As magnification increases, the amount of light (image brightness) decreases. This happens because the same amount of light is spread over a larger image area, making the image appear dimmer. Specifically, image brightness is inversely proportional to the square of the magnification (∝1M2\propto \frac{1}{M^2}∝M21) in transmitted light microscopy. Thus, when magnification increases, brightness decreases rapidly if other factors remain constant
. However, image brightness also depends on the numerical aperture (NA) of the objective lens. Brightness is proportional to the square of the numerical aperture (∝NA2\propto NA^2∝NA2), so objectives with higher NA gather more light and produce brighter images even at higher magnifications
. In epi-illumination (reflected light), brightness behaves somewhat differently: it depends on the fourth power of the numerical aperture and inversely on magnification, but less strongly than in transmitted light. This means brightness can sometimes increase with magnification in epi-illumination at lower magnifications but generally decreases at very high magnifications
. Because brightness decreases with increasing magnification, it is common practice to increase the illumination intensity (e.g., by opening the iris diaphragm or increasing light source voltage) to compensate and maintain good image visibility at higher magnifications
. Summary:
- Increasing magnification reduces image brightness because light is spread over a larger area.
- Image brightness ∝NA2M2\propto \frac{NA^2}{M^2}∝M2NA2 in transmitted light.
- Higher numerical aperture objectives gather more light and produce brighter images.
- In epi-illumination, brightness depends more strongly on numerical aperture and less on magnification.
- To maintain brightness at higher magnifications, illumination intensity should be increased.
This explains why, as magnification increases, the amount of light reaching the eye or camera decreases unless compensated by increasing illumination
