Optical Microscopy

This page provides a summary of my research in high resolution optical microscopy and image restoration. The 4Pi-confocal microscope I built at Max-Planck Institute for Biophysical Chemistry was the first one capable of routinely recording 3D images of biological specimen at a resolution far beyond the diffraction limit. The resources provided in the publications list give a complete description of the optomechanical measures that allowed to control the experimental setup with the required precision in the nanometer range. The focus of a 4Pi-confocal microscope consists of 3 distinct spheres. This leads to ghost images of the specimen that need to be removed using image restoration techniques. Therefore I had to investigate under which circumstances the object can be unambigously reconstructed from the recorded raw images. The in-depth study on this was published in the Journal of the Optical Society of America.

Breaking the resolution barrier

In 1996 I joined the group "High Resolution Optical Microscopy" of Stefan W. Hell at Max-Planck Institute for Biophysical Chemistry in Göttingen where I stayed until 2000. After moving from the former location in Turku, Finland to Göttingen, we spent the first years building some of the advanced light microscopes invented by Dr. Hell. Since the micropscopes were unique in resolution, we soon started cooperation with several biologists of the institute to employ the new technique to biological research. My project was the 4Pi-confocal microscope. After several prototypes of this micropscope have been built by S.W. Hell, M. Schrader and K. Bahlmann, I designed a new version where improved optomechanical components, new electronics and my image acquisition software allowed to routinely record 3D images of biological specimen.

4Pi-confocal Microscopy

The highest "conventional" optical resolution is obtained by employing high aperture lenses to focus laser light into a fluorescent specimen and using a pinhole in the detection path to obtain a detection focus. This technique is named confocal microscopy because the illumination and detection foci overlap. To record 3D images, either the focus can be moved through the sample or vice versa. The ideal focus for 3D images would be a perfect sphere. However, due to the limited aperture angle of even the best lenses, the PSF (point spread function) of a confocal microscope is considerably elongated in direction of the optical axis. Consequently, the axial resolution of a confocal microscope is 3-4 times worse than its lateral resolution. The 4Pi-confocal microscope uses two opposing lenses to overcome this limitation and obtain an almost spherical PSF. The challenge of this technique is to make the two illumination foci and the one or two detection foci overlap while maintaining a fixed phase relation between the interfering light beams.

Never trust a Standing Wave

In addition to the 4Pi-confocal microscope there are several other approaches (I5M, Standing Wave) that use two opposing lenses to increase resolution in light microscopy. All of these microscopes, including the 4Pi, exhibit PSF's that consist of several distinct foci. This leads to additional "ghost images" of the object that need to be removed by either deconvolution or image restoration. To learn which microscopy technique delivers faithful images of the objects being captured, I investigating the imaging process from the acquisition of the raw image to the deconvolved or restored final result. I found that, apart from the 4Pi-confocal microscope, any of the other approaches leads to artefacts that can not be removed by any image reconstruction technique.

The Cover story

One of the images of the microtubule network which I captured in January 1998 became a famous cover artwork for the Max-Planck Society. The comparison of the confocal and 4Pi-confocal images pictures scientific progress in a very intuitive way. Therefore it was used not only for the cover of the "Jahrbuch 1999" but also for various brochures regarding science in Europe.

Publications

Hell, S. W. and M. Nagorni (1998). "4Pi-confocal microscopy with alternate interference."
Opt. Lett. 23(20): 1567-1569
Nagorni, M. and S. W. Hell (1998). "4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution."
J. Struct. Biol. 123: 236-247
Schmidt, M., M. Nagorni and S. W. Hell (2000). "Subresolution axial distance measurements in far-field fluorescence microscopy with precision of 1 nanometer."
Rev. Sci. Instrum. 71(7): 2742-2745
Nagorni, M. and S. W. Hell (2001). "Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. I. Comparative study of concepts."
J. Opt. Soc. Am. A 18(1): 36-48
Nagorni, M. and S. W. Hell (2001). "Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. II. Power and limitation of nonlinear image restoration."
J. Opt. Soc. Am. A 18(1): 49-54
Kano, H., S. Jakobs, M. Nagorni and S. W. Hell (2002). "Dual-color 4Pi-confocal microscopy with 3D-resolution in the 100 nm range."
Ultramicr. 90: 207-213
Matthias Nagorni: Implementierung von X Window Applikationen zur Bildaufnahme und Steuerung am Heidelberger Wellenfeldmikroskop
Diplomarbeit, Universität Heidelberg 1996
Matthias Nagorni: Auflösungserhöhung in der Fluoreszenzmikroskopie durch kohärente Superposition der fokussierten Lichtwellen zweier sich gegenüberstehender Objektive: Theorie und Experiment
Dissertation, Universität Heidelberg 2000