draw cells from microscope slides and photomicrographs

Microscope in Cell Studies (Cambridge 9700 – 1.1)

Learning Outcomes

By the end of this topic students will be able to:

• Identify all main parts of a compound light microscope and explain their functions.
• Prepare reliable temporary (wet‑mount) slides of plant and animal cells.
• Set the illumination and focus correctly for each magnification and record observations.
• Draw accurate, labelled sketches of cells from microscope slides and from photomicrographs.
• Calculate total magnification and determine the actual size of structures from drawings, photomicrographs and electron‑microscope images.
• Interpret photomicrographs, recognise common artefacts and relate image features to cell function.
• Apply safe handling and care procedures for microscopes, slides and mounting media.

1. Types of Microscopes Used in A‑Level Biology

2. Parts of the Compound Light Microscope

3. Magnification, Resolution and Size Calculations

3.1 Total magnification

\( \text{Total magnification} = \text{ocular magnification} \times \text{objective magnification} \)

3.2 Resolution limit

The smallest distance that can be distinguished is limited by the wavelength of light (λ) and the numerical aperture (NA) of the objective:

\( d = \dfrac{0.61\,\lambda}{\text{NA}} \)

With visible light (λ ≈ 550 nm) the practical limit is ≈ 0.2 µm.

3.3 Calibrating an ocular micrometer with a stage micrometer

1. Place the stage micrometer on the stage and focus with the low‑power objective (4 ×).
2. Switch to the ocular micrometer and note how many ocular divisions correspond to a known distance on the stage micrometer (e.g., 10 µm).
3. Calculate the calibration factor:
   1 ocular division = (known distance) ÷ (number of ocular divisions) (e.g., if 10 µm = 25 ocular divisions, then 1 ocular division = 0.40 µm).
4. Record the factor for the objective you are using; repeat for each objective if required.

3.4 Determining actual size from a hand‑drawn sketch

1. Measure the feature on the sketch with a ruler (mm).
2. Using the calibration factor from 3.3, convert the measured length to micrometres:
   Actual size (µm) = measured length (mm) × (10 000 µm ÷ 1 m) ÷ (magnification) ÷ (calibration factor)
3. Include a clearly drawn scale bar on the sketch (e.g., 10 µm) and note the conversion used.

3.5 Determining actual size from a photomicrograph (digital image)

1. Measure the feature in pixels (using image‑analysis software or the ruler tool).
2. Read the scale bar on the image: e.g., 50 µm = 200 px.
3. Conversion factor: \(1\;\text{px} = \dfrac{50\;\mu\text{m}}{200\;\text{px}} = 0.25\;\mu\text{m/px}\).
4. Actual size = measured pixels × conversion factor.

3.6 Size calculations for electron‑microscope images

• SEM: Shows a 3‑D surface view; scale bars are usually given in µm. Use the same pixel‑to‑µm conversion as for light‑microscope images.
• TEM: Provides a 2‑D projection of a thin slice; scale bars are often in nm. Convert accordingly:
  \(1\;\text{px} = \dfrac{\text{scale bar (nm)}}{\text{pixels of scale bar}}\).

4. Making Temporary (Wet‑Mount) Preparations

1. Place a clean glass slide on a flat surface.
2. Using a pipette, add a single drop (≈ 2 µL) of distilled water, saline, or an appropriate mounting medium to the centre of the slide.
3. Transfer a small fragment of the specimen (e.g., onion epidermis, cheek cell smear) onto the drop.
4. Hold a cover slip at a 30° angle and gently lower it onto the drop. Capillary action will spread the liquid and minimise air bubbles.
5. If the preparation begins to dry, add a second tiny drop of water at the edge of the cover slip and re‑centre the specimen.
6. Secure the slide with slide clips, label with specimen name, date and intended magnification.

5. Observing Cells – Practical Procedure

1. Start with the lowest‑power objective (4 ×). Use the coarse focus knob to obtain a rough focus.
2. Center the field of view with the mechanical stage controls.
3. Adjust the iris diaphragm for a bright, even illumination.
4. Switch to the next higher objective (10 ×) and use the fine focus knob to sharpen the image.
5. Repeat for the high‑power objective (40 ×). If an oil‑immersion lens (100 ×) is required:
   • Place a single drop of immersion oil on the slide directly under the objective.
   • Rotate the oil‑immersion objective into place and use only the fine focus knob.
6. Record the total magnification for each observation and note all visible structures (cell wall, membrane, nucleus, nucleolus, vacuole, chloroplasts, etc.).

6. Sketching Cells from Slides and Photomicrographs

Hand‑drawn sketches are a core assessment skill. Follow this systematic approach:

1. Set up your drawing area: Use a sharp HB pencil, a ruler and a 1 cm‑scale bar template.
2. Determine the scale:
   • Calculate the conversion factor using the total magnification or the calibrated ocular micrometer (see Section 3.3).
   • Example: 400 × total magnification, ocular micrometer calibrated at 0.40 µm per division → 1 mm on paper represents 40 µm in the specimen.
3. Draw the outline: Keep proportions accurate; use light, smooth strokes.
4. Add a scale bar: Draw a line of the appropriate length (e.g., 10 µm) and label it.
5. Label structures: Use standard abbreviations (N, CW, Cyt, Chl, Vac, etc.) and draw arrows pointing to each feature.
6. Show shading/hatching: Indicate dense regions such as the nucleolus or stacked chloroplasts.
7. Include a caption: State specimen, magnification, staining (if any) and date.

7. Interpreting Photomicrographs (and Electron Micrographs)

1. Check that a scale bar is present; note its length and unit.
2. Convert pixel measurements to real dimensions using the method in Section 3.5 (or 3.6 for SEM/TEM).
3. Identify the same structures you would label in a hand‑drawn sketch; compare to ensure consistency.
4. Look for common artefacts and record them:
   • Air bubbles, precipitated stain, torn cells, dust particles (light‑microscope).
   • Charging artefacts or sputter‑coating irregularities (SEM).
   • Sectioning knife marks, heavy metal precipitation (TEM).
5. Discuss any specialisation evident in the image (e.g., guard cells with stomatal pores, palisade vs. spongy mesophyll, mitochondrial cristae in TEM).

8. Safety and Care of the Microscope

• Never force the coarse focus knob when a high‑power objective is in use – the lens may contact the slide.
• When using oil‑immersion lenses, apply a single drop of immersion oil; clean excess oil from the lens with lens paper after use.
• Cover the microscope with a dust‑proof lid when not in use.
• Handle slides by the edges; dispose of broken glass in a designated container.
• Turn off the light source and unplug the microscope at the end of the session.
• Store prepared slides in labelled slide boxes; keep wet mounts in a humid chamber if they must be examined later.

9. Summary Checklist

• Identify all microscope parts and state their functions.
• Calculate total magnification correctly for any ocular/objective combination.
• Calibrate the ocular micrometer with a stage micrometer for each objective.
• Prepare a clean, stable wet‑mount slide; know how to rescue a moving or drying specimen.
• Adjust illumination and focus for each objective, using coarse then fine focus.
• Produce a labelled sketch with an accurate scale bar and appropriate caption.
• Calculate actual dimensions from sketches, photomicrographs and electron‑microscope images.
• Analyse a photomicrograph, recognising artefacts and relating image features to cell function.
• Apply safety and care procedures throughout.

10. Sample Assessment Questions

1. Explain why the resolution of a light microscope is limited to about 0.2 µm.
2. Calculate the total magnification when using a 10 × ocular and a 40 × objective.
3. Describe three common sources of error when preparing a wet‑mount slide and how to minimise each.
4. A photomicrograph shows a 50 µm scale bar that measures 200 pixels. If a cell measures 80 pixels, what is its actual size?
5. Using a calibrated ocular micrometer (0.01 mm per division) and a stage micrometer (10 µm per division), you count 25 ocular divisions across a nucleus. What is the actual diameter of the nucleus?
6. Identify two artefacts that may appear in a TEM image of a plant cell and explain how they could affect interpretation.
7. For an SEM image the scale bar reads 5 µm = 150 px. A stomatal pore measures 45 px. Calculate its real width.
8. State the purpose of the condenser and iris diaphragm when observing a stained animal cell.