Understanding Light Microscope Resolution: Key Concepts Explained

now this is where it gets a little bit confusing

what we did earlier was we talked about magnification and we said that magnification is just

basically to enlarge the specimen size and we also said earlier

that light has a wavelength of about 400 nanometers to 700 nanometers just a bit of revision 400 nanometer is

violet 700 nanometer is red light always never lose sight of that you must

remember that now comes the most i would say for this chapter at least the most confusing

concept which is the concept of resolution a lot of students especially when i'm

teaching them they'll look at a textbook definition and they'll be like yeah i don't understand that what does

that even mean now resolution in the textbook basically says the ability

to distinguish or separate between two separate points and a lot of students just basically

go okay what does that even mean uh oh another definition that you can also include in the exam

is the sharpness or detail of an image so what so let's understand or let's

explore this concept a little bit a very important thing to understand about the

resolution of light microscopes is basically this the resolution of light microscope

or i'm just going to put resolution las equals to the wavelength of the electromagnetic

wave divided by two so if they ask you to calculate the resolution

okay the resolution using violet light it basically means 400 divided by 2

equals to 200 nanometers that's a very simple calculation but it does not answer the question what

does it even mean that it has a resolution of 200 nanometers

and for resolution the smaller the number the better i'll explain why

let's imagine a situation over here and the situation is as such you are viewing a specimen under a

microscope and the specimen is as such the actual specimen

looks very uh simple it is just a two simple dots and the two simple dots over here are

500 nanometers apart we're going to ask a very simple question over here

what do we see yes this is very important okay what do we see do we see as two

separate spots or do we see it as one spot if you're using a light microscope yes

you will still see it as two separate points now why do you see it as two separate points

the reason you see it as two separate points is because light

if you remember has a maximum resolution it's going to put max os of 200 nanometers

so anything 200 nanometers and above light is able to distinguish between the two

separate spots over here now if you don't understand this let's try another example

the actual specimen is now ha this is where it becomes interesting the actual specimen is now 100

nanometers above but what do we see using in our eyepiece when we are viewing it under the

eyepiece over here we are looking at it do we see it as two separate spots or do we see it as one spot

see here's where it becomes a little odd you will only be able to see it as a single blob over here now why is that

so why don't you see two separate spots is that detailed no it's not detailed

because this is the detailed image but this one over here what you see is not detailed at all

what gives why does this happen this happens because the two spots are less than

200 nanometers apart and because they are less than 200 nanometers apart what happens

light waves are unable to resolve the image therefore in this case it is less sharp or basically less

detailed that's what that means so we have a problem problem number one light microscope over

here it's unable to visualize extremely small specimens

an example of this extremely small specimen that i told you before was the ribosome because why couldn't it

resolve the ribosome because the ribosome was too small and remember the light wavelength

it just basically the ribosome is in this case the ribosome is unable to interrupt light

so we have the first problem over here number two it's unable to resolve

any specimen smaller than 200 nanometers now a lot of students think that if you're not able to resolve

anything smaller than 200 nanometers it means you're not able to see it uh it does not mean that you might still

be able to see something like for example let's say let's say in this case you

have two specimens over here you have two specimens over here under the microscope the only issue is the two

specimens are 180 nanometers apart but the problem is when you're using the light microscope

what we might see is we might just see it as a single blob over here so it is less detailed so it's

not sharp you're not able to study the specimen in detail that's the problem with light

microscopes they they work they have some advantages by the way i'm not i'm not like you know bashing light

microscopes there is a time and place to use light microscopes definitely

however they do have their own shortcomings so we're going to be talking about

the differences in resolution between red light and violet light because

sometimes students get a little bit confused when they're comparing the resolution between

two different colors now this is a bit of reference for you what is the meaning of resolution by the way

resolution is just basically the ability to distinguish between two separate points that's what we

discussed earlier and remember red light will have a resolution of 350 nanometers

how did i get 350 nanometers if you remember red light has a wavelength of 700

nanometers and it's divided by two that is the formula so what does it mean by

a resolution of 350 nanometers this means that red light has the ability to distinguish

two separate points which are 350 nanometers and higher violet light however has a resolution of

200 nanometers and if you remember violet light has a wavelength of 400 nanometers divided by 2 and you

will get a resolution of 200 nanometers basically it means that

violet light is able to distinguish separate points of 200 nanometers and higher

and i did ask uh in an earlier question which light has a better resolving power or which light

provides better resolution and i did say that violet light is better than red light

in producing a more detailed image under the microscope how is that so let's see

here we have two circles and the circles basically show us our eyepiece of the light

microscope this is our eyepiece of the light microscope and we will be viewing a particular

image based on a natural specimen now the actual specimen is let's for

example just say there are two bacteria over here the bacteria are those orange color

circles over there okay there you can see two bacteria and for example these two bacteria uh

i'm just gonna put a distance these two bacteria are 500 nanometers apart if

we view this bacteria under the light microscope using red light will we see

two bacteria or will we see a shapeless blob we will see two bacteria over here for

the red light and we will also see two bacteria for the violet light so this is

say to be a detailed image why does this mean that this is a detailed image because

in reality there were two bacteria which are 500 nanometers apart and when we viewed it under the

microscope we are able to see the two bacteria so the image is clear now why are we able to see two bacteria

using red light or using violet light the reason is because like i said earlier

if you remember red light has a this ability red light has a ability a red light has an ability

to distinguish between points which are 350 nanometers and higher so because the bacteria is

500 nanometers apart red light has the ability to distinguish them so is violet light by the way

what if we have two bacteria viewed under the microscope and the bacteria are actually 300 nanometers apart this

is where it becomes interesting if you were viewing this specimen under red light

you will not be able to see the two bacteria instead you will see a shapeless mass

you're unable to distinguish the two separate points over here so you will not be sure of what you're

exactly looking at are you looking at one bacterium or are you looking at two bacteria in

this case reason is because the actual distance of the bacteria is

300 nanometers and red light can only resolve things which are 350 nanometers and higher

anything below 350 nanometers it like becomes a little bit useless you're still able to see something but

you're not exactly sure what you're looking at that's why we say it's less detailed however with violet

light you're still able to see it as two separate points you are able to see hey there are two bacteria over there

because their distance is 300 nanometers apart and violet light can distinguish

anything 200 nanometers apart and higher so in this case red light gives you a less detailed image

violet light gives you a more detailed image even though both of them are being

viewed on the same magnification and let's try one last experiment over here now we have two bacteria

being viewed under the microscope and the two bacteria are about 100 nanometers apart

if we were to view this bacteria under red light we will see it as one shapeless blob

and if we were to view it under violet light we will also see it as one shapeless blob

we are not exactly sure what we are looking at why is that so because the actual

distance of the two bacteria are 100 nanometer apart red light has a resolution of 350

nanometers and higher violet light has a resolution of 200 nanometers and higher

both these lights are unable to resolve anything lower than 200 nanometers and therefore

they are unable to distinguish two separate points which are 100 nanometers apart

so in this situation because the distance of the bacteria is 100 nanometers apart using light

microscope will give you a less detailed image and scientists in this case will be

concerned because they won't they will be wondering am i actually looking at

one bacterium am i looking at two bacteria what exactly am i looking at light microscope has now reached its

limit and we need a better microscope we need a better solution