Two-photon-like microscopy with orders-of-magnitude lower illumination intensity via two-step fluorescence

Nature Communications
Maria IngaramoGeorge Patterson

Abstract

We describe two-step fluorescence microscopy, a new approach to non-linear imaging based on positive reversible photoswitchable fluorescent probes. The protein Padron approximates ideal two-step fluorescent behaviour: it equilibrates to an inactive state, converts to an active state under blue light, and blue light also excites this active state to fluoresce. Both activation and excitation are linear processes, but the total fluorescent signal is quadratic, proportional to the square of the illumination dose. Here, we use Padron's quadratic non-linearity to demonstrate the principle of two-step microscopy, similar in principle to two-photon microscopy but with orders-of-magnitude better cross-section. As with two-photon, quadratic non-linearity from two-step fluorescence improves resolution and reduces unwanted out-of-focus excitation, and is compatible with structured illumination microscopy. We also show two-step and two-photon imaging can be combined to give quartic non-linearity, further improving imaging in challenging samples. With further improvements, two-step fluorophores could replace conventional fluorophores for many imaging applications.

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Citations

Feb 15, 2018·Optics Letters·Xiang-Dong ChenFang-Wen Sun
Feb 3, 2019·Nature Communications·Jes DreierIlaria Testa
May 7, 2021·Science Advances·Miao-Ping ChienAdam E Cohen
Aug 18, 2020·Journal of the American Chemical Society·Carlos Benitez-MartinJoakim Andréasson

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Methods Mentioned

BETA
fluorescence microscopy
imaging techniques
fluorescence imaging

Software Mentioned

rsCherry
Padron
SOFI
MPDI
pcSOFI
MPAI
Python
rsPIM
CSM

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