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

Ingaramo M, York AG, Andrade EJ, Rainey K, Patterson GH - Nat Commun (2015)

Bottom Line: Both activation and excitation are linear processes, but the total fluorescent signal is quadratic, proportional to the square of the illumination dose.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.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Two-step fluorescence principle and two-step imaging using Padron.(a) Activation and excitation probabilities are proportional to illumination intensity for an ideal two-step fluorophore. Fluorescence is confined to a small region, the product of the activation and excitation regions. (b) We activate and excite Padron with point-focused 488 nm light, and image the fluorescence onto a camera. (c) The signal collected is approximately quadratic in the 488 nm dose, because (d) the degree of activation is approximately linear in the 488 nm dose for our typical dosage. (e) Next we deactivate Padron with an unfocused beam of 405 nm light, to mimic the spontaneous deactivation of an ideal two-step fluorophore. (f) The activated fraction decays exponentially with increasing 405 nm dose. (g) Finally we reposition the illumination pattern, and repeat the process. The scan pattern is coarse to avoid exciting previously activated Padron, and (h) we interleave multiple coarse scans to achieve a fine scan. Note that panels (c,d,f) are conceptual illustrations; for quantitative information, see Supplementary Fig. 4 and ref. 16.
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f1: Two-step fluorescence principle and two-step imaging using Padron.(a) Activation and excitation probabilities are proportional to illumination intensity for an ideal two-step fluorophore. Fluorescence is confined to a small region, the product of the activation and excitation regions. (b) We activate and excite Padron with point-focused 488 nm light, and image the fluorescence onto a camera. (c) The signal collected is approximately quadratic in the 488 nm dose, because (d) the degree of activation is approximately linear in the 488 nm dose for our typical dosage. (e) Next we deactivate Padron with an unfocused beam of 405 nm light, to mimic the spontaneous deactivation of an ideal two-step fluorophore. (f) The activated fraction decays exponentially with increasing 405 nm dose. (g) Finally we reposition the illumination pattern, and repeat the process. The scan pattern is coarse to avoid exciting previously activated Padron, and (h) we interleave multiple coarse scans to achieve a fine scan. Note that panels (c,d,f) are conceptual illustrations; for quantitative information, see Supplementary Fig. 4 and ref. 16.

Mentions: An ideal two-step fluorophore would have two states, active and inactive, and would rapidly equilibrate to the inactive state. Illumination activates the fluorophore, and also excites the active state, causing fluorescence proportional to the degree of activation multiplied by the degree of excitation, as illustrated in Fig. 1a and Supplementary Fig. 1. If both activation and excitation probabilities are proportional to illumination intensity, and neither activation nor excitation approaches 100%, then the signal is quadratic, proportional to the illumination squared.


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

Ingaramo M, York AG, Andrade EJ, Rainey K, Patterson GH - Nat Commun (2015)

Two-step fluorescence principle and two-step imaging using Padron.(a) Activation and excitation probabilities are proportional to illumination intensity for an ideal two-step fluorophore. Fluorescence is confined to a small region, the product of the activation and excitation regions. (b) We activate and excite Padron with point-focused 488 nm light, and image the fluorescence onto a camera. (c) The signal collected is approximately quadratic in the 488 nm dose, because (d) the degree of activation is approximately linear in the 488 nm dose for our typical dosage. (e) Next we deactivate Padron with an unfocused beam of 405 nm light, to mimic the spontaneous deactivation of an ideal two-step fluorophore. (f) The activated fraction decays exponentially with increasing 405 nm dose. (g) Finally we reposition the illumination pattern, and repeat the process. The scan pattern is coarse to avoid exciting previously activated Padron, and (h) we interleave multiple coarse scans to achieve a fine scan. Note that panels (c,d,f) are conceptual illustrations; for quantitative information, see Supplementary Fig. 4 and ref. 16.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559865&req=5

f1: Two-step fluorescence principle and two-step imaging using Padron.(a) Activation and excitation probabilities are proportional to illumination intensity for an ideal two-step fluorophore. Fluorescence is confined to a small region, the product of the activation and excitation regions. (b) We activate and excite Padron with point-focused 488 nm light, and image the fluorescence onto a camera. (c) The signal collected is approximately quadratic in the 488 nm dose, because (d) the degree of activation is approximately linear in the 488 nm dose for our typical dosage. (e) Next we deactivate Padron with an unfocused beam of 405 nm light, to mimic the spontaneous deactivation of an ideal two-step fluorophore. (f) The activated fraction decays exponentially with increasing 405 nm dose. (g) Finally we reposition the illumination pattern, and repeat the process. The scan pattern is coarse to avoid exciting previously activated Padron, and (h) we interleave multiple coarse scans to achieve a fine scan. Note that panels (c,d,f) are conceptual illustrations; for quantitative information, see Supplementary Fig. 4 and ref. 16.
Mentions: An ideal two-step fluorophore would have two states, active and inactive, and would rapidly equilibrate to the inactive state. Illumination activates the fluorophore, and also excites the active state, causing fluorescence proportional to the degree of activation multiplied by the degree of excitation, as illustrated in Fig. 1a and Supplementary Fig. 1. If both activation and excitation probabilities are proportional to illumination intensity, and neither activation nor excitation approaches 100%, then the signal is quadratic, proportional to the illumination squared.

Bottom Line: Both activation and excitation are linear processes, but the total fluorescent signal is quadratic, proportional to the square of the illumination dose.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.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.

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.

No MeSH data available.


Related in: MedlinePlus