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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 improves image resolution by narrowing the excitation point-spread function.One-photon excitation images of BL21(λ)DE3 Escherichia coli bacteria expressing Padron using (a) one-step or (b) two-step fluorescence imaging. White arrows indicate the region expanded in (c,d). Intensity profiles measured along the white lines in (c,d) show that two-step fluorescence imaging ((e) open circles) narrows the apparent width of fine structures in excitation images when compared with one-step fluorescence imaging ((e) closed circles), consistent with our expectation that two-step fluorescence shrinks the excitation point-spread function. See Supplementary Fig. 7 for additional examples. The corresponding emission images (f,g) respectively, show almost no change in apparent width between one-step ((h) closed circles) and two-step ((h) open circles) imaging, consistent with our expectation that two-step fluorescence does not affect the emission point-spread function. Scale bars are 1 μm for (a,b) and 0.5 μm for (c,d,f,g).
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f4: Two-step fluorescence improves image resolution by narrowing the excitation point-spread function.One-photon excitation images of BL21(λ)DE3 Escherichia coli bacteria expressing Padron using (a) one-step or (b) two-step fluorescence imaging. White arrows indicate the region expanded in (c,d). Intensity profiles measured along the white lines in (c,d) show that two-step fluorescence imaging ((e) open circles) narrows the apparent width of fine structures in excitation images when compared with one-step fluorescence imaging ((e) closed circles), consistent with our expectation that two-step fluorescence shrinks the excitation point-spread function. See Supplementary Fig. 7 for additional examples. The corresponding emission images (f,g) respectively, show almost no change in apparent width between one-step ((h) closed circles) and two-step ((h) open circles) imaging, consistent with our expectation that two-step fluorescence does not affect the emission point-spread function. Scale bars are 1 μm for (a,b) and 0.5 μm for (c,d,f,g).

Mentions: We expect quadratic non-linearity to slightly shrink the excitation volume, improving the lateral resolution of an ‘excitation image' (total intensity emitted from a single illumination spot versus illumination spot position). One-step (Fig. 4a) versus two-step (Fig. 4b) one-photon imaging of Escherichia coli expressing Padron qualitatively confirms this prediction. As expected, two-step imaging (Fig. 4d) produces a sharper excitation image than one-step imaging (Fig. 4c), narrowing the apparent width of fine structures (Fig. 4e, Supplementary Fig. 7 and Supplementary Software 1). Since the shape of the bacteria is three-dimensional, this effect could be due to either improved sectioning or improved lateral resolution. However, the corresponding emission images (total intensity collected at each camera pixel, Fig. 4f,g) show no apparent difference in width (Fig. 4h), implying that sectioning alone does not lead to narrowing. Stereotyped structures like microtubules or beads would be better for quantifying resolution, but these measurements are tricky because Padron's two-step behaviour is sample-dependent.


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 improves image resolution by narrowing the excitation point-spread function.One-photon excitation images of BL21(λ)DE3 Escherichia coli bacteria expressing Padron using (a) one-step or (b) two-step fluorescence imaging. White arrows indicate the region expanded in (c,d). Intensity profiles measured along the white lines in (c,d) show that two-step fluorescence imaging ((e) open circles) narrows the apparent width of fine structures in excitation images when compared with one-step fluorescence imaging ((e) closed circles), consistent with our expectation that two-step fluorescence shrinks the excitation point-spread function. See Supplementary Fig. 7 for additional examples. The corresponding emission images (f,g) respectively, show almost no change in apparent width between one-step ((h) closed circles) and two-step ((h) open circles) imaging, consistent with our expectation that two-step fluorescence does not affect the emission point-spread function. Scale bars are 1 μm for (a,b) and 0.5 μm for (c,d,f,g).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Two-step fluorescence improves image resolution by narrowing the excitation point-spread function.One-photon excitation images of BL21(λ)DE3 Escherichia coli bacteria expressing Padron using (a) one-step or (b) two-step fluorescence imaging. White arrows indicate the region expanded in (c,d). Intensity profiles measured along the white lines in (c,d) show that two-step fluorescence imaging ((e) open circles) narrows the apparent width of fine structures in excitation images when compared with one-step fluorescence imaging ((e) closed circles), consistent with our expectation that two-step fluorescence shrinks the excitation point-spread function. See Supplementary Fig. 7 for additional examples. The corresponding emission images (f,g) respectively, show almost no change in apparent width between one-step ((h) closed circles) and two-step ((h) open circles) imaging, consistent with our expectation that two-step fluorescence does not affect the emission point-spread function. Scale bars are 1 μm for (a,b) and 0.5 μm for (c,d,f,g).
Mentions: We expect quadratic non-linearity to slightly shrink the excitation volume, improving the lateral resolution of an ‘excitation image' (total intensity emitted from a single illumination spot versus illumination spot position). One-step (Fig. 4a) versus two-step (Fig. 4b) one-photon imaging of Escherichia coli expressing Padron qualitatively confirms this prediction. As expected, two-step imaging (Fig. 4d) produces a sharper excitation image than one-step imaging (Fig. 4c), narrowing the apparent width of fine structures (Fig. 4e, Supplementary Fig. 7 and Supplementary Software 1). Since the shape of the bacteria is three-dimensional, this effect could be due to either improved sectioning or improved lateral resolution. However, the corresponding emission images (total intensity collected at each camera pixel, Fig. 4f,g) show no apparent difference in width (Fig. 4h), implying that sectioning alone does not lead to narrowing. Stereotyped structures like microtubules or beads would be better for quantifying resolution, but these measurements are tricky because Padron's two-step behaviour is sample-dependent.

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