Predicted Spatial Resolution of Super-Resolving Fluorescence Microscopy Using Two-Color Fluorescence Dip Spectroscopy

摘要

The super-resolving technique is among the most important subjects in optical microscopy. An aim of this technique is to overcome the optical diffraction limit, i.e., the Rayleigh limit. Since an optical microscope directly allows plain visual images of samples without causing any damage, it has long been an indispensable measurement tool for biology. However, due to the described restrictions, the limit of the spatial resolution is roughly the same as the half-wavelength of the light; a green probe light typically gives a resolution of about 250 nm. This value is not sufficient to explore the inside of a cell because most of the contents are much smaller than the wavelength of the light, such as the several tens of nanometers size of cell organelles (for example, lysosome and endoplasmic reticulum). To reveal phenomena inside a living cell, biologists have needed an optical microscope with a much higher resolution beyond the diffraction limit. Under this circumstance, we have proved that a fluorescence depletion process induced by two-color irradiation (two-color fluorescence dip spectroscopy) is able to break the resolution limit of a laser scanning microscope. Figure 1 shows the excitation scheme of two-color fluorescence dip spectroscopy. It is now a well-established technique used to observe the higher excited states of molecules. The first laser (pump beam with a wavelength of λ_(1)) excites a molecule from the ground state (S_(0)) to an S_(1) state, where fluorescence light is emitted. The second laser (erase beam with a wavelength of λ_(2)) further upconverts the S_(1) molecule to a higher excited state (S_(n)). Opening various relaxation channels from the S_(n) states, such as internal conversion (inter-system crossing and so on), the molecule in the S_(n) state can decay to the ground state without fluorescence, i.e., a fluorescence depletion process. Thus, when the wavelength matches that of the S_(n)←S_(1) transitions, the fluorescence from the molecule is depleted. This fluorescence depletion process was developed to detect the S_(n)←S_(1) transition in molecular spectroscopy. Perceiving this optical property, we have proposed a novel microscopy. If the erase beam is condensed onto some part of a focused pump beam area on a dyed sample, the fluorescence depletion process takes place in the area where the two beams overlap. We have proposed a first-order Bessel beam with a doughnut shape for the erase beam. Using this beam, the fluorescence area shrinks without losing the fluorescence intensity in the center region. As shown in Fig. 2, the area becomes narrower than the diffraction limit, i.e., this is a super-resolving microscopy.