### Microscope Equipment

- Many biological structures are
**too small**to be seen by the**naked eye** **Optical microscopes**are an invaluable tool for scientists as they allow for**tissues**,**cells**and**organelles**to be seen and studied- Light is directed through a thin layer of
**biological material**(containing the tissue(s), cell(s) or organelle(s) to be observed) that is supported on a**glass slide** - This light is focused through several
**lenses**so that an image is visible through the eyepiece

#### Apparatus

- The key components of an optical microscope you will need to use are:
- The eyepiece lens
- The objective lenses
- The stage
- The light source
- The coarse and fine focus

- Other apparatus used:
- Forceps
- Scissors
- Scalpel
- Coverslip
- Slides
- Pipette

*The components of an optical microscope*### Calibration, Sample Prep & Setup

#### Preparing specimens & samples

**Specimens**must be**prepared**on a**microscope slide**to be observed under a light microscope- This must be done
**carefully**to avoid**damaging**the biological specimen and the structures within it - The most common specimens to observe under a light microscope are
**cheek cells**(animal cells) and**onion cells**(plant cells) - Preparing a slide using a
**liquid specimen**:- Add a few drops of the sample to the slide using a
**pipette** - Cover the liquid/smear with a coverslip and gently press down to
**remove air bubbles** **Wear gloves**to ensure there is no cross-contamination of foreign cells

- Add a few drops of the sample to the slide using a
- Preparing a slide using a
**solid specimen**:- Use scissors to cut a small sample of the tissue
- Peel away or cut a
**very thin layer**of cells from the tissue sample to be placed on the slide (using a scalpel or forceps) - Some tissue samples need to be treated with chemicals to kill/make the tissue rigid
- Gently place a coverslip on top and press down to
**remove any air bubbles** - A
**stain**may be required to make the structures visible depending on the type of tissue being examined.- Commonly used stains include
**methylene blue**to stain**cheek cells**and**iodine**to stain**onion cells**

- Commonly used stains include
- Take care when using sharp objects and wear gloves to prevent the stain from dying your skin

- Preventing the dehydration of tissue:
- The thin layers of material placed on slides can
**dry up rapidly** - Adding a drop of water to the specimen (beneath the coverslip) can prevent the cells from being damaged by dehydration

- The thin layers of material placed on slides can

*Care must be taken to avoid smudging the glass slide or trapping air bubbles under the coverslip*#### Viewing the specimen

- When using an optical microscope always
**start with the low power objective lens**:- It is
**easier to find**what you are looking for in the field of view - This helps to
**prevent damage**to the lens or coverslip in case the stage has been raised too high

- It is
- Unclear or blurry images:
- Switch to the lower power objective lens and try using the
**coarse focus**to get a clearer image - Consider whether the specimen sample is
**thin enough**for light to pass through to see the structures clearly - There could be
**cross-contamination**with foreign cells or bodies

- Switch to the lower power objective lens and try using the

#### A calibrated graticule must be used to take measurements of cells

- A
**graticule**is a small disc that has an engraved**scale.**It can be placed into the eyepiece of a microscope to act as a ruler in the field of view - As a graticule has no fixed units it must be
**calibrated**for the objective lens that is in use. This is done by using a scale engraved on a microscope slide (**a stage micrometer**) - By using the two scales together the number of micrometers each graticule unit is worth can be worked out
- After this is known the graticule can be used as a
**ruler**in the field of view

*The stage micrometer scale is used to find out how many micrometers each graticule unit represents*#### Limitations of microscopy

- The size of cells or structures of tissues may appear inconsistent in different specimen slides
- Cell structures are
**3D**and the different tissue samples will have been**cut at different planes**resulting in inconsistencies when viewed on a**2D slide**

- Cell structures are
- Optical microscopes do not have the same magnification power as other types of microscopes and so there are some structures that cannot be seen
- The treatment of specimens when preparing slides could alter the structure of cells

#### Using units in microscopy

- You may be given a question in your Biology exam where the measurements for a magnification calculation have
**different units** - You need to ensure that you
**convert them both into the same unit**before proceeding with the calculation (usually to calculate the magnification) - Remember the following to help you convert between mm (millimetres), µm (micrometres) and nm (nanometres):

**Converting between mm (millimetres), µm (micrometres) and nm (nanometres)**- If you are given a question with
**two different units**in it, make sure you make a conversion so that**both**measurements have the**same**unit before doing your calculation

### Calculation of Magnification

#### Higher Tier Only

- Magnification is calculated using the following equation:

**Magnification = Drawing size ÷ Actual size**

- One way to remember the equation is using an
**equation triangle**:

**An equation triangle for calculating magnification**

- Rearranging the equation to find things other than the magnification becomes easy when you remember the triangle –
**whatever you are trying to find, place your finger over it and whatever is left is what you do**, so:- Magnification = image size ÷ actual size
- Actual size = image size ÷ magnification
- Image size = actual size × magnification

- Remember magnification
**does not have any units**and is just written as ‘X 10’ or ‘X 5000’

#### Worked Example

An **image** of an animal cell is 30 mm in size and it has been **magnified** by a factor of X 3000. What is the **actual** size of the cell?

To find the **actual** size of the cell:

**Worked example using the equation triangle for magnification**

- You may also be asked to calculate the
**total magnification**of a light microscope if given the magnification of the**eyepiece****lens**and the magnification of the**objective lens** - As these are two separate parts of a light microscope, each with its own magnifying power, you can simply
**multiply the two values**to calculate the total magnification:

**Magnification of light microscope = Magnification of eyepiece lens × Magnification of objective lens**

#### Standard form

- When doing calculations and unit conversions, it is common to come across
**very big**or**very small**numbers - Standard form can be useful when working with these numbers
- Standard form is a way of writing very big and very small numbers using
**powers of 10**

#### How to use standard form

- Using standard form, numbers are always written as follows:
**a × 10**^{n} - The rules:
**1 ≤ a < 10**(the number 'a' must always be between 1 and 10)**n > 0**for LARGE numbers ('n' = how many times 'a' is multiplied by 10)**n < 0**for SMALL numbers ('n' = how many times 'a' is divided by 10)

#### Using standard form to convert between units

- For example, you can write
**1 metre**in**millimetres**using standard form:- 1 m = 1000 mm
- So, 1 m = 1 mm × 1000
- So, 1 m = 1 mm × 10 × 10 × 10
- So, as we had to
**multiply**1 mm by 10**three times**to get 1 m, we write this as: - 1 m = 1
**× 10**mm^{3 }

- Writing
**1 millimetre**in**metres**using standard form is also possible and is just the**opposite**:- 1 mm = 0.001 m
- So, 1 mm = 1 m ÷ 1000
- So, 1 mm = 1 m ÷ 10 ÷ 10 ÷ 10
- So, as we had to
**divide**1 m by 10**three times**to get 1 mm, we write this as: - 1 mm = 1
**× 10**m^{-3 }

- Exactly the same process can be used if you needed to convert
**micrometres**into**millimetres**. For example:- 1 µm = 0.001 mm
- So, 1 µm = 1 mm ÷ 1000
- So, 1 µm = 1 mm ÷ 10 ÷ 10 ÷ 10
- So, as we had to
**divide**1 mm by 10**three times**to get 1 µm, we write this as: - 1 µm = 1
**× 10**mm^{-3 }

#### Examples of using standard form in conversion calculations

- You could be asked to state 45 centimetres in millimetres using standard form:
- 1 cm = 10 mm
- So, 45 cm = 450 mm
- So, 45 cm = 4.5 mm × 10 × 10
- So, as we had to
**multiply**4.5 mm by 10**two times**to get 45 cm, we write this as: - 45 cm = 4.5
**× 10**mm^{2 }

- You could also be asked to state 250 micrometres in millimetres using standard form:
- 1 µm = 0.001 mm
- So, 250 µm = 0.25 mm
- So, 25 µm = 2.5 mm ÷ 10
- So, as we had to
**divide**2.5 mm by 10 just once to get 250 µm, we write this as: - 250 µm = 2.5
**× 10**mm^{-1 }

#### Worked Example

**Step One: Convert units**

Remember that 1 mm = 1000 µm

So to get from µm to mm you need to divide by 1000

**Step Two: Calculate the thickness of the leaf in mm**

2000 ÷ 1000 = 2, so the actual thickness of the leaf is 2 mm and the drawing thickness is 50 mm

**Step Three: Put these values into the equation for calculating magnification **

Magnification = image size ÷ actual size

= 50 ÷ 2

= 25

= 50 ÷ 2

= 25

So the magnification is

**x 25**#### Exam Tip

It is easy to make silly mistakes with magnification calculations. To ensure you do not lose marks in the exam:

**Always look at the units**that have been given in the question – if you are asked to measure something, most often you will be expected to measure it in millimetres NOT in centimetres – double-check the question to see!**Learn the equation triangle**for magnification and always write it down when you are doing a calculation – examiners like to see this!