Biophotonics_Concepts_to_Applications

Example 8.6Consider two microscope setups being used at a 700 nm

wavelength. Suppose for one setup NAobj1= 0.80 and for the other setup

NAobj2= 1.25. Which setup yields afiner resolution?

Solution:FromEq.(8.6), R 1 =0.61λ/NAobj= 0.61(700 nm)/0.80 = 533 nm.

For the second setup, R 2 = 0.61(700 nm)/1.25 = 341 nm. Therefore the second

setup has afiner resolution.

8.3 Confocal Microscopy

When using the conventional microscopes described in Sect.8.1, the entire spec-
imen being examined, or a large portion of it, is illuminated and viewed simulta-
neously. In this method, the viewer concentrates on the segment of the specimen
that is located at the focal point of the objective lens of the microscope. The
simplicity of this setup imposes a limitation on the quality of the image, because
unfocused light from other portions of the specimen, plus other unwanted scattered
light, also will show up in the image. The result is that the image can be blurred or
some details can be obscured.
These limitations are mitigated through the use of confocal microscopy [ 12 – 14 ].
A confocal microscope creates sharp images of a specimen that normally appear
blurred when viewed with a conventional microscope. This image-enhancing
characteristic is achieved by an optical method that excludes most of the light that
does not come from the focal plane of the specimen.
The key feature of a confocal microscope is the use of two spatialfilters with
pinhole apertures, as Fig.8.10shows. One aperture is located near the light source

Camera or

photodetector

Disks with

pinhole

Objective lens

Focal plane

Specimen

Laser

Solid blue lines:

Illumination lines

Solid green lines:

Observed image lines

Dashed green lines:

Blocked image lines

Dichroic mirror

Fig. 8.10 Basic concept of
confocal microscopy

8.2 Resolution and Diffraction Limit 247