Understanding Microorganisms: Types of Microscopes and Their Applications

microorganisms are way too small to be seen with the unaided eye instead we'll need to use a microscope to view them

microscope is a term that is derived from latin we can break it down into micro which means small

in scope to look at we're going to take a look at the different types of microscopes that

exist those that are ancient using those single lenses to view and magnify

organisms to those that can use electron beams to magnify them instead when it comes to

units of measurement we use the metric system in the sciences especially in the medical field and of

course the major advantage here of the metric system is that it relates to the other

unit and factors by 10 which means that when we move from one unit to the next we're usually

multiplying or dividing by a factor of 10 either by 10 100

a thousand so on so forth so if we take a look at meter which is the basic unit for length

and we try to use that in the context of measuring the size of these different organisms we

will be able to measure them in the micrometer range and some even in the nanometer

so microorganisms are measured in very small units micrometers and nanometers so

how does that compare to meters well a micrometer one micrometer equals ten to the negative six meters the

prefix micro indicates that this unit meter should be divided by 1 million

or 10 to the power 6. if we take a look at nanometers then we need to divide the meter unit by

a billion or 10 to the power 9. so in this case we're using very small units and prefixes to describe the size

of microorganisms one micrometer for bacteria

and in the nanometers for viruses we use microscopes to aid us in viewing those microorganisms

and we have different types so in general we either have simple microscopes that

use one lens or compound microscopes that use more than one

lens the simplest microscope used by van leuven hoax had only one lens it was kind of similar to a magnifying glass

but it had much better magnification then after that we had robert hook who built compound microscopes those

that have multiple lenses and then several after kept building and making those microscopes better

of course those early microscopes were of poor quality and were not used to see bacteria

it wasn't until the 1830s until better microscopes were developed by the likes of lister

and much improvements were made to these microscopes to allow for the viewing

of microscopic organisms like bacteria but our focus here are compound microscopes

that use multiple lenses to view specimen and under compound microscopes we have

several different types we have light microscopes two photon microscopes scanning acoustic

of course we then have the electron microscopes and the scan probe microscopy compound microscopes are also

known as light microscopes and we're going to focus on several examples

of this type of microscope first and then discuss the rest

so starting with light microscopy light microscopy we have several different

subtypes that use light visible light to observe specimen any kind of this type of microscope is

classified under light microscope so compound like microscopes dark field face contrast

differential interference fluorescence and confocal microscopy are all examples of light microscopes

the term compound microscopes again refer to the fact that they used two lenses

to view the organism and it's probably the one that you're most familiar with or have seen in the

lab the compound light microscopes lenses are housed in the objective area

and in the ocular piece and the setup inside is magnify once using the objective lenses

magnify another time using the ocular lens and that magnification is multiplied

so if you have here an objective that magnifies your image by four times and then the

ocular piece magnifies the image by ten times then the total magnification

needs to be multiplied four times ten the total magnification of your image would be 4dx

and that's what compound light microscopes are they use more than one lens

to magnify the image the specimen is placed on a stage that can be lowered or raised depending

on what objective we're using and the light source is

on the bottom so the light source will direct the visible light through a condenser and a

diaphragm that focuses the light into the specimen in the slide through the lens

of the objective specific lens that you're using and then through this whole area

and then the last set of lenses which are the oculars and then allows you see to see the image

through the eyepiece in a compound microscope the image that forms is magnified like i said twice so

the total magnification we'll need to consider the objective lens

times the ocular lens one term that you hear frequently when talking about microscopes is resolution

what does this mean resolution refers to the ability of the lens to distinguish two points

and you can see here with this image that as we go through the different images

we are able to see the letter more clearly this magnification

and this image has great resolution what does this mean so if you think about

let's say a highway or you're driving on a road that has two-way traffic and you're driving at night and you're

able to see the cars that are coming towards you in your direction

and then passing you as you drive on this two-way street when the car is still far

away you might see the headlights as just one big

blob of light coming towards you but as you get closer and closer to the car you can see that there are two distinct

headlights as you come closer to it and that's kind of the same idea if a microscope has better resolution

you're going to be able to differentiate different spots in your specimen as distinct

areas if it has poor resolution then you're just not going to be able to distinguish those two items as separate

items instead they're just going to look like one blurry image and

we're going to see here with different types of microscopes different magnifications and different

types are going to have different types of resolution another thing that i want to point out when it

comes to compound light microscopes is the refractive index the refractive index measures

the light bending ability of a medium you've probably seen this experiment where you have water in a clear glass

and then you put a pencil in that glass and you look at the interface where

water and air meet you try to focus on the image of the pencil you will see at that point that the

image is a little distorted as soon as you look at the image in the water it's going to look like the

pencil is towards the side not continuous and that is due to the light

moving from one medium to the other which is air into liquid or liquid into air

and in this case light bends as it moves through this these different media

and the same is going to happen when the light is moving from the light source through the air through the glass

through the air again and into the objective lens there's going to be

some bending of the light and when light bends and you're not able to capture it that's going to make

the specimen blurry when does this become a problem this becomes a problem with magnifications

that are pretty high 100x for example is the highest magnification

on a bright field compound light microscope and at that point the refractive index

is pretty big you can take a look here how much light bends when we're using the 100x

lens so the light will not be able to pass through here as efficiently and we're going to

see a blurry image so how do we fix this we add a drop of oil drop this drop of

oil is going to reduce the bending ability of the medium and in this case the light

will be able to continue to pass through the objective lens

and make the image a little bit clearer take a look here in the air when light is passing through this solid

to this air medium the refraction is great light will not be able to pass through

however adding immersion oil will allow the light to bend less and continue forward through

the objective lens allowing us to see a better image so this is the basic setup for a

compound light microscope we're going to see several different examples that have

slight variations so what i've been talking about so far is the compound light microscope that can

use bright field elimination the path of light in bright field microscopy

is just using regular visible light from the bottom so going back here this is the light source through the

specimen and then the light is collected through the objective lens

now as the light passes through the dark objects will be visible against the bright background

light will be reflected of the specimen and the light that is reflected does not enter

the objective lens so all in all characteristics of this type of microscope

is that it uses visible light as a source of elimination and the best resolution we can get is

around 0.2 micrometers and what you're going to see is a specimen

that appears against a bright background so the specimen will look dark and the surrounding will look bright

some advantages of this microscope is that it's pretty inexpensive it's easy to use

we use it to observe stained organisms in the lab however it does not allow us to view

very small specimens like viruses the next major type of microscope light

microscope is the dark field microscope this one uses a special condenser with an opaque disc

that illuminates all light in the center of the beam what does this mean

it means that light is reflected by the specimen and will enter the objective lens what we will see

is in this case a specimen that looks light and the background will look dark

so if you're using this microscope your problem this phase of the microscope is because

the microorganism that you're viewing might be

live or if you were to use a regular microscope it might look invisible because of how light

it is you cannot stain a microorganism maybe or it could be a ruined if you do stain

it so what this microscope does is it uses an

opaque disc and when i say opaque it means black dark and so we insert that disc

and we allow the light to pass the disc is going to block that light and it's not going to enter the objective

lens directly the only light that is going to be reflected is going to be reflected

from the specimen and that's going to enter the objective lens

because there's no direct background light the specimen will look light

against that black background the next example here is to show you what the opaque disc does

and how it blocks the light from entering the objective directly and instead all the light that is entering

is from the specimen itself and you can see it here this is the light source this is the

opaque disk and this is a specimen so really this is mostly dark the

background is going to be dark the light that is reflected off of the specimen is collected

through the objective next one is face contrast face contrast uses a special condenser

and it has a ring-shaped diaphragm the diaphragm's function is to allow direct light to

pass through the condenser focusing the light on the specimen and then a diffraction plate

is also used in the objective lens we have two types of light rays we have direct light rays

and reflected light rays they're both brought together to produce an image why would we use this type of microscope

this allows us to see detailed examination of the internal structure of

living specimen and you can see that with the image right here so

the main function of this microscope is to bring together two sets of light rays direct light rays and reflected or

diffracted and the image that is collected will allow us to examine

detailed inner structures so if you look at the phase contrast type of microscope setup

you can see that there are different phases between the light that is passing

through the object and the background and we superimpose the light rays producing a contrast so the light source

is here this is the diaphragm ring that i was mentioning

and in here we have different types of light that are passing

through the specimen and then the specimen will reflect certain type of light the light that is

traveling directly from the condenser lens and the light that is traveling from the

specimen are going to be out of phase and when that happens

that image that is collected will allow for viewing of internal structures

the fourth major type of microscope under the light microscope category is differential interference contrast

this is similar to phase contrast however it uses differences in refractive indices

to produce images so it uses two beams of light that are separated by prism so the

specimen will appear like it's colored because of this prism effect and what's cool about this microscope is

it allows you to see the three-dimensional image of the specimen that you're

viewing so the major idea here is that it uses two beams of light and there's a prism

in there it's going to split those light beams giving more contrast and color to the

specimen and allowing you to view this in 3d the last type of microscopy under light

category is a fluorescence microscopy and this type of a microscope uses an ultraviolet source of illumination

that will cause fluorescent compounds in the specimen to emit light so those fluorescent substances are

usually fluorescent dyes that can stain the cell especially if the cells do not naturally

fluoresce and using this microscope the dyes will give off color

when viewed under this microscope and the uses here are for rapid detection and identification of

microorganisms in clinical specimen or tissue for example those

fluorochromes those dyes they have special attraction to different organisms and

as an example one of them glows yellow when we expose it to uv light and this type of

fluorochrome is absorbed by a bacteria we call mycobacterium tuberculosis the bacteria that causes tuberculosis

and when we apply this dye to the sample especially if we suspect

that the bacteria is there um it's going to allow us to see a bright yellow

organism against a dark background another one is the organism bacillus and thrices

the causative agent for anthrax it appears apple green when we stain it with another

fluorochrome so far the different types of microscopes that we mentioned are under

the light microscopy category we're going to move to the next category which discuss confocal microscopy

confocal microscopy is similar to the last one which we just talked about the fluorescence

microscope and this one specifically also uses fluorochrome dyes

however the microscope employs the use of a single photon to illuminate a plane of specimen at a time what that

means is it's going to kind of section it and then it's going to illuminate every

section or each plane of the specimen and it will allow us to see the three-dimensional shape

especially that it also emits those fluorescent colors what would we use this for

this would be used to obtain 3d dimensional images of cells especially for biomedical

applications it allows us to view biofilms as you're seeing with this image

and we can use them to directly stain different types of bacteria that cause infection

like niceria gonorrhea the next type of microscope is a two photon microscope currently

using those microscopes is limited to very advanced research and clinical labs because of

how much they cost and a single two photon microscope usually costs between three hundred

thousand to five hundred thousand dollars because it uses those lasers to

excite the dyes that we use on the specimen for this specific type of microscope

similar to confocal microscopy the specimen is stained with those fluorochromes those fluorescent dyes

however the two-photon microscope uses two types of photons uses a long wavelength light

and the two photons that are then used will excite the dyes to emit light

this allows us to view the depth of living cells that are around

one millimeter so it actually allows us to see cells activity in

real time for example cells of our immune system have been observed using this microscope

as they respond to an antigen the next category are the super resolution light microscopes

and those microscopes use two laser lights to illuminate one nanometer at a time it also uses

fluorescent molecules to glow the specimen and the second wavelength is going to cancel all the fluorescence

except for that one nanometer that it's supposed to illuminate

the major purpose of this type of microscopy after putting all the images together

is to observe the location of the different molecules inside the cell next up we have the

scanning acoustic microscopes also called sam this basically consists of interpreting the action of sound

waves that are sent through the specimen so instead of actually sending light or photons

sound waves are sent through they have specific frequency they travel through the specimen

and some of it is reflected and when it hits the interface within the material then

all of that is collected and translated into an image using a computer the this type of microscope is used to

study living cells that are attached to a surface especially cancer cells or artery

plaques and even bacterial biofilm another category of microscopes are electron

microscopes these types of microscopes use electron beams

instead of visible light to view the specimen especially objects that are smaller than

0.2 micrometers like viruses and internal structures of cells all of that can be viewed using this

microscope and these free electrons travel in waves are collected by the use of a magnet

so if you take a look at the internal setup of an electron microscope and compare it to a light microscope

you'll notice that these electron microscopes use magnets to focus the electron beams

very similar to how light microscopes use lenses to focus light it's important to

note that there are two main types of electron microscopes transmission electron microscopes

and scanning there are differences between the two in the electron microscope specifically

the transmission one the beam of electrons is focused using an electron gun and it will

pass through the specimen that is prepared using a very ultra thin section

of that specimen the beam is then focused on a small area of the specimen through a condenser lens

directing the electron beams in a straight line to illuminate the specimen

so you can see the electron gun here the condenser we have a second condenser we have the specimen and then the

electrons are focused by an electromagnetic projector lens onto

a screen final image that we call a micrograph will appear as many light and dark areas

depending on how many electrons are absorbed by the different areas of the specimen

the transmission electron microscope has very high resolution it allows us to see the

organism or the specimen in a 2d manner so if you're looking for that information you'll find it on this slide

and in order to use the transmission electron microscope and view the specimen the specimen has to be stained

with metal salts and the magnification is between 10 000 to 10 million

for the scanning electron microscope it uses this beam of electrons to scan the surface of the entire specimen

the scanning electron microscope will use a beam of electrons that is produced from an electron gun uh

it will use this electromagnetic lens to direct the electron through the surface of the

specimen and the the image that is produced will be viewed on a screen

now what's uh special about the scanning electron microscope is it allows you to see the 3d

structure of the organism because it's only going to direct those electrons to the surface and it

allows the magnification to be around 1000 to 500 000. so the the main purpose of the scanning

is to study the surface features of cells and viruses the transmission electron microscope

allows you to see the inside and will need to use very thin slices so you can compare the two

images that you can see so for example this is the image of a cell in a biofilm you can see

some internal structures because of how the transmission electron microscope works

however the scanning only scans the surface so you can see the 3d structure of staphylococcus aureus

in this image the last category of microscopes that i want to discuss are the scanning probe microscopes since

the early 1980s several of these microscopes have been developed

they use probes to examine the surface of a specimen using electric currents they don't

modify the organism or expose it to any damaging radiation

these microscopes can get very close to the molecular shapes inside the cell it can characterize our

chemical properties it can determine temperature in different areas of the cell and we

have two major kinds we have the the tunneling microscope and the atomic force

microscope the tunneling one specifically uses a tungsten probe that scans

the specimen and produces an image that reveals the bumps and the depressions of the atoms on the surface

and basically giving us great details about its surface the resolution here is very powerful

it's even greater than that of an electron microscope it can resolve features that are about 1

over 100 the size of an atom take a look here this is an image produced by the scanning tunneling

microscope of pure gold surface we're looking at the atoms of gold

and how they are arranged this image is of an atomic force microscope

the atomic force microscope uses a metal and diamond probe that is gently forced down onto the

specimen and it moves along the surface and the movements are recorded we can see the 3d

image that is produced this image here shows long stranded like molecules

of cellulose that is derived from from plant fibers all of these different types of microscopes that we

talked about can be used in microscopy we've discussed them in the order of increasing

resolution our most used microscopes are the light microscopes especially in science labs

and the rest are for more clinical purposes and are more expensive