Biomedical subjects

Marcus Dyba

Publications and source records attributed to Marcus Dyba.

4 recordsLinked to original sources

PubMed2004

Concepts for nanoscale resolution in fluorescence microscopy.

Spatio-temporal visualization of cellular structures by fluorescence microscopy has become indispensable in biology. However, the resolution of conventional fluorescence microscopy is limited by diffraction to about 180 nm in the focal plane and to about 500 nm along the optic axis. Recently, concepts have emerged that overcome the diffraction resolution barrier fundamentally. Formed on the basis of reversible saturable optical transitions, these concepts might eventually allow us to investigate hitherto inaccessible details within live cells.

Animals↗

PubMed2003

Immunofluorescence stimulated emission depletion microscopy.

We report immunofluorescence imaging with a spatial resolution well beyond the diffraction limit. An axial resolution of approximately 50 nm, corresponding to 1/16 of the irradiation wavelength of 793 nm, is achieved by stimulated emission depletion through opposing lenses. We have demonstrated not only that an antibody-tagged label is stable enough to be recorded in this microscopy mode, but also that subdiffraction resolution can be obtained using a standard immunofluorescence preparation.

Cells, Cultured↗

PubMed2003

Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission.

Saturated stimulated-emission depletion (STED) of a fluorescent marker has been shown to break the diffraction barrier in far-field fluorescence microscopy and to facilitate spatial resolution down to a few tens of nanometers. Here we investigate the photostability of a fluorophore that, in this concept, is repeatedly excited and depleted by synchronized laser pulses. Our study of bacteria labeled with RH-414, a membrane marker, reveals that increasing the duration of the STED pulse from approximately 10 to 160 ps fundamentally improves the photostability of the dye. At the same time the STED efficiency is maintained. The observed photobleaching of RH-414 is due primarily to multiphoton absorption from its ground state. One can counteract photobleaching by employing STED pulses that range from 150 ps to approximately half of the lifetime of the excited state. The results also have implications for multiphoton excitation microscopy.

Absorption↗

PubMed2002

Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution.

We report spots of excited molecules of 33 nm width created with focused light of lambda = 760 nm wavelength and conventional optics along the optic axis. This is accomplished by exciting the molecules with a femtosecond pulse and subsequent depletion of their excited state with red-shifted, picosecond-pulsed, counterpropagating, coherent light fields. The lambda/23 ratio constitutes what is to our knowledge the sharpest spatial definition attained with freely propagating electromagnetic radiation. The sub-diffraction spots enable for the first time far-field fluorescence microscopy with resolution at the tens of nanometer scale, as demonstrated in images of membranes of bacillus megaterium.

Journal Article↗