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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.


One-step, two-photon versus two-step, two-photon imaging of an artificial sample with substantial of out-of-focus fluorophores.Fixed U2OS cells with Padron-labelled mitochondria immersed in Padron solution, imaged with (a) one-step, two-photon and (b) two-step, two-photon fluorescence (see also Supplementary Fig. 5). Scale bars, 1 μm.
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f3: One-step, two-photon versus two-step, two-photon imaging of an artificial sample with substantial of out-of-focus fluorophores.Fixed U2OS cells with Padron-labelled mitochondria immersed in Padron solution, imaged with (a) one-step, two-photon and (b) two-step, two-photon fluorescence (see also Supplementary Fig. 5). Scale bars, 1 μm.

Mentions: We expect quartic non-linearity to give even better sectioning than quadratic non-linearity, so we investigate the combination of two-step with two-photon fluorescence. We found that short pulses of infrared light (950 nm central wavelength, 140 fs pulse duration and 12.5 ns repetition time) efficiently activate and excite Padron (Supplementary Fig. 4(c)), so we compare one-step two-photon versus two-step two-photon imaging. We use a similar artificial sample to that shown in Fig. 2, and a similar imaging procedure except we substitute focused, pulsed infrared light for focused blue light. The initial sample is a single layer of fixed U2OS cells expressing Padron fused to the mitochondrial targeting sequence of the precursor of subunit VIII of human cytochrome c oxidase. As expected, one-step two-photon imaging gives a high-quality image in this case (Supplementary Fig. 5(e)). Next, we add a drop of Padron protein above the cells. As shown in Fig. 3a, one-step two-photon imaging fails to capture a useful image in this challenging sample, while two-step two-photon imaging performs well (Fig. 3b), presumably because two-step two-photon fluorescence further reduces out-of-focus excitation (Supplementary Fig. 6), reducing noise enough to allow imaging.


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, two-photon versus two-step, two-photon imaging of an artificial sample with substantial of out-of-focus fluorophores.Fixed U2OS cells with Padron-labelled mitochondria immersed in Padron solution, imaged with (a) one-step, two-photon and (b) two-step, two-photon fluorescence (see also Supplementary Fig. 5). Scale bars, 1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: One-step, two-photon versus two-step, two-photon imaging of an artificial sample with substantial of out-of-focus fluorophores.Fixed U2OS cells with Padron-labelled mitochondria immersed in Padron solution, imaged with (a) one-step, two-photon and (b) two-step, two-photon fluorescence (see also Supplementary Fig. 5). Scale bars, 1 μm.
Mentions: We expect quartic non-linearity to give even better sectioning than quadratic non-linearity, so we investigate the combination of two-step with two-photon fluorescence. We found that short pulses of infrared light (950 nm central wavelength, 140 fs pulse duration and 12.5 ns repetition time) efficiently activate and excite Padron (Supplementary Fig. 4(c)), so we compare one-step two-photon versus two-step two-photon imaging. We use a similar artificial sample to that shown in Fig. 2, and a similar imaging procedure except we substitute focused, pulsed infrared light for focused blue light. The initial sample is a single layer of fixed U2OS cells expressing Padron fused to the mitochondrial targeting sequence of the precursor of subunit VIII of human cytochrome c oxidase. As expected, one-step two-photon imaging gives a high-quality image in this case (Supplementary Fig. 5(e)). Next, we add a drop of Padron protein above the cells. As shown in Fig. 3a, one-step two-photon imaging fails to capture a useful image in this challenging sample, while two-step two-photon imaging performs well (Fig. 3b), presumably because two-step two-photon fluorescence further reduces out-of-focus excitation (Supplementary Fig. 6), reducing noise enough to allow imaging.

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.