Digital photography has been in the clutches of pixel mania for years now – and there is no end in sight. In microscopic applications, however, the camera with the most pixels is not necessarily the best one. The application and optical power of the microscope are the factors determining which camera will ultimately produce the best imaging results. The key criterion for microscopic resolution is the numerical aperture (NA), i.e. the light-gathering power of an optical system.
Ten-metre thick microscopes?
The light-gathering power of cameras or telescopes can be increased by using larger lenses with more diameter. The world record is held by the new 10.4 metre diameter mirror at the astronomy observatory in Las Palmas, Spain. However, this is not possible with microscopic lenses. You can increase the light gathering power effectively by interposing a medium with a high refraction index between lens and specimen, but in general the NA of a good dry lens is limited to about 1.0 and a good immersion oil lens to about 1.45. The NA for stereo microscopesis somewhere between 0.01 and 0.2 depending on the zoom setting.
Building stereo objectives with even higher NA is extremely difficult as you have to stay within the 24 mm stereo base to avoid altering the geometry of the stereoscopic system. With innovative FusionOptics, however, Leica Microsystems succeeded in setting a new world record for stereoscopic resolution and depth of field.
Applying the formula of 3000 x NA you can easily calculate how many pixels are actually available at the sensor of the camera taking into account the actual magnification and the sensor size. At low magnification, the microscope is usually able to deliver more details to the camera than it can capture. At high magnification however, it is the optical system that limits the amount of detail that a camera can capture. At 1x magnification the instrument delivers about 14.3 megapixels of information to the camera, while at 16x this figure drops to 2.6 megapixels.
How do you explain this apparently inverse effect? It has to do with the limited field of view. At high magnification or zoom settings, the field of view is relatively small. Looking at the round and bright circle on your specimen when using coaxial illumination clearly indicates that the higher you magnify, the smaller the bright spot becomes. You can resolve more details when you zoom into a detail or switch to a lens with higher NA.
If you work mostly at very high magnifications, the optical system is limited to about 3–5 megapixels that can be transferred to the sensor of a camera. Setting the camera to a high resolution of, say, 12 megapixels would produce a larger image, but you would not gain any additional information. If you use the microscope at low magnification on the other hand, then you definitely need a high resolution digital camera to capture all the details that your microscope can deliver – even such details in your specimen that you cannot see with the naked eye at that magnification.