Multi-Photon-Like Fluorescence Microscopy Using Two-Step Imaging Probes
George Patterson,
Investigator,
National Institutes of Health
Multi-photon microscopy provides deeper tissue imaging capacity than most linear fluorescence imaging techniques but often suffers from decreased resolution. Implementation of Multifocal Structured Illumination Microscopy (MSIM) withmulti-photon excitation takes advantage of specimen penetration afforded bylonger wavelengths but provides resolution doubled images with better sectioning and contrast in thick scattering samples. However, one disadvantage with multi-photon microscopy is the relatively high illumination intensities required for nonlinear excitation. We will discuss “two-step” fluorescence microscopy, a new approach to nonlinear imaging requiring orders of magnitude less light than conventional multi-photon excitation. This technique is based on positive reversible photoswitchable fluorescent probes, such asPadron, which display behaviors necessary for the “two-step” approach. These require that the fluorescent molecule photoswitch or ideally rapidly equilibrate to a non-fluorescent state, that the probe photoswitch to an active fluorescent state, and that the same wavelength used to turn on the probe also excite the active state to produce fluoresce. Since both activation and excitation are linear processes requiring sequential absorption processes, the total fluorescent signal is proportional to the square of the illumination dose. Thus, two-step microscopy is similar in principle to two-photon microscopy but with orders-of-magnitude better cross-section. We will show that the quadratic nonlinearity from two-step Padron fluorescence using blue light excitation improves resolution, reduces out-of-focus signal, and is compatible with structured illumination microscopy. While no ideal two-step probe is known, improvements in the characteristics discussed earlier could lead to increased resolution and optical sectioning for many forms of conventional and super-resolution microscopy techniques.
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