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. 2012 Aug 1;3(8):1955-63.
doi: 10.1364/BOE.3.001955. Epub 2012 Jul 27.

Focal Switching of Photochromic Fluorescent Proteins Enables Multiphoton Microscopy With Superior Image Contrast

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Free PMC article

Focal Switching of Photochromic Fluorescent Proteins Enables Multiphoton Microscopy With Superior Image Contrast

Ya-Ting Kao et al. Biomed Opt Express. .
Free PMC article

Abstract

Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes.

Keywords: (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy; (190.4180) Multiphoton processes.

Figures

Fig. 1
Fig. 1
Fundamental imaging-depth limit of multi-photon fluorescence microscopy. (A) Depth-dependent two-photon optical sections of a tissue phantom made of 5% intralipid, 2% agarose gel and fluorescent beads (diameter 0.9 µm) under a constant laser power excitation. Fluorescence signal quickly attenuates with the imaging depth. (B) Depth images of the same sample using a compensative higher laser power to maintain the signal strength. The resulting images can reach deeper than (A), but their contrast deteriorates as the out-of-focus background begins to dominate. The fundamental imaging-depth limit is defined when the in-focus signal and the out-of-focus background are equal to each other.
Fig. 2
Fig. 2
One-photon and two-photon induced photoswitching of Dronpa-3 protein. (A) Upon irradiation at 405 and 488 nm, Dronpa-3 switches between dark and bright states in a reversible manner (cyan and purple dashed lines), where the downward black arrow and green arrow indicates the non-radiative relaxation from the excited dark state and florescence decay from the excited bright state, respectively. (B) Time-lapse (in seconds) two-photon images (at 920nm) of Dronpa-3 expressing E. coli cells undergoing bright-to-dark switching upon the same 920nm irradiation. A two-times higher 920nm laser power leads to notably faster switching-off kinetics (lower panel). (C) Time-lapse (in seconds) two-photon images (at 920nm) of Dronpa-3 expressing E. coli cells undergoing dark-to-bright switching upon 800nm irradiation (applied between adjacent images). A two-times higher 800nm laser power leads to notably faster switching-on kinetics (lower panel).
Fig. 3
Fig. 3
Principles of multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI) with RSFPs. (A) MPDI. For the regular pre-switching image, when imaging deep into the scattering sample, substantial laser intensity is distributed out of focus, generating background that is comparable to the in-focus signal. In the post-switching image, in-focus RSFPs are switched off much more than those out of focus, creating a disparity of dark-bright states in space. The resulting difference image leads to significantly improved contrast. (B) MPAI. RSFPs which are originally in the bright state will be completely switched off into the dark state. The subsequent multiphoton activation will switch on a higher percentage of RSFPs at focus than those out of focus. This spatial disparity of dark-bright transitions leads to a significantly decreased background in the final multiphoton imaging step.
Fig. 4
Fig. 4
Experimental demonstration of multiphoton deactivation and imaging (MPDI) on tissue phantoms. (A) For Dronpa-3 expressing E. coli cells packed in 3D, the regular pre-switching image (at 920nm) is overwhelming at a depth of 250 µm. After performing a slow deactivation scanning, the post-deactivation image is dimmer. The difference image (after auto-scaled) offers a much improved image contrast. (B) Similar contrast improvement is observed for HEK 293T cells (transfected by H2B-Dronpa plasmids) placed on a dense layer of scattering E. coli cells expressing Dronpa-3.
Fig. 5
Fig. 5
Experimental demonstration of multiphoton activation and imaging (MPAI) on tissue phantoms. (A) For Dronpa-3 expressing E. coli cells mixed with polystyrene beads packed in 3D, the background in the bright-state image (at 920nm) is overwhelming at a depth of 140µm. A brief 488nm laser illumination converted Dronpa-3 in the volume of interest completely to the dark state. Afterwards, a relatively weak 800nm pulsed laser was used to activate Dronpa-3 prior to the subsequent two-photon imaging at 920nm. The resulting MPAI image reveals features that are buried in the original image of the bright state. (B) Similar contrast improvement is observed for HEK 293T cells (transfected by H2B-Dronpa-3 plasmids) placed on a 120µm-thick layer of Dronpa-3 expressing E. coli cells.

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