A new imaging method for confocal microscopy

摘要

Confocal laser microscope (CLM) is indispensable today in the biology research field as the tool to clarify three dimensional structure and temporal transformations of living cells. The biggest advantage of CLM is to obtain "Optical slice images" in the direction of depth. The fluorescence from specimen is detected by a photo-detector in CLM through the small aperture called "Pinhole". The smaller the diameter of the pinhole is, the thinner the optical slice becomes. However, there is a problem that the contrast degrades because the images darken as the pinhole gets smaller, while the out-of-focus light increases as the pinhole is enlarged. To solve the problem, we developed a new detection method. In this method named "VAAS", it provides with the detector that captures light that doesn't pass through the pinhole in addition to the detector that captures light passes through the pinhole. Both detectors convert the light into electric signals at the same time. This method enables to eliminate out-of-focus light from the bright images acquired with large pinhole. In addition, quantitative experiments and analysis has proved that the contrast would be improved about 10dB compared with conventional CLM. VAAS is expected to be applied widely in the field of research to observe living cells where the reduction of optical toxicity is required in the future.