo TEM
M = 500,000 R = 0.5 nm
how does a SEM work?
- beam of electrons fired form cathode and focused by magnets
- electrons sent across surface of specimen
- secondary electrons reflected and collected
advantages of SEM
- 3D images produced
disadvantages of SEM
- specimen must be dehydrated and metallic salt stains placed
- sample preparation causes distortion
- lower resolution
magnification/resolution of SEM
M = 200,000 R = 3-10 nm
general disadvantages of EM ?
- grayscale images, digital false colour can be added
- generally dead
- expensive
- complex, need skill/training
- vacuum required
- ## sample preparation may cause distortion of image
magnification equaiton?
M=I/A
purpose of differential staining?
- different organelles absorb different amounts of stain
- thus we can differentiate between them
all purpose stain (stains all)
methylene blue
stain that binds to DNA and stains chromosomes what colour
- acetic orcein
- dark red
stains cytoplasm?
- eosin
stains lipids?
- sudan red
stains cellulose in plant walls WHAT COLOUR and starch granules WHAT COLOUR
- iodine in potassium iodide
- culluose walls yellow
- starch granules blue/black: violet under microscope
what is done by experts to specimens before use in labs?
- specimens dehydrated
- embedded in wax to prevent distortion during slicing
- sliced using microtomes (knives) to make VERY THIN slices called SECTIONS
- fixing using formaldehyde to stop specimens in near natural state
how to you use/calibrate an eyepeice graticule using a stage micrometer?
- clip in stage micrometer
- place EPG into eyepeice lens
- align, measure from 2 intervals
- MATHSSSS
types of mounts?
types of slides?
- wet mount
- temporary mount
- dry mount
+ smear slide
+ squash slide
how is each mount/slide prepared?
- dry: specimen sliced using microtome
- wet: specimen immersed in liquid, cover slip at 45
- temporary mount: in liquid evaporates, cover slip
- squash: soft specimen, wet mount squashed using cover slip
- smear: edge of cover slip used to smear, dropped at 45
