05/19/2016

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HyVolution - Super-resolution Imaging with a Confocal Microscope

Dr. Rolf T. Borlinghaus Kontakt, Dr. Constantin Kappel, Leica Microsystems GmbH



Since the invention of the microscope, there has been continual discussion about the possibility of showing more detailed features of specimens as compared to just magnifying them. Ernst Abbe was the first to explore the issue in depth and found there was a limit to the size of details visible in ordinary light microscopes.

Ernst Abbe formulated the standard

This "Abbe limit" depends on the wavelength λ (color) of the light applied and the aperture NA of the objective in use.

In Abbe's words: "... so folgt, dass, wie auch das Mikroskop in Bezug auf die förderliche Vergrößerung noch weiter vervollkommnet werden möchte, die Unterscheidungsgrenze für centrale Beleuchtung doch niemals über den Betrag der ganzen, und für äusserste schiefe Beleuchtung niemals über den der halben Wellenlänge des blauen Lichts um ein Nennenswerthes hinausgehen wird". The limit of differentiation ... will not substantially surpass half the wavelength of blue light in case of most oblique illumination. The "most oblique illumination" would be the use of a condenser with an NA that at least matches the NA of the objective lens. Keep in mind that around 1850 AD, a "microscope" typically employed transmitted light brightfield in the visible range.

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