# Microscopy

## Microscopy Revision

**Microscopy**

**Cells** are too small to see with the human eye, but thanks to the development of technology, we are able to view cells and even their **subcellular structures** under **microscopes**.

**Microscopes**

**Light microscopes** use light and lenses to create a magnified image of a specimen. Their development enabled scientists to view **individual cells** and their **larger subcellular structures** such as nuclei. Light microscopes have been further developed, with improved **magnification** and **resolution**, but typically have a maximum magnification of only \times 1500 and resolutions of up to 0.2\text{ μm}. This means that the amount of detail that can be seen via a light microscope is limited.

**Electron microscopes** have much **greater magnification **and **resolving power** because they use electron beams instead of light which have a much smaller wavelength. The development of the electron microscope allowed scientists to see cells in much **more detail** including the internal structures of mitochondria, chloroplasts and nuclei, and tiny structures like ribosomes and plasmids.

**Note:**

‘Resolution’ or ‘Resolving power’ is just the ability to distinguish between two points. You can think of resolution in the context of a photograph – if you enlarge the image, after a certain point you will not be able to see any more detail and it just becomes more blurry.

**Magnification**

The magnification of an image can be calculated by a simple formula:

**\text{Magnification} = \dfrac {\text{Size of the image}}{\text{Actual size of object}}**

This formula can be **rearranged** to find the size of an image or an object.

An easy way to do this is by using this **formula triangle**:

Just cover up the part of the triangle you want to find and follow the equation that is left behind.

**Example**:** A student measures an image of a cell using a ruler. It is 55\text{ mm} wide and the image has been magnified by a factor of \times 5000. What is the actual width of the cell in \text{μm}?**

Use the **formula triangle** to work out what calculation to do. Cover up the ‘actual size’ portion of the triangle and notice that what remains is image size divided by magnification.

\text{Actual size =}\dfrac{\text{Image size}}{\text{Magnification}}

**Substitute** the numbers from the question into the equation:

\text{Actual size} =\dfrac{55}{5000} =0.011\text{ mm}

To **convert the answer into μm** you need to times by 1000 because there are 1000 μm in a mm (more on this in other topics).

0.011\times 1000 = 11 \text{ μm}

**Standard Form**

Questions may also ask you to give an answer in **standard form**. This is just an easier way of writing a number when it is really big or really small and so has lots of zeros in it.

In order to convert to standard form you **move the decimal point**, left or right, until it is a number between 1 and 10. The number of places the decimal point has moved is represented by a power of 10. If the decimal point moves **left** the the power will be **positive** and if it moves to the **right **it will be **negative**.

**Example:**

0.00023 in standard form would be 2.3\times 10^{-4} as get to 2.3 the decimal point moves 4 places to the right.

46000000 in standard form would be 4.6\times 10^{7} as to get to 4.6 the decimal point moves 7 places to the left.

**Required Practical**

**Using a light microscope to draw and label a cell with a magnification scale.**

**Prepare the slide**

- Add a drop of
**water**to a clean slide. - Carefully
**extract**the cells of interest and place them on the slide, in the water – common choices are human cheek cells (animal) and onion epidermal cells (plant). - Highlight the cells using an appropriate
**stain**(Iodine for onion cells and methylene blue for cheek cells). - Finally, place a
**cover slip**over the top of the specimen.

**View slide under the light microscope**

- Carefully place the slide onto the
**stage**and clip it in place. - Select the
**objective lens**with the lowest power and therefore lowest magnification. - While looking down the
**eyepiece**, move the stage up and down using the**coarse adjustment knob**until the image becomes more focussed. - Use the
**fine-adjustment knob**to further focus the image until it is clear. - Switch to a higher powered objective lens and refocus if greater magnification is required.

**Drawing your findings**

- Biological drawings should be scientific so draw what you see down the microscope with
**clear unbroken lines**and in the**correct proportions**. - Label the different
**cell structures**with clear straight lines, add a**title**and state the**magnification**that the cell was observed under.

**Create and add a scale bar to the drawing**

- Clip an
**eyepiece graticule**to the top of the slide and select the \times 100 objective lens on the microscope. - Line the cells up and count how many fit along 1\text{ mm} on the eyepiece graticule.
- Divide 1 (\text{mm}) by the number of cells counted to find the length of one cell and add this to the drawing.

## Microscopy Example Questions

**Question 1: **Name an advantage of using an electron microscope over a light microscope.

**[1 mark]**

Electron microscopes have much **greater magnification and resolving power** so** smaller subcellular structures can be viewed in great detail and studied** e.g. ribosomes, plasmids and the internal structures of mitochondria, chloroplasts and nuclei.

**Question 2: **A specimen is 38 \text{ μm} long. It is viewed under a microscope with a magnification of \times 100. Calculate the length of the image produced in \text{mm}.

**[2 marks]**

\text{Image size} = 100 \times 38= 3800\text{ μm}

3800\text{ μm} = 3.8 \text{ mm}

**Question 3:** Describe how a student would prepare a slide of onion cells, ready to be viewed under a light microscope.

**[3 marks]**

Add a **drop of water** to a slide then **carefully extract the onion cells** and place them in the water on the slide. Then **stain the cells with iodine** to make them more visible and place a** cover slip** over the top.