How to study cells - Microscopes, magnification and calibrating the eyepiece graticule

Summary

In this educational video, Mr. Ik explores the world of microscopes, focusing on their types, magnification, and calibration. He explains the difference between optical and electron microscopes, highlighting the latter's superior resolution and magnification for detailed cellular observation. The tutorial covers the magnification formula, unit conversion, and the calibration process of the eyepiece graticule using a stage micrometer. This comprehensive guide is aimed at students seeking to understand microscopes for their A-Level biology studies.

Takeaways

• 🔬 The video covers the topic of microscopes, focusing on magnification calculation, unit conversion, and calibration of the eyepiece graticule.
• 🌟 Mr. Ik discusses three key types of microscopes: optical or light microscopes, and two types of electron microscopes - transmission and scanning electron microscopes.
• 🔍 Magnification in microscopes refers to the size increase of the image compared to the actual object size, while resolution is the minimum distance between two objects that can still be viewed as separate.
• 🌈 Optical microscopes have lower resolution and magnification but allow for color images and the use of living samples.
• 🖤 Electron microscopes offer higher resolution and magnification but produce black and white images and require samples to be in a vacuum, thus not suitable for living samples.
• 📏 The formula for calculating magnification is image size equals actual size times magnification, which can be rearranged to find either value.
• 📐 Unit conversion is necessary when measuring image sizes in millimeters and actual sizes in micrometers, with millimeters converted to micrometers by multiplying by 1000.
• 📏 The eyepiece graticule is a tool for measuring the size of objects under the microscope, but it requires calibration using a stage micrometer.
• 🔧 Calibration of the eyepiece graticule involves aligning it with a stage micrometer, counting divisions, and calculating the value per division at different magnifications.
• 🔬 The video concludes with a summary of key points, including the differences between microscope types, the use of the magnification formula, and the importance of calibrating the eyepiece graticule.

Q & A

What are the three key types of microscopes mentioned in the script?

The three key types of microscopes mentioned are the optical or light microscope, and the electron microscopes, which include the transmission electron microscope and the scanning electron microscope.

What is the definition of magnification in the context of microscopes?

Magnification refers to how many times larger the image that you look at is compared to the actual size of the object.

What is the definition of resolution or resolving power in microscopy?

Resolution or resolving power is the minimum distance between two objects which they can still be viewed as separate, meaning they do not blur into looking like one single point.

Why do electron microscopes have a higher resolving power and magnification than optical microscopes?

Electron microscopes have a higher resolving power and magnification because electrons have a much shorter wavelength than light, which allows for higher detail and magnification in the images they produce.

What is the difference between a transmission electron microscope and a scanning electron microscope?

A transmission electron microscope passes a beam of electrons through a very thin specimen, creating a 2D image with varying shades of white and black based on electron absorption. A scanning electron microscope, on the other hand, scatters and reflects electrons off the surface of the specimen, creating a 3D image that provides details on texture and depth.

Why can't living samples be viewed with an electron microscope?

Living samples cannot be viewed with an electron microscope because the electron beam would be absorbed by air, and the samples need to be in a vacuum for the electrons to reach and interact with the specimen to create an image.

What is the formula used to calculate magnification from a microscope image?

The formula used to calculate magnification from a microscope image is Image Size (I) = Actual Size (A) × Magnification (M).

How do you convert millimeters to micrometers when measuring a microscope image?

To convert millimeters to micrometers, you multiply the number of millimeters by a thousand, since one millimeter is equal to one thousand micrometers.

What is an eyepiece graticule and why is it necessary to calibrate it?

An eyepiece graticule is a glass disk with a scale etched onto it, located within the eyepiece of a microscope. It is necessary to calibrate it because the value of each division on the graticule changes with different magnifications, and calibration ensures accurate measurements of the specimen.

How do you calibrate an eyepiece graticule using a stage micrometer?

To calibrate an eyepiece graticule, you place a stage micrometer on the microscope stage and align it with the eyepiece graticule. You then count how many divisions on the eyepiece graticule fit into one division on the stage micrometer, and use the known value of the stage micrometer's divisions to determine the value of each division on the eyepiece graticule at the current magnification.

Outlines

00:00

🔬 Introduction to Microscopes and Their Types

This paragraph introduces the topic of microscopes, focusing on their types and applications in cell biology. Mr. Ik begins by addressing the request for help with microscopes, particularly in magnification calculation, unit conversion, and calibration of the eyepiece graticule. The session is part of a broader study of cell methods. The three key types of microscopes are mentioned: the optical or light microscope and the electron microscopes, which include transmission and scanning types. The paragraph also covers the basic definitions of magnification and resolution, which are crucial for understanding the capabilities of microscopes. Optical microscopes are limited by the longer wavelength of light, resulting in lower resolution and magnification but allowing for color images and the use of living samples. In contrast, electron microscopes use electron beams with shorter wavelengths, providing higher resolution and magnification but only in black and white and requiring vacuum conditions, thus precluding the observation of living samples.

05:01

📊 Magnification Calculation and Unit Conversion

The second paragraph delves into the mathematical aspects of microscope usage, specifically the calculation of magnification and the conversion of units. The formula for image size in relation to actual size and magnification is introduced, and a refresher is provided on how to rearrange the formula to solve for magnification or actual size. The importance of unit conversion is emphasized, as measurements taken with a microscope are often in millimeters, which need to be converted to micrometers to match the scale of cellular structures. The process of converting millimeters to micrometers and vice versa is explained, and a worked example is provided to illustrate how to calculate the magnification of a micrograph image using a scale bar.

10:04

📏 Calibration of the Eyepiece Graticule

This paragraph explains the use and calibration of the eyepiece graticule, a tool for measuring the size of structures observed under a microscope. The eyepiece graticule is a glass disk with a scale etched onto it, used to measure objects by counting the number of graticule divisions they cover. However, because different objective lenses provide different magnifications, the graticule must be calibrated each time a new lens is used. Calibration involves using a stage micrometer, a glass slide with a known scale in micrometers. The process of aligning the graticule with the stage micrometer, counting the divisions, and calculating the value of each division at a given magnification is described. An example is given to demonstrate how to determine the actual size of a cell nucleus by counting the number of graticule divisions it covers and multiplying by the calibrated value per division.

15:06

🔍 Summary of Key Points on Microscopes

The final paragraph summarizes the key points covered in the session on microscopes. It highlights the differences between optical and electron microscopes, emphasizing the superior resolving power and magnification of electron microscopes, which allow for detailed observation of organelles. The paragraph reiterates the importance of the magnification formula and its application in determining the actual size of structures from microscope images. It also stresses the necessity of calibrating the eyepiece graticule for accurate size measurements at different magnifications. The session concludes with a reminder of the related videos available for further study and an invitation to subscribe for updates on new content.

Keywords

💡Microscope

A microscope is an instrument used to view objects that are too small to be seen by the naked eye. In the video, it is the central tool used to explore the internal structures of cells, with a focus on different types, such as optical and electron microscopes. These tools are essential in the study of cells, as they allow scientists to magnify samples and study cellular details like organelles.

💡Magnification

Magnification refers to how many times larger the image of an object appears compared to its actual size. In the video, magnification is key to understanding the clarity and size of the images produced by microscopes. It is involved in the calculation of how big a specimen appears under different types of microscopes, as explained through examples such as the image size formula.

💡Resolution

Resolution, or resolving power, is the ability of a microscope to distinguish between two closely spaced objects. In the video, resolution is discussed in terms of how well optical and electron microscopes can differentiate details within cells, like organelles. Higher resolution allows for clearer, more detailed images, which is why electron microscopes are preferred for viewing small cell structures.

💡Optical Microscope

An optical or light microscope uses visible light to magnify images of specimens. In the video, it is described as the common type of microscope used in schools, providing color images but having lower magnification and resolution compared to electron microscopes. This type is useful for observing living samples, though it cannot show organelles in great detail.

💡Electron Microscope

Electron microscopes use a beam of electrons instead of light to produce highly magnified images. The video explains that there are two types: transmission and scanning. These microscopes offer greater magnification and resolution than optical microscopes but can only show black-and-white images and require a vacuum, making them unsuitable for observing living specimens.

💡Transmission Electron Microscope (TEM)

A TEM passes electrons through a thin specimen to produce a 2D image of its internal structures. The video highlights that this microscope type is used to see details inside organelles like chloroplasts. It produces black-and-white images based on how many electrons pass through or are absorbed by different parts of the specimen.

💡Scanning Electron Microscope (SEM)

An SEM scans the surface of a specimen with electrons, producing a detailed 3D image. The video explains that this microscope is used to view surface textures and structures in cells. Unlike TEM, it does not require thin slices of specimens and gives detailed depth information through its images.

💡Eyepiece Graticule

An eyepiece graticule is a small glass disc with a scale etched onto it, placed in the eyepiece of a microscope to measure specimen sizes. The video explains that it must be calibrated using a stage micrometer at different magnifications, as its scale changes with varying lens strengths. It allows for precise measurement of cell components, like the size of a nucleus.

💡Stage Micrometer

A stage micrometer is a glass slide with a precise scale, used to calibrate the eyepiece graticule of a microscope. In the video, it is used to determine the value of each division on the eyepiece graticule by aligning it with the scale on the micrometer. This ensures accurate measurements at different magnifications.

💡Unit Conversion

Unit conversion is the process of changing measurement units to ensure they are consistent in scientific calculations. In the video, this is crucial when calculating magnification, as image sizes are often measured in millimeters while actual cell sizes are in micrometers. Understanding conversions between these units is important for accurate magnification calculations.

Highlights

Introduction to microscopes and their importance in studying cells.

Explanation of magnification calculation and its significance in microscope usage.

Conversion of units between millimeters and micrometers for accurate measurements.

Calibration of the eyepiece graticule for precise cell measurements.

Types of microscopes: optical or light microscope, and electron microscopes (transmission and scanning).

Definition of magnification and resolution in the context of microscopes.

The role of wavelength in determining the resolution of optical and electron microscopes.

Advantages of optical microscopes: color images and the ability to use living samples.

Limitations of optical microscopes in resolving small organelles and internal structures.

Electron microscopes' requirement for vacuum and the impact on sample preparation.

Detailed explanation of transmission electron microscopes and their imaging process.

Scanning electron microscopes' ability to provide 3D images and texture details.

Mathematical formula for calculating image size and its rearrangement for different measurements.

Practical example of calculating magnification using a scale bar on a micrograph.

Step-by-step guide on calibrating the eyepiece graticule using a stage micrometer.

Summary of key points for understanding microscopes and their applications in biology.

Link to the next video on self-fractionation and ultracentrifugation for further learning.

Transcripts

hello and welcome to learn a level

biology for free with mr. ik today we're

gonna be going through microscopes and

this is one of the topics that I

frequently get requests in my tutoring

to have help with going through the

magnification calculation the conversion

of units and mainly the calibration of

the eyepiece grass cue so we'll be

covering all of that in today's session

so if you want get some paper at the

ready for some of the questions and make

notes as we go so this all falls under

topic 2 within the methods of studying

cells and once we've gone through the

cells it's knowing how scientists

discovered that these are the internal

structures our cells have and this has

been worked out through using

microscopes and self fractionation and

ultra Gatien now this video we're just

going to be focusing on the types of

microscopes magnification and

calibration of the eyepiece graticule

and but for the link to the next video

on self fractionation and ultra

centrifugation that'll be at the end of

this one so you can just click the link

to that one say microscopes this is

something that you did cover at GCSE and

there are three key types of microscopes

you have the optical or light microscope

and the electron microscopes but there's

two types of electron microscope you

have a transmission or a scanning

electron microscope now the first bit of

recap to have a go at is can you still

remember from GCSE the definition of

what magnification means and resolution

or resolving power so pause the video at

this point to see can you come up with a

definition for those to write the

definition for magnification is the

magnification of the microscope is

referring to how many times larger the

image that you look at is compared to

the actual size of the object the

resolution of a microscope is the

minimum distance between two objects

which they can still be viewed as

separates so by that we mean if you are

looking at your sample through the

eyepiece under the microscope the

minimum distance in which you can still

see two different parts of the cell has

been separate rather than then they have

just blurred into look like one single

point so the resolution or the resolving

power in an optical microscope is

determined by the wavelength of light

and in an electron microscope it's the

same idea it's the wavelength of the

beam of electrons so let's have a look

at these microscopes in a bit more

detail at this point I've just split it

into the two types generically so the

optical microscope will be the type of

microscope you're familiar with this is

what schools and colleges will have

access to and in this case you have a

beam of light being released by your

lamp or sometimes it's a mirror to

reflect a plugged in lamp and that light

then gets shown up and is condensed and

that is what creates the image now you

have a much lower resolution on light

microscopes compared to electron

microscopes because the light has a

longer wavelength and that is what

determines the resolution of microscopes

it also has a lower magnification in

comparison but you can get color images

so what you're viewing you will see

those real colors and you can use living

samples in contrast the electron

microscope whether it's scanning or

transmission for both of these this time

the source is going to be missing a beam

of electrons and those will be condensed

using electromagnets and that is what is

going to create the image electrons have

a much shorter wavelength so you get a

higher resolution or resolving power

higher magnification but you can only

get black and white images and the

samples that you've you have to be in a

vacuum so completely vacuum no air and

for that reason you can't view living

sample

so a bit more about the optical

microscope we've talked about in fact it

has a lower resolution because of that

longer wavelength what that means in

terms of its application is you can't

see the inside of organelles in detail

and some of the small organelles you

can't actually see at all so I've got an

example over here where we're looking at

mitosis in root tips and you can see

that there is an outer layer you can't

actually see the difference between the

cell membrane and the cell wall we know

that this liquid in the middle must be

the cytoplasm and stained purple are the

chromosomes so we can see where the

nucleus is but that is the level of

detail you can see with the

magnification and resolving power of an

optical microscope the electron

microscopes so going back to what I was

saying the specimen has to be in a

vacuum and the reason for that is the

electron beam that is released those

electrons would be absorbed by the air

and never even reach the sample or

specimen to create the image so for that

reason has to be prepared as a vacuum

and that's why you can't view living

samples and you only get black and white

images so a bit more detail the

transmission electron microscope so

transmission means to pass through and

that is what is happening in a

transmission electron microscope that

beam of electrons some will be absorbed

by the specimen some will pass through

and that's why you get these different

shades of white and the black and the

darker is the more electrons have been

absorbed and that's how you get this

detailed image below so it's a 2d image

it's only black and white but you can

see this here is our chloroplasts inside

of a plant cell and you can see the

internal structures you can see these

thylakoid membranes you can see the

grana those stacks of the membranes as

well scanning electron microscope you

don't have to have these very very thin

samples like

with the transmission this time it's not

going to be transmitting so passing

through the specimen instead it will

scatter and reflect off the surface and

because of the different depths of the

specimen that will affect the scattering

of the electrons and that then creates

this 3d image so the scanning electron

microscope will give you details on the

image to do with the texture and the 3d

depths of either your cells or your

organelles so moving on then

magnification is one of the math skills

linked to microscopes and it's used with

optical microscope images so the formula

again straight from GCSE image size

equals actual size times magnification

now I've deliberately written it this

way rather then is the magnification

formula because I think this is the

easiest way to remember it I am so I

beam image a is the actual M is the

magnification and then once you can

remember I am you can rearrange the

formula to work out magnification or

actual size now that is a skill from

GCSE maths but if you can't remember

that I'll link up the top here just

click to see one of the GCSE videos I

have on rearranging the formula just to

recap to get your aid of a math skills

up scratch so the other thing that

you'll need to be able to do is one of

the math skills is converting units and

that's because your image size so this

is when you're going to be measuring

your microscope image which is also

called a micrograph you'll be using a

millimeter ruler so your image size will

be recorded in millimeters the actual

size of cells and organelles is much

smaller it's going to be micrometers and

in order to use this formula you have to

have both of those sizes in the same

unit so if you have recorded your image

size in millimeter then you'll need to

convert it into micrometers so you have

the same units

to go from millimeters to micrometers

you multiply however many millimeters

you have by a thousand so if you

measured two millimeters that means you

have two thousand micrometers all you

could do the conversion the other way

round if your actual size is in

micrometers you can convert your image

size into micrometers as well and in

which case if you had two thousand

micrometers to convert that back into

millimeters they'd be divided by a

thousand and you have two millimeters so

just to go through a worked example

we've got here it's a bit blurry but

I'll put in all the details you could be

asked to work out what is the

magnification of this micrograph image

and they've given you a scale bar and if

you've shown a scale bar what that means

it is the length of that bar is

representing an actual size of 50

micrometers on this image so our scale

bar is the actual size and that has 50

micrometers what you then need to do to

work out the magnification we noted need

to know the image size and you don't

need to measure any cells for this you

are measuring what is the image size of

that scale bar so you'd line up your

ruler and measure how many millimeters

long your scale bar is and in this

example it's 20 millimeters and I

measured it so now we know we'd be doing

our image size which is 20 millimeters

divided by our actual size of the scale

bar which is 50 micrometers but we need

to get those into the same units so I'm

going to convert 20 millimeters into the

micrometer units to match the scale bar

so I need to do 20 times a thousand so

that gives me 20,000 that is our image

size divided by our actual size is 50 so

our magnification of this image is 400

times magnification so finally the last

skill

is the use of an eyepiece graticule and

how you calibrate it so I'm just going

to show you this image here of the

microscope first of all just so you can

see where the eyepiece graticule is

located so this eyepiece graticule is a

glass disk which is within the eyepiece

and that glass disc has a scale

scratched or etched onto it and that is

so you can line it up on top of whatever

you're visualizing to see how many

divisions on your eyepiece graticule

does the nucleus cover for example and

that can then be used to measure the

size the actual size of the objects that

you're viewing under the microscope

rather than using the formula that we

saw on the previous slide however it's

not quite as straightforward as that

because as you're using your light

microscope you will be potentially

moving between these different objective

lenses and each lens is a different

magnification and what that means is the

divisions on this eyepiece graticule

scale will be worth different distances

depending on how magnified the image is

and that's why we have to calibrate the

eyepiece graticule each time we use it

at a new magnification so to calibrate

it you need to use a second scale which

is called a stage micrometer and this is

a glass slide looks quite like a glass

microscope sight and it's called a stage

micrometer because this is the scale

that you're place on the stage and it's

measuring distances in micrometers so

the scale on it now mine isn't quite to

scale but that scale that you have

scratched onto this piece of glass is

two millimeters long and each single

division is worth 10 micrometers now

I've only done every 10 divisions on

this that's all I could fit in on the

diagram so one division is worth 10

micro

meters so 10 of these divisions is 100

micrometers long so if we go through

step by step then how you would use the

stage micrometer and the eyepiece

graticule to calibrate the graticule

it's a step 1

you'd place your stage micrometer on the

stage look through your eyepiece and

this is what you should see you have the

eyepiece graticule scale and then line

that up directly next to your stage

micrometer scale so step 1 line them up

so they're next to each other as you can

see in this image step 2 you need to

count how many divisions on the eyepiece

graticule scale fit into one division on

your micrometer scale now you might find

that easier to work out how I'm doing in

this world example we can see here that

we have 20 divisions from the eyepiece

graticule scale fit into 10 divisions on

the stage micrometer scale so I've got

20 fitting into 10 or in other words 2

of the divisions from the eyepiece

graticule scale fit into 1 division of

the stage micrometer scale so we have a

ratio of 2 divisions to 1 so now we've

got that we can link it back to what we

said about stage micrometers on our

stage micrometer 1 division is always

worth 10 micrometers so you can use that

to then work out at this magnification

what is one division worth on the

eyepiece graticule so we said one

division on the micrometer scale is 10

micrometers we said two divisions fit

into one on the micrometer and if one

division is worth 10 but to fit in each

time is 10 divided by 2 and we know then

that on the eyepiece graticule one

division is worth 5 micrometers at the

current magnification so now you can

take out your stage micrometer

put in whatever slide you want to use to

measure the distances or size of some of

the cells organelles so I've gone back

to the slide that we saw earlier on and

in this case I'm going to measure the

distance or the length of the nucleus in

this example now you would have all of

the subdivisions I've just not shown it

in this image so where we've got 40 to

50 50 to 60 you would have an extra 10

divisions so you could see 41 42 43 and

so on so we've said that we've worked

out that our eyepiece graticule

at this magnification 1 division is

worth 5 micrometers so I'm now going to

measure to see how many divisions the

nucleus covers and I'm estimating that

is 13 divisions we've got 10 and there

may be 3 more so the nucleus is 13

divisions long 1 division is worth 15 so

multiply that by our 5 that's 1

divisions worth 5 and 13 divisions so

that means in total the nucleus actual

size this magnification we've worked out

it's 65 micrometers now it will be that

distance or length even at every

magnification the only thing that would

change is what one division on the

eyepiece graticule is worth at different

magnifications so that's it for

microscopes so just to go over again a

summary of some of the key points you

need to know you need to say the

differences between optical and electron

microscopes and the electron microscope

has a much higher resolving power and

magnification and that's why the

electron microscopes can be used to see

the details inside of organelles the

formula I am so image size equals actual

size times magnification can be used to

work out the actual size of a structure

or the magnification from your

microscope image and lastly the actual

size of structures can be

measured using the microscope if you

have an eyepiece graticule in the

eyepiece but you have to calibrate it

first with the stage micrometer it's

take into account the different

magnifications and that's it for

microscopes for a level bulging now

there are quite a lot of linking videos

to this so if you want to just go over

some of the content or the math skills

over here are linked to the rearranging

the formula as well as you've got your

eukaryotic cells and prokaryotic cell

structures and last if you aren't

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