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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.

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Related in: MedlinePlus

One-step versus two-step imaging of an artificial sample with substantial out-of-focus fluorophores.Fixed U2OS cells expressing F-tractin-Padron immersed in Padron solution, imaged with (a) one-step and (b) two-step fluorescence (see also Supplementary Fig. 2). Scale bars, 5 μm.
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f2: One-step versus two-step imaging of an artificial sample with substantial out-of-focus fluorophores.Fixed U2OS cells expressing F-tractin-Padron immersed in Padron solution, imaged with (a) one-step and (b) two-step fluorescence (see also Supplementary Fig. 2). Scale bars, 5 μm.

Mentions: We expect quadratic non-linearity to improve image sectioning, so we compare one-step and two-step imaging of an artificial sample with and without out-of-focus fluorophores. Initially the sample is a single layer of fixed U2OS cells expressing an F-tractin-Padron fusion protein labelling the actin cytoskeleton1819. As expected, one-step fluorescence produces high-quality images of this thin, sparsely fluorescent sample (Supplementary Fig. 2(e)). Next, we add an optically thick layer of Padron protein above the cells. As shown in Fig. 2a, one-step imaging becomes almost useless in this thick, densely fluorescent sample. However, two-step imaging recovers a high-quality image despite very unfavourable conditions (Fig. 2b).


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)

One-step versus two-step imaging of an artificial sample with substantial out-of-focus fluorophores.Fixed U2OS cells expressing F-tractin-Padron immersed in Padron solution, imaged with (a) one-step and (b) two-step fluorescence (see also Supplementary Fig. 2). Scale bars, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: One-step versus two-step imaging of an artificial sample with substantial out-of-focus fluorophores.Fixed U2OS cells expressing F-tractin-Padron immersed in Padron solution, imaged with (a) one-step and (b) two-step fluorescence (see also Supplementary Fig. 2). Scale bars, 5 μm.
Mentions: We expect quadratic non-linearity to improve image sectioning, so we compare one-step and two-step imaging of an artificial sample with and without out-of-focus fluorophores. Initially the sample is a single layer of fixed U2OS cells expressing an F-tractin-Padron fusion protein labelling the actin cytoskeleton1819. As expected, one-step fluorescence produces high-quality images of this thin, sparsely fluorescent sample (Supplementary Fig. 2(e)). Next, we add an optically thick layer of Padron protein above the cells. As shown in Fig. 2a, one-step imaging becomes almost useless in this thick, densely fluorescent sample. However, two-step imaging recovers a high-quality image despite very unfavourable conditions (Fig. 2b).

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