The Microscope In Cell Studies

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レッスンノート

Key Microscopy Terms

Magnification is how many times larger the image is than the actual object.
Resolution is the ability to distinguish two close objects as separate.
Light microscopes are limited by low resolution and magnification.
Electron microscopes use a beam of electrons, giving a shorter wavelength and higher resolution.
Magnification does not have units.

Optical (Light) Microscopes

Use light to form images.
Maximum resolution is around 0.2 μm (200 nm).
Maximum magnification is around ×1500.
Can view the nucleus, mitochondria and chloroplasts.
Cannot view ribosomes, ER or lysosomes.
Can observe live specimens and produce colour images.

Electron Microscopes

Use a beam of electrons to form the image.
Maximum resolution is around 0.0002 μm (0.2 nm).
Maximum magnification is around ×1,500,000.
Can view smaller organelles such as ribosomes and ER.
Produce black and white images and specimens must be dead.
Two types: Transmission electron microscopes (TEMs) and Scanning electron microscopes (SEMs).

Transmission Electron Microscopes (TEMs)

Electrons pass through the specimen.
Give high-resolution, 2D images of internal structures.
Specimens must be very thin.
Cannot view live cells.
May introduce artefacts.

Scanning Electron Microscopes (SEMs)

Electrons scan the specimen surface.
Produce 3D images of external surfaces.
Have lower resolution than TEM.
Cannot view live specimens.

Comparing Microscopes

Light microscope: radiation = light; resolution ~0.2 μm; magnification up to ×1500; 2D colour image; living or dead; simple preparation; thick sample acceptable; shows nucleus, mitochondria, chloroplasts; inexpensive and common in schools.
TEM: radiation = electrons; resolution ~0.0002 μm; magnification up to ×1,500,000; 2D black and white image; dead only (due to vacuum); complex preparation, may introduce artefacts; sample must be very thin; shows internal structures and organelles; expensive and specialised.
SEM: radiation = electrons; resolution ~0.002 μm; magnification up to ×500,000; 3D black and white image; dead only (due to vacuum); complex preparation, may introduce artefacts; sample can be thick or 3D; shows surface details and external structures; expensive and specialised.

Using an Optical Microscope

Place the slide containing a tissue specimen on the microscope stage.
Turn the objective lens so that the lowest power lens is in use.
Use the coarse focus wheel to lower the lens as far down towards the stage as possible.
Adjust the fine focus to raise the lens until the specimen is in focus.
If the specimen is too small, turn the objective lens to the next-highest power and refocus.
Repeat with the highest power objective lens if necessary.

Using a Graticule and Stage Micrometer

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

Observing Starch Grains in Plant Cells

Starch is the storage polysaccharide of plants.
Sugars formed during photosynthesis are stored as starch inside starch grains.
Starch grains are commonly found in the stroma of chloroplasts, storage organs such as potato tubers, and the seeds of cereals and legumes.
Starch grains are large enough to be seen with an optical microscope but require staining to be seen easily.
Iodine (I₂) in potassium iodide (KI) solution can be used to stain starch grains.
Add a drop of potassium iodide onto the specimen before placing a coverslip over the top.
This makes the starch grains darker in colour, making them easier to see.
Iodine in potassium iodide solution has a light orange-brown colour but turns blue-black in the presence of starch.

Scientific Drawings

Biological drawings are line pictures that show specific features observed when the specimen was viewed.
Add a title and magnification.
Use plain paper and a sharp HB pencil.
Draw clear, single lines – no shading.
Make it large, proportional, and well-defined.
Use straight label lines (no arrows or crosses), on one side, drawn with a ruler.
Only draw what you see, not what you think you see.

Magnification Calculations

Magnification is the number of times larger an image appears compared to the specimen's actual size.
Light microscopes use an eyepiece lens (commonly ×10) and several objective lenses with varying magnifications.
Total magnification = eyepiece magnification × objective magnification.
Magnification (M) can be calculated if both the size of the image (I) and the actual size of the specimen (A) are known: M = I / A.
The size of cells is typically measured using the micrometre (μm) scale, with cellular structures measured in micrometers (μm) or nanometers (nm).
When doing calculations, all measurements must be in the same units; it is best to use the smallest unit of measurement shown in the question.
To convert units, multiply or divide depending if the units are increasing or decreasing.
1000 nanometers (nm) = 1 micrometre (μm); 1000 micrometres (μm) = 1 millimetre (mm); 1000 millimetres (mm) = 1 metre (m); 1 cm = 10 mm.

Cell Fractionation & Ultracentrifugation

Cell fractionation is a process used to isolate specific organelles from cells for closer study.
The process has three main stages: homogenisation, filtration, and ultracentrifugation.
Homogenisation: cells are broken up using a homogeniser (blender), breaking the plasma membrane and releasing organelles into a solution called the homogenate.
Homogenisation is carried out in a cold, isotonic, buffered solution: cold slows enzyme activity; isotonic prevents osmotic damage to organelles; buffered maintains pH to avoid protein/enzyme denaturation.
Filtration: the homogenate is filtered through a gauze to remove large debris; organelles remain in the filtered solution (the filtrate).
Ultracentrifugation: the filtrate is placed into a tube and spun in a centrifuge at increasing speeds.
Heaviest organelles form a pellet at the bottom; the rest stay suspended in the solution above the pellet, known as the supernatant.
The supernatant is re-spun at higher speeds to isolate lighter organelles.
Order of separation (heaviest to lightest): Nuclei, Chloroplasts (in plants), Mitochondria, Lysosomes, Endoplasmic reticulum, Ribosomes.
This process is repeated at increasing speeds until all the different types of organelle present are separated out.

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練習問題

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1.Which of the following best defines the term 'resolution' when used in microscopy?
Easy
- AThe ability to distinguish two close objects as separate
- BHow many times larger the image is than the actual object
- CThe maximum useful magnification of the microscope
- DThe colour of the image produced by the microscope
2.What is the maximum resolution of a light (optical) microscope?
Easy
- A0.0002 μm (0.2 nm)
- B0.002 μm (2 nm)
- C0.2 μm (200 nm)
- D2 μm (2000 nm)
3.Which of the following structures cannot be viewed using an optical (light) microscope?
Easy
- ANucleus
- BMitochondria
- CChloroplasts
- DRibosomes
4.Which type of microscope uses a beam of electrons to produce a 3D image of the surface of a specimen?
Easy
- AOptical microscope
- BTransmission electron microscope (TEM)
- CScanning electron microscope (SEM)
- DConfocal microscope
5.Which of the following is a limitation of using a transmission electron microscope (TEM)?
Easy
- AIt can only view live specimens
- BIt produces images with lower resolution than a light microscope
- CSpecimens must be very thin
- DIt cannot show internal structures
6.A student is using a microscope with a ×10 eyepiece lens and a ×40 objective lens. What is the total magnification?
Medium
- A×4
- B×50
- C×400
- D×4000
7.A cell is viewed using a light microscope. The actual diameter of the cell is 50 μm. The image of the cell is 500 μm across. What is the magnification?
Medium
- A×10
- B×100
- C×1000
- D×0.1
8.Which of the following is the correct order of organelle separation during ultracentrifugation, from heaviest (first to form a pellet) to lightest (last to form a pellet)?
Medium
- ANuclei → mitochondria → lysosomes → ribosomes
- BRibosomes → lysosomes → mitochondria → nuclei
- CNuclei → ribosomes → mitochondria → lysosomes
- DMitochondria → nuclei → ribosomes → lysosomes

もっと問題

9.Which of the following statements about cell fractionation are correct? (Select all that apply.)
Medium
- AHomogenisation breaks the plasma membrane and releases organelles into solution.
- BThe homogenate is filtered through a gauze to remove large debris.
- CUltracentrifugation separates organelles by spinning at increasing speeds.
- DThe supernatant is the pellet of heaviest organelles at the bottom of the tube.
- EA cold, isotonic, buffered solution is used to slow enzyme activity and prevent osmotic damage.
10.Which of the following are advantages of using a transmission electron microscope (TEM) compared with a light microscope? (Select all that apply.)
Medium
- AHigher resolution
- BHigher maximum magnification
- CCan view live specimens
- DProduces colour images
- ECan view smaller organelles such as ribosomes and ER
11.Which of the following are standard conventions for biological drawings? (Select all that apply.)
Medium
- AUse a sharp HB pencil and plain paper
- BDraw clear, single lines with no shading
- CUse straight label lines drawn with a ruler
- DInclude a title and magnification
- EUse arrows for label lines
12.Electron microscopes can be used to view living specimens.
Easy
True or false?
13.A light microscope can produce colour images.
Easy
True or false?
14.An eyepiece graticule has fixed units engraved on it, so it does not need to be calibrated.
Easy
True or false?
15.Iodine in potassium iodide solution turns blue-black in the presence of starch.
Easy
True or false?
16.Match each type of microscope with its correct description.
Medium
- Light microscope
- Transmission electron microscope (TEM)
- Scanning electron microscope (SEM)
- Uses light to form a 2D colour image; can view live specimens
- Passes electrons through a thin specimen to give a 2D black and white image of internal structures
- Scans electrons across the specimen surface to give a 3D black and white image of external structures
17.Match each term with its correct definition.
Medium
- Magnification
- Resolution
- Homogenisation
- Ultracentrifugation
- The number of times larger the image is than the actual object
- The ability to distinguish two close objects as separate
- Breaking cells open to release organelles into solution
- Spinning a filtrate at increasing speeds to separate organelles by density
18.Place the steps of using an optical microscope to view a specimen in the correct order.
Medium
- Place the slide on the microscope stage
- Turn the objective lens to the lowest power
- Use the coarse focus wheel to lower the lens towards the stage
- Adjust the fine focus to bring the specimen into focus
- Turn to a higher power objective lens and refocus if needed
19.Arrange the following organelles in the order they are separated during ultracentrifugation, from first (heaviest) to last (lightest).
Medium
- Nuclei
- Mitochondria
- Lysosomes
- Ribosomes
20.Which structure would be visible in this cell using the optical microscope?
Medium
- ARibosome
- BEndoplasmic reticulum
- CChloroplast
- DLysosome
21.Which organelle will form a pellet first during ultracentrifugation?
Medium
- ANuclei
- BMitochondria
- CLysosomes
- DRibosomes
22.Match each microscope with the correct radiation used to form its image.
Medium
- Optical (light) microscope
- Transmission electron microscope
- Scanning electron microscope
- Light
- Electrons passed through the specimen
- Electrons scanned across the specimen surface
23.Match each microscope with the type of image it produces.
Medium
- Light microscope
- Transmission electron microscope
- Scanning electron microscope
- 2D colour image
- 2D black and white image
- 3D black and white image
24.Which of the following statements about the resolution of microscopes are correct? (Select all that apply.)
Medium
- AResolution is the ability to distinguish two close objects as separate
- BThe maximum resolution of a light microscope is around 0.2 μm
- CElectron microscopes have higher resolution because electrons have a shorter wavelength than light
- DLonger wavelengths of radiation give higher resolution
- EThe maximum resolution of an electron microscope is around 0.0002 μm
25.Which of the following organelles can be seen using an optical (light) microscope? (Select all that apply.)
Medium
- ANucleus
- BMitochondria
- CChloroplasts
- DRibosomes
- ELysosomes
26.Which of the following are limitations of using a transmission electron microscope (TEM)? (Select all that apply.)
Medium
- ASpecimens must be very thin
- BLive cells cannot be viewed
- CImages are always 3D
- DArtefacts may be introduced during preparation
- EIt uses light to form the image
27.Place the steps of viewing a specimen with an optical microscope into the correct order.
Medium
- Place the slide on the microscope stage
- Turn the lowest power objective lens into position
- Lower the lens towards the stage using the coarse focus wheel
- Adjust the fine focus until the specimen is in focus
- Turn to the next-highest power objective lens and refocus
28.Arrange the following stages of cell fractionation into the order in which they are carried out.
Medium
- Homogenisation
- Filtration
- Ultracentrifugation
29.An eyepiece graticule has no fixed units and must be calibrated using a stage micrometer for the objective lens in use.
Easy
True or false?
30.A transmission electron microscope can be used to observe living specimens.
Easy
True or false?
31.Iodine in potassium iodide solution is light orange-brown but turns blue-black in the presence of starch.
Easy
True or false?
32.Which of the following are advantages of using a scanning electron microscope (SEM) rather than a transmission electron microscope (TEM)? (Select all that apply.)
Hard
- AIt produces a 3D image
- BIt shows surface details and external structures
- CSpecimens can be thick or 3D
- DIt has a higher resolution than a TEM
- EIt can be used to view living specimens
33.Match each term used in cell fractionation with its correct meaning.
Medium
- Homogenate
- Filtrate
- Supernatant
- The solution of released organelles produced by breaking up cells
- The solution that passes through the gauze after large debris is removed
- The liquid remaining above the pellet after centrifugation
34.A student views a specimen under a light microscope and cannot see the ribosomes or the endoplasmic reticulum. Which statement best explains why?
Medium
- AThe resolution of a light microscope is too low to separate points closer than about 200 nm.
- BThe magnification of a light microscope cannot be increased beyond ×1500, so small organelles are blurred.
- CRibosomes and endoplasmic reticulum do not absorb light, so they are invisible.
- DThe specimen was dead, so ribosomes and endoplasmic reticulum had been destroyed.
35.A photomicrograph of a cell was taken using a microscope with a resolution of 0.2 nm. The image shows internal membranes and ribosomes but no surface detail. Which type of microscope was used?
Medium
- ATransmission electron microscope
- BScanning electron microscope
- COptical microscope
- DConfocal light microscope
36.A cell is magnified ×4000. The image of its nucleus measures 8 mm across. What is the actual diameter of the nucleus in micrometres (μm)?
Medium
- A2 μm
- B0.5 μm
- C20 μm
- D2000 μm
37.A student is comparing images produced by different microscopes. Which of the following statements are correct? (Select all that apply.)
Medium
- AA TEM produces 2D black-and-white images of internal structures.
- BAn SEM produces 3D images of external surfaces.
- CAn optical microscope can produce colour images of living specimens.
- DAn SEM has a higher resolution than a TEM.
- EAn optical microscope has a higher resolution than an SEM.
38.An electron microscope can resolve structures as small as 0.2 nm because electrons have a shorter wavelength than visible light.
Easy
True or false?
39.Match each observation to the most likely microscope used.
Medium
- 3D image of the surface of a pollen grain
- 2D image of ribosomes inside a cell
- Colour image of living onion cells
- Image showing chloroplasts and nuclei only
- Scanning electron microscope
- Transmission electron microscope
- Optical microscope
40.Place the following steps of preparing a plant cell slide to view starch grains into the correct order.
Medium
- Add a drop of iodine in potassium iodide solution to the specimen
- Place the specimen on a microscope slide
- Lower a coverslip slowly at an angle
- View under the microscope
41.Complete the sentence about cell fractionation.
Easy
During homogenisation, cells are broken up in a cold, ____ solution to prevent osmotic damage to organelles.
42.Complete the sentence about centrifugation.
Easy
After centrifugation, the heaviest organelles form a pellet at the bottom, and the liquid above the pellet is called the ____.
43.Name the three main stages of cell fractionation in order.
Medium
The three main stages of cell fractionation are ____, ____, and ____.
44.An image of a cell is 30 mm in size and has been magnified by a factor of ×3000. What is the actual size of the cell in micrometres (μm)?
Medium
45.A mitochondrion is viewed under an electron microscope. The image length is 6.4 cm and the magnification is ×20,000. Calculate the actual length of the mitochondrion in micrometres (μm).
Medium
46.A chloroplast is magnified ×250. Its image length on a micrograph is 50 mm. Calculate the actual length of the chloroplast in micrometres (μm).
Medium
47.Name the stain used to make starch grains visible under an optical microscope.
Medium
48.What is the name of the solution that remains above the pellet after ultracentrifugation?
Medium
49.Calculate the magnification of the image.
Medium
50.What is the name of the liquid remaining above the pellet after the first centrifugation?
Medium
51.An image of a cell measures 30 mm and has been magnified ×3000. Calculate the actual size of the cell in micrometres (μm).
Medium
52.A mitochondrion has an image length of 6.4 cm on a micrograph taken at a magnification of ×20,000. Calculate the actual length of the mitochondrion in micrometres (μm).
Hard
53.Describe how a transmission electron microscope (TEM) uses electrons to generate an image.
Hard
54.Explain why a cold, isotonic, buffered solution is used during homogenisation in cell fractionation.
Hard
55.Give two conventions that must be followed when producing a biological drawing of a specimen viewed under a microscope.
Hard
56.A scientist homogenises liver tissue in a cold, isotonic, buffered solution before ultracentrifugation. Explain why each of these three conditions is necessary.
Hard
57.An image of a cell is 30 mm in size and has been magnified by a factor of ×3000. What is the actual size of the cell in micrometres (μm)?
Medium
- A9 μm
- B10 μm
- C12 μm
- D11 μm
58.A mitochondrion is viewed under an electron microscope. The image length is 6.4 cm and the magnification is ×20,000. Calculate the actual length of the mitochondrion in micrometres (μm).
Medium
- A2.2 μm
- B3.2 μm
- C4.2 μm
- D5.2 μm
59.A chloroplast is magnified ×250. Its image length on a micrograph is 50 mm. Calculate the actual length of the chloroplast in micrometres (μm).
Medium
- A200 μm
- B220 μm
- C240 μm
- D180 μm
60.Calculate the magnification of the image.
Medium
- A22
- B24
- C20
- D18
61.An image of a cell measures 30 mm and has been magnified ×3000. Calculate the actual size of the cell in micrometres (μm).
Medium
- A9 μm
- B11 μm
- C12 μm
- D10 μm
62.A mitochondrion has an image length of 6.4 cm on a micrograph taken at a magnification of ×20,000. Calculate the actual length of the mitochondrion in micrometres (μm).
Hard
- A5.2 μm
- B2.2 μm
- C3.2 μm
- D4.2 μm

復習ノート

## The Microscope in Cell Studies ### Key definitions - **Magnification**: how many times larger the image is than the actual object - **Resolution**: the ability to distinguish two close objects as separate ### Optical (light) microscopes - Use light to form images; max resolution ~**0.2 μm (200 nm)**; max magnification ~**×1500** - Can view **nucleus, mitochondria, chloroplasts** — **not** ribosomes, ER or lysosomes - Can observe **live specimens** and produce **colour** images ### Electron microscopes - Use a **beam of electrons** → shorter wavelength → higher resolution - Max resolution ~**0.0002 μm (0.2 nm)**; max magnification ~**×1,500,000** - Images are **black and white**; specimens must be **dead** - **TEM**: electrons **pass through** the specimen → high-resolution **2D** images of **internal** structures. Limitations: specimens must be thin, cannot view live cells, may introduce **artefacts** - **SEM**: electrons **scan the specimen surface** → **3D** images of **external** surfaces. Lower resolution than TEM; cannot view live specimens ### Measuring cells - **Graticule**: a small disc with an engraved scale placed in the eyepiece, acting as a ruler in the field of view - It has no fixed units, so it must be **calibrated** using a **stage micrometer** (a scale engraved on a microscope slide) - **Total magnification = eyepiece magnification × objective magnification** - **Magnification = image size ÷ actual size** (magnification has no units) - Convert units first: **1000 nm = 1 μm; 1000 μm = 1 mm; 1000 mm = 1 m** (and 1 cm = 10 mm) ### Observing starch grains - Starch is the **storage polysaccharide of plants**; found in the **stroma of chloroplasts**, storage organs (e.g. potato tubers) and seeds of cereals and legumes - Starch grains need **staining** to be seen easily: **iodine (I₂) in potassium iodide (KI)** — light orange-brown, turns **blue-black** with starch - Add a drop of potassium iodide to the specimen before lowering the coverslip **at an angle** to avoid air bubbles (artefacts) ### Biological drawings - Add a **title and magnification**; use plain paper and a sharp **HB pencil** - Draw **clear, single lines – no shading**; make it large, proportional and well-defined - Use **straight label lines (no arrows or crosses), on one side, drawn with a ruler** - Only draw what you see, not what you think you see ### Cell fractionation Three stages: **homogenisation, filtration, ultracentrifugation** - **Homogenisation**: cells broken up in a homogeniser, releasing organelles into the **homogenate**. Carried out in a **cold, isotonic, buffered** solution: - Cold: slows enzyme activity - Isotonic: prevents osmotic damage to organelles - Buffered: maintains pH to avoid protein/enzyme denaturation - **Filtration**: homogenate filtered through gauze to remove large debris; organelles remain in the **filtrate** - **Ultracentrifugation**: filtrate spun at increasing speeds; heaviest organelles form a **pellet**, the rest stay in the **supernatant**, which is re-spun at higher speeds - Order of separation (heaviest to lightest): **nuclei → chloroplasts (in plants) → mitochondria → lysosomes → endoplasmic reticulum → ribosomes**

Magnification: How many times larger the image is than the actual object.
Resolution: The ability to distinguish two close objects as separate.
Optical (light) microscope: Uses light to form images; maximum resolution around 0.2 μm (200 nm) and maximum magnification around ×1500; can view live specimens in colour.
Transmission electron microscope (TEM): Electrons pass through the specimen, giving high-resolution, 2D images of internal structures; specimens must be thin, live cells cannot be viewed and artefacts may be introduced.
Scanning electron microscope (SEM): Electrons scan the specimen surface, producing 3D images of external surfaces; has lower resolution than a TEM and cannot view live specimens.
Artefact: A structure seen under a microscope that is not part of the specimen, e.g. dust, air bubbles or fingerprints.
Graticule: A small disc with an engraved scale placed into the eyepiece of a microscope to act as a ruler in the field of view.
Stage micrometer: A scale engraved on a microscope slide, used to calibrate the eyepiece graticule for the objective lens in use.
Total magnification: Eyepiece magnification × objective magnification.
Magnification equation: Magnification = image size ÷ actual size (magnification has no units).
Unit conversions: 1000 nm = 1 μm; 1000 μm = 1 mm; 1000 mm = 1 m; 1 cm = 10 mm.
Starch: The storage polysaccharide of plants; sugars formed during photosynthesis are stored as starch inside starch grains.
Iodine test for starch: Iodine (I₂) in potassium iodide (KI) solution is light orange-brown but turns blue-black in the presence of starch.
Biological drawing conventions: Add a title and magnification; use plain paper and a sharp HB pencil; draw clear single lines with no shading; make it large, proportional and well-defined; use straight label lines (no arrows or crosses) on one side, drawn with a ruler.
Cell fractionation: A process used to isolate specific organelles from cells for closer study, with three stages: homogenisation, filtration and ultracentrifugation.
Homogenisation: Cells are broken up using a homogeniser, breaking the plasma membrane and releasing organelles into a solution called the homogenate.
Homogenate: The solution containing released organelles produced by homogenisation.
Isotonic: Prevents osmotic damage to organelles during homogenisation.
Filtration: The homogenate is filtered through gauze to remove large debris; organelles remain in the filtered solution (the filtrate).
Ultracentrifugation: The filtrate is spun in a centrifuge at increasing speeds; heaviest organelles form a pellet at the bottom while the rest stay suspended in the supernatant.
Supernatant: The solution above the pellet, containing the organelles that have not yet been separated; it is re-spun at higher speeds to isolate lighter organelles.
Order of separation: Heaviest to lightest: nuclei, chloroplasts (in plants), mitochondria, lysosomes, endoplasmic reticulum, ribosomes.

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