Confocal microscopy

Light and acoustic microscopy (Structural & Morphology Characterization)

CM is used to determine the surface contours and profiles of micro-structured components and to quantify the height and depth of features; it is also able to build a 3-D image with structure information below the surface.

CM offers several advantages over conventional optical microscopy, including shallow depth of field, elimination of out-of-focus glare, and the ability to collect serial optical sections from thick specimens.

In conventional microscopy, the sample is placed on the microscope stage and the entire field of the specimen is illuminated and visualized. Although the highest intensity is at the focal point of the objective lens, other parts of the sample are illuminated generating background “noise” which compromises the quality of the image. However, in confocal microscopy, the incoming light is focused through the microscope objective on a small spot inside the microstructure. The same objective gathers the reflected or fluorescent light coming back from the sample. A small pinhole aperture in a screen is used so that only the light emitting from the desired focal spot is can pass through. The screen blocks any light scattered outside the focal plane. In optical terms, the pinhole is placed in a conjugate focal plane of the sample (hence the designation “confocal”). A sensitive light detector, such as a photomultiplier tube, on the other side of the pinhole to detects the confocal light. Thus the specimen can be imaged one “point” at a time.

To create virtual-sectioned images of the microstructure, the focal spot is serially scanned in the X–Y plane. As the scanning progresses, signal from the detector is fed to a computer that collects all the “point images” of the sample and serially constructs the image one pixel at a time. Because the sample is not actually sectioned, it is possible to image and display a “stack” of virtual, confocal images a video monitor and achieve a high-contrast image that could not be acquired by conventional microscopy.

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