Optical conjugate plane

A method to detect diffusion coefficients of single molecules in solution is confocal microscopy. A confocal microscope uses point illumination, which is achieved by a pinhole in front of the objective (in an optically conjugated plane to the sample). The illuminated voliune is in the order of 1 femtoliter and. 

Parfocal objectives par- fo-k9l-9b- jek-tiv n. Objectives which are mounted so that only small adjustment of the bodytube and stage is necessary to focus after changing from one objective to any of the others. They are mounted in such a way that the second conjugate plane is in the same position on the optical axis of the microscope for each objective. Objectives used on a rotating nosepiece are usually parfocal. Eyepieces are also parfocal within any given manufacturer s series. 

Planes constructed at the focal points perpendicular to the optical axis are called focal planes. Parallel rays, even if not parallel to the axis, intersect in the focal plane. Corresponding points in the object and image space— points that satisfy an object-image relation—are called conjugate points. Planes within the points are conjugate planes. The distances between conjugate points and the surface are called conjugate distances [2]. 

Conjugate planes is the term given to planes in the optical system which are equivalent. An object placed in one of them will appear as a sharp image in the other subsequent planes of the series. There are two separate series of conjugate planes in the compound microscope (Fig. 11.4) ... 

The schlieren microscope is able to detect refractive index variations to six decimal places. Any small difference in optical path (index difference, film thickness, etc) is very precisely detected by the schlieren microscope, especially in the Dodd modification. It is, in effect, a darkfield method. The specimen is illuminated with light in a portion of the illuminating cone and that direct light is masked in the conjugate back focal plane of the objective (Fig. 3). The only light to pass through this plane is refracted, reflected, or diffracted by the specimen. 

In real curvature sensors, a vibrating membrane mirror is placed at the telescope focus, followed by a collimating lens, and a lens array. At the extremes of the membrane throw, the lens array is conjugate to the required planes. The defocus distance can be chosen by adjusting the vibration amplitude. The advantage of the collimated beam is that the beam size does not depend on the defocus distance. Optical fibers are attached to the individual lenses of the lens array, and each fiber leads to an avalanche photodiode (APD). These detectors are employed because they have zero readout noise. This wavefront sensor is practically insensitive to errors in the wavefront amplitude (by virtue of normahzing the intensity difference). 

Compared to conventional microscopy, confocal microscopy delivers superior optical sectioning by using a pinhole in a plane conjugate with the focal (xy) plane. It allows a crisp 3D image to be built from a stack of 2D images even for somewhat turbid samples, but each 2D image is acquired by scanning, which imposes limits on acquisition speed. The technique has been described in detail before [82]. 

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