In an optical microscope experiment, objective and eyepiece lenses are combined to magnify and observe cells and tissues that are difficult to see with the naked eye.
During observation, it is important to understand not only total magnification but also field diameter, depth of field, resolution, calibration of the eyepiece micrometer, and the actual size of the specimen.
This article assumes an experiment in which onion epidermal cells are observed and their size is measured using an eyepiece micrometer and a stage micrometer. It is organized in the order of introduction, precautions, items to record in the results, sample experimental values, calculations, sources of error, an example results section, and discussion examples.
Note:
This article is a reference for learning purposes. The experimental conditions and measured values shown are illustrative examples and are not actual measurement results.
When carrying a microscope, hold the pillar or arm with one hand and support the base with the other. When using a high-power objective lens, take care not to let the tip of the lens touch the prepared slide.
Clean lenses with lens paper, and handle staining solutions according to the laboratory manual and the instructions of the instructor.
- Basics of the Optical Microscope
- Field Diameter and Magnification
- Resolution
- Calibrating the Eyepiece Micrometer
- Main Items to Record in the Results
- Sample Experimental Conditions
- Sample Experimental Values
- Calculation Methods
- Main Sources of Error
- Example of How to Write the Results
- Discussion Example
- Summary
Basics of the Optical Microscope
In an optical microscope, light passing through the specimen is magnified by the objective lens and then magnified further by the eyepiece lens for observation.
Total magnification is calculated as the product of the eyepiece magnification and objective magnification.
Total magnification = Eyepiece magnification × Objective magnification
Field Diameter and Magnification
When the same eyepiece is used, the field diameter is approximately inversely proportional to the objective magnification.
D1M1 = D2M2
Resolution
Resolution is the ability to distinguish two closely spaced points as separate. Simply increasing magnification does not reveal details beyond the resolving power of the microscope.
d ≈ 0.61λ/NA
Calibrating the Eyepiece Micrometer
The divisions on an eyepiece micrometer do not have a length unit by themselves. They are superimposed on a known length of a stage micrometer to determine the length represented by one eyepiece division for each objective lens.
Length per eyepiece division = Length on stage micrometer ÷ Number of matching eyepiece divisions
Main Items to Record in the Results
- Type of microscope
- Specimen observed
- Whether staining was used and the staining solution
- Eyepiece magnification
- Objective magnification
- Total magnification
- Field diameter
- Number of eyepiece micrometer divisions
- Corresponding distance on the stage micrometer
- Length per eyepiece division
- Long and short dimensions of the specimen
- Number of specimens measured
- Mean value
- Structures observed
- Orientation of the image
- Brightness and contrast
- Sketch or captured image
Sample Experimental Conditions
| Item | Sample condition |
|---|---|
| Observation instrument | Bright-field optical microscope |
| Eyepiece lens | 10× |
| Objective lenses | 4×, 10×, 40× |
| Specimen | Onion epidermal cells |
| Staining solution | Iodine solution or aceto-orcein |
| Stage micrometer | 1 mm divided into 100 divisions; 10 µm per division |
| Number of cells measured | 10 |
Sample Experimental Values
Magnification and Field Diameter
| Eyepiece magnification | Objective magnification | Total magnification | Field diameter |
|---|---|---|---|
| 10× | 4× | 40× | 4.50 mm |
| 10× | 10× | 100× | 1.80 mm |
| 10× | 40× | 400× | 0.45 mm |
Example Eyepiece Micrometer Calibration
| Objective magnification | Eyepiece divisions | Corresponding distance | One eyepiece division |
|---|---|---|---|
| 4× | 25 divisions | 1.00 mm | 40 µm |
| 10× | 50 divisions | 1.00 mm | 20 µm |
| 40× | 100 divisions | 0.50 mm | 5.0 µm |
Example Measurements of Onion Epidermal Cells
| Cell No. | Long-axis divisions | Long dimension | Short-axis divisions | Short dimension |
|---|---|---|---|---|
| 1 | 48 | 240 µm | 14 | 70 µm |
| 2 | 52 | 260 µm | 15 | 75 µm |
| 3 | 50 | 250 µm | 13 | 65 µm |
| 4 | 46 | 230 µm | 14 | 70 µm |
| 5 | 54 | 270 µm | 16 | 80 µm |
| 6 | 49 | 245 µm | 15 | 75 µm |
| 7 | 51 | 255 µm | 14 | 70 µm |
| 8 | 47 | 235 µm | 13 | 65 µm |
| 9 | 53 | 265 µm | 15 | 75 µm |
| 10 | 50 | 250 µm | 14 | 70 µm |
Sample Observed Structures
| Structure | Observation |
|---|---|
| Cell wall | Clearly observed along the outer boundary of the cells |
| Cytoplasm | Observed as a thin layer inside the cell wall |
| Nucleus | Observed as a darkly stained structure in some cells after staining |
| Vacuole | Observed as a bright region occupying most of the cell interior |
| Chloroplasts | Not clearly observed in onion bulb-scale epidermis |
Calculation Methods
Total Magnification
10 × 40 = 400×
Field Diameter
If the field diameter is 1.80 mm with a 10× objective, the field diameter with a 40× objective is calculated as follows.
1.80 × 10 = D × 40
D = 0.45 mm
Length per Eyepiece Division
If 100 eyepiece divisions correspond to 0.50 mm, the calculation is as follows.
0.50 mm = 500 µm
500 ÷ 100 = 5.0 µm/division
Cell Size
Long dimension = 48 divisions × 5.0 µm = 240 µm
Short dimension = 14 divisions × 5.0 µm = 70 µm
Mean Values
Mean long dimension = 250 µm
Mean short dimension = 71.5 µm
Estimating Size from the Number of Cells in the Field of View
If approximately six cells are arranged across a field diameter of 0.45 mm, the estimate is as follows.
0.45 mm ÷ 6 = 0.075 mm = 75 µm
Approximate Resolution
For a wavelength of 550 nm and a numerical aperture of 0.65, the calculation is as follows.
d ≈ 0.61 × 550 nm ÷ 0.65 ≈ 516 nm ≈ 0.52 µm
Main Sources of Error
| Source of error | Effect | Improvement |
|---|---|---|
| Micrometer calibration error | Produces a systematic error in all measurements | Calibrate for each objective lens |
| Misreading the scale | Overestimates or underestimates cell size | Use consistent criteria for the start and end points |
| Unclear cell boundaries | Increases variation in measured values | Adjust staining, illumination, and focus |
| Tilted prepared slide | Makes the apparent length shorter | Mount the specimen flat |
| Compression by the coverslip | Deforms cells | Do not press down strongly |
| Drying of the specimen | Causes cells to shrink | Observe before the specimen dries |
| Insufficient or excessive staining | Makes nuclei or boundaries difficult to see | Standardize staining time |
| Incorrect focus | Blurs outlines | Focus using the fine-adjustment knob |
| Incorrect objective selection | Causes the calibration value to differ by several-fold | Confirm the objective magnification being used |
| Dirty lenses | Reduces image contrast | Clean with lens paper |
| Inappropriate illumination | Makes cell boundaries difficult to see | Adjust the diaphragm and light intensity |
| Too few cells measured | Increases the influence of individual variation | Measure many cells in multiple fields of view |
Example of How to Write the Results
Onion epidermal cells were observed using an optical microscope. A 10× eyepiece and 4×, 10×, and 40× objective lenses were used, giving total magnifications of 40×, 100×, and 400×, respectively.
As magnification increased, details such as the cell wall and nucleus became clearer, while the field diameter decreased from 4.50 mm to 0.45 mm.
With the 40× objective lens, one eyepiece micrometer division corresponded to 5.0 µm. Ten onion epidermal cells were measured, giving a mean long dimension of 250 µm and a mean short dimension of 71.5 µm.
In the observed cells, the cell wall, cytoplasm, and vacuole were identified, and nuclei were observed in some cells after staining.
Discussion Example
In this experiment, onion epidermal cells were observed with an optical microscope to investigate differences in appearance with magnification and to measure cell size.
At low magnification, a wide area could be observed, making it easier to examine the arrangement and overall shape of many cells. At high magnification, the field of view became narrower, but details such as cell walls and nuclei could be observed more clearly.
When the objective magnification was increased from 10× to 40×, the field diameter decreased from 1.80 mm to 0.45 mm. This is because the field diameter is approximately inversely proportional to the objective magnification.
At high magnification, the depth of field became shallow, and even small differences in specimen thickness caused the image to go out of focus. Fine adjustment with the fine-focus knob was therefore necessary.
With the 40× objective, 100 eyepiece divisions corresponded to 500 µm, so one eyepiece division was calculated to be 5.0 µm. The mean long dimension of ten onion epidermal cells was 250 µm, and the mean short dimension was 71.5 µm.
The variation in measured values was probably caused not only by natural differences among cells but also by differences in the observation position, cell tilt, and the exact positions chosen for reading the cell boundaries.
The cell wall was clearly visible, and most of the cell interior appeared as a bright region corresponding to the vacuole. After staining, nuclei were observed as dark structures in some cells.
Chloroplasts were not clearly observed in the epidermal cells of the onion bulb scale because the bulb scale is underground and normally performs little photosynthesis.
One source of error is incorrect calibration of the eyepiece micrometer. An incorrect calibration value produces a systematic error in the same direction in all measured cell sizes.
In addition, drying of the specimen or compression by the coverslip can deform cells and produce long and short dimensions that differ from their actual values.
Measurement accuracy can be improved by confirming the calibration for each objective lens, selecting the same type of cells from multiple fields of view, increasing the number of measurements, and standardizing staining and illumination conditions.
These results show that increasing magnification narrows the field of view and reduces depth of field while allowing finer details of cellular structures to be observed. They also show that calibrating an eyepiece micrometer makes it possible to quantitatively measure the actual size of cells.
Discussion Example When the Measured Value Is Larger Than the Reference Value
Possible reasons why the measured cell size was larger than the reference value include overestimating the length represented by one eyepiece division, measuring beyond the outer edge of the cell wall, and preferentially selecting larger cells.
Discussion Example When the Measured Value Is Smaller Than the Reference Value
Possible reasons why the measured cell size was smaller than the reference value include drying or compression of the specimen, tilting of the cells, and underestimating the calibration value of the eyepiece micrometer.
Discussion Example When the Nucleus Was Difficult to See
Possible reasons why the nucleus was difficult to see include insufficient staining time, inadequate penetration of the staining solution, a focal plane that did not coincide with the nucleus, and excessive illumination.
Summary
In an optical microscope, total magnification is calculated as the product of the eyepiece and objective magnifications. Increasing magnification makes fine details easier to observe, but it narrows the field of view and reduces depth of field.
By calibrating the eyepiece micrometer with a stage micrometer, the actual size of cells and tissues can be measured.
In the discussion, it is important to relate magnification, field of view, resolution, depth of field, staining, specimen deformation, and calibration errors to the observations and deviations in measured values.
