Microscope objectives detection efficiency

Figure 3. Confocal optical detection channel demonstrating the concept of spatial filtering. A microscope objective lens collect the light emitted from a point light source or a single molecule. The image appears as a diffraction pattern (Airy pattern, see insert). The diameter of the pinhole placed in the image plane is such that only light from the bright central spot can pass onto the detector. Radiation from an out-of-focus light source in the sample is efficiently discriminated.

The collection efficiency of the optical system increases with the square of the effective numerical aperture of the microscope objective lens. A good lens is therefore essential in order to obtain a high coincidence rate. If the sample is transparent the light can be collected from both sides, either by the condenser lens of the microscope or by a second microscope lens. Theoretically, the collection efficiency can be doubled and the coincidence rate increased by a factor of four. Moreover, in a microscope with two aligned microscope lenses exciting and detecting from both sides of the sample, the focal volume can be considerably decreased [64, 448]... 

The ideal collection optic would collect fluorescence from a Ansr solid angle around the detection zone with 100% efficiency, while being completely inefficient at collecting scattered laser light. Obviously, there exists no such optic, but there are various approaches reported in the literature taken to emulate this ideal. In most cases, either a high numerical aperture microscope objective or a fiber-optic system is used to collect the fluorescence and transmit it to the signal transducer. 

The efficiency of AFM depends on several parameters pertaining to apparatus. Essential prerequisites for a good z-resolution include the flexibility of the cantilever and a sensitive detection of its movement. An accurate positioning and the sharpness of the microscopic tip, on the other hand, are substantial contributions to the x,y-resolution. Upon optimal adjustment of all parameters, the method is able to provide pictures with atomic resolution. The size and the shape of the microscopic tip constitute the limiting factor to the resolution of small objects, and especially of narrow, deep slits. Normally, tips made of silicon are used in atomic force microscopy. They are pyramidal in shape and exhibit a diameter of ca. 10 nm on their apex. These tips are unsuitable to examining deep grooves and, due to their brittleness, their mechanical resistance leaves much to be desired (Figure 3.106). Moreover, the resolution decreases when the tip breaks. Carbon... 

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