Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging

Abstract

We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as β-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quantification of proteins in the inner mitochondrial membrane with ∼20 nm optical resolution.

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Introduction

Far-field fluorescence microscopy combined with efficient fluorescent probes allows the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to the diffraction barrier of light which imposes an optical resolution limit of approximately 200 nm in the imaging plane. In the recent past¹ new techniques emerged that break the diffraction barrier and enable structural investigations with so far unmatched resolution. Most of them are based on reversible saturable optical fluorescence transitions (RESOLFT)² by switching fluorophores between a detectable fluorescent and a nonfluorescent (dark) state upon illumination with light of different wavelength. Resolution enhancement is achieved by using a light intensity distribution of a deactivation laser beam featuring a local zero in the laser focus by the use of a phase mask, overlaid with an excitation laser beam and scanning of the sample or laser beams.^3,4 Alternatively, stochastic photoactivation and deactivation of single fluorophores and precise position localization of each individual fluorescent spot can be used in combination with wide-field fluorescence microscopy to reconstruct a superresolution image.^5–11 Both approaches control the fluorescence emission of fluorophores in time either at the ensemble level switching fluorophores in the outer parts of the laser focus into the nonfluorescent state or at the single molecule level by switching only a subset of fluorophores to the fluorescent state. Therefore, all these methods critically rely on the availability of efficient molecular optical switches and are generalized under the denotation “photoswitching microscopy”.^12,13

Molecular optical switches comprise fluorescent proteins that can be photoactivated.^14,5,6 or reversibly photoswitched,^4,15 organic fluorophores such as carbocyanine dyes Cy5 and Alexa647 in the presence^7,16 or absence^17,11,18 of an activator fluorophore, or photochromic molecules that undergo a photochemical reaction to enter or leave a fluorescent state.¹⁹ The increased interest and attention of researchers in biological fields is in particular reasoned by a stepwise development of experimental conditions that are compatible with biological samples, and simplifications of subdiffraction-resolution fluorescence imaging methods themselves. This is in particular true for organic fluorophores. While there is an ongoing development of fluorescent proteins that can be photoactivated or reversibly photoswitched, their use is still largely limited to target proteins they are genetically fused to. The advantage of organic fluorophores acting as photoswitches lies in the variability of their use: they can be conjugated to small receptor-binding peptides, to short DNA or RNA fragments or hairpins, whole proteins, and even small drug molecules. Therefore, standard organic fluorophores that can undergo reversible photoswitching are of special interest as they can be easily attached to target molecules and photoswitching under various experimental conditions has been demonstrated.^{19,20,13,21,22}

The carbocyanine derivatives Cy5 and Alexa647 are the first fluorophores that were used successfully for high resolution microscopy in methods termed STORM (stochastic optical reconstruction microscopy)^7,16 and (direct) dSTORM^17,11,18, respectively. Experimentally, a sample that has been densely labeled with the fluorescent photoswitch, e.g. via immunocytochemistry, is prepared such that most fluorophores are in their dark states, and only a subset of fluorophores is fluorescent at a time. Here, one has to make sure that individual fluorophores are at least spaced by a distance larger than the diffraction limit, to allow unambiguous identification of single molecules. In a next step, the emission profiles of single emitters are localized with high precision through the approximation with a Gaussian fit, which can be done with an accuracy of a few nanometers.²³ The repetitive cycling of photoactivation, localization and deactivation of individual fluorophores allows reconstructing images that break the diffraction barrier of light with an experimental resolution down to ∼20 nm.^16,11,18 Typical recording times are from tens of seconds to minutes, and high-resolution images of cytoskeletal structure,^16,11 as well as quantitative protein distribution in mitochondria,¹⁸ have been demonstrated. However, the use of carbocyanine dyes is only compatible with fixed cell experiments, as the underlying switching mechanism requires oxygen to be removed and a reducing agent, typically a thiol-reagent such as β-mercaptoethylamine (cysteamine) at a concentration of ∼100 mM, has to be added.^17,7 Furthermore, switching of carbocyanines requires the use of two laser wavelengths simultaneously or in alternation, as the reverse transition from the nonfluorescent dark state of the fluorophores is light-induced.

Here, we demonstrate that standard fluorophores with different absorption and emission wavelengths, e.g. ATTO520 and ATTO655, can be used advantageously for multicolor photoswitching microscopy with an optical resolution of ∼20 nm. Upon intersystem crossing the triplet state of the fluorophore is selectively reduced by thiol-containing reducing agents, such as β-mercaptoethylamine or the intracellular tripeptide glutathione to form a long-lived radical anion. The fluorescent state (singlet manifold) of the fluorophore is repopulated by reaction of a thermally stable radical anion with oxygen naturally present in aqueous solvents at concentrations of ∼250 μM²⁴ at room temperature. Provided that the redox properties of the fluorophore are matched with the redox potential of the reducing reagent and its concentration (10–100 mM), radical anions can be generated that exhibit a longer survival time (lifetime) than the lifetime of the fluorescent state. As such, conventional fluorophores can be used as molecular photoswitches at the single molecule level, and largely extend the list of fluorescent probes that are suited for multicolor photoswitching microscopy with subdiffraction resolution. Since oxygen does not have to be removed, in contrast to all alternative approaches of super-resolution fluorescence microscopy with standard fluorophores,^17,10,13,21 and it relies only on the presence of millimolar thiol-containing reducing agents, the method we describe provides access to measurements in living cells.

Materials and methods

Cell culture and immunocytochemistry

African green monkey kidney cells were plated in Labtek 8-well chambered cover glass (Nunc). After 12 to 24 h, the cells were fixed using 3.7% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min. The fixed cells were washed five times with PBS, permeabilized (PBS containing 0.5% v/v Triton X-100) for 10 min, and treated with blocking buffer (PBS containing 5% w/v normal goat serum (NGS; Sigma) for 30 min. Microtubules were stained with mouse monoclonal anti-β-tubulin antibodies (clone: TUB 2.1, Sigma) for 60 min. For single tagged samples cells were stained for 60 min with ATTO655 labeled goat anti-mouse F(ab′)₂ fragments or ATTO520 labeled goat anti-mouse F(ab′)₂ fragments (Invitrogen, M35000), respectively. For double tagged ATTO655/ATTO520 microtubule filaments the cells were treated with equal concentrations of labeled goat anti-mouse F(ab′)₂ fragments, simultaneously. Three washing steps using PBS containing 0.1% v/v Tween 20 were performed after each staining step. The ATTO-dyes were purchased as NHS derivatives (Atto-Tec GmbH) and labeled to F(ab′)₂ fragments separately and were purified on a NAP 5 column (Sephadex G-25 DNA Grade, GE Healthcare). The degree of labeling of F(ab′)₂ fragments was determined to 1–2 dependent on the fluorophore, respectively.

For multicolor photoswitching microscopy of microtubule filaments and enzymes of the respiratory chain, mouse anti-β-tubulin and sheep anti-cytochrome c (C9616, Sigma) antibodies were used as primary set. Here ATTO520 modified goat anti-mouse F(ab′)₂ fragments and ATTO655 modified rabbit anti-sheep antibodies served as secondary set. Three washing steps using PBS containing 0.1% v/v Tween 20 were performed after each staining step. The ATTO-dyes were coupled to F(ab′)₂ fragments and IgG (rabbit anti-sheep) separately and were purified on a NAP 5 column (Sephadex G-25 DNA Grade, GE Healthcare).

Photoswitching microscopy

Fluorescence imaging was performed on an inverted microscope (Olympus IX71) applying an objective type total internal reflection fluorescence (TIRF) configuration equipped with an oil-immersion objective (PlanApo 60x, NA 1.45, Olympus).^11,18 Photoswitching microscopy was performed with an argon–krypton laser (Innova 70C, Coherent) using an appropriate excitation wavelength for the individual fluorophore used, i.e. 514 nm for ATTO520,564 nm for ATTO565, and 647 nm for ATTO655, ATTO680, and ATTO700. Typical illumination intensities of 20 to 40 mW (1–2 kW cm⁻²) were applied.

Using appropriate filter sets the fluorescence was recorded on an EMCCD camera (Andor Ixon+ DV897DCS-BV). Typically, 8000–16000 frames at frame rates of 10–100 Hz were used. For photoswitching microscopy the concentration of β-mercaptoethylamine or glutathione (Sigma) was adjusted to 10–100 mM in phosphate buffer saline (pH 7.4) without applying an oxygen scavenger system. According to the standard deviation of the PSF, σ, and the number of photons detected, N, the localization accuracy can be approximated to σ/√N.²³ Typically, several thousand photons can be detected for a single fluorophore per image, thus predicting a theoretical localization precision of <10 nm. Images were generated as described previously.²² Briefly, fluorescent spots of single fluorophores were identified in each image frame applying an intensity threshold, and were fit to a Gaussian function to determine their centre of mass. All localization events were summed up in a two dimensional histogram with binning widths between 8.5 to 17 nm.

Results

In fact photoswitching microscopy only requires reversible switching of a fluorophore between a fluorescent and a nonfluorescent or dark state provided that the survival time or lifetime of the dark state is substantially longer than the lifetime of the fluorescent state to ensure the isolation of the fluorescence emission of individual fluorophores in time. Thus, also triplet states which are naturally present in virtually all fluorophores might be used for photoswitching microscopy.^20,10 However, the triplet state lifetimes of standard organic fluorophores are short and vary between less than 1 μs up to a few hundred μs in air-saturated solution, with triplet quantum yields Φ_isc < 0.1%.^25,22 To realize photoswitching microscopy based on triplet states of standard fluorophores the lifetime of the fluorescent state, i.e. the time the fluorophore is repeatedly cycled between its singlet ground and excited state, has to be shortened substantially applying high excitation intensities. Simultaneously the lifetime of the dark state has to be prolonged upon oxygen removal because oxygen is known as very potent triplet quencher. Thus, either oxygen has to be removed, e.g. by enzymatic oxygen scavenging systems,²⁶ or immobilization of the sample in PVA, a polymer with very low oxygen permeability, to enable photoswitching microscopy with subdiffraction-resolution.¹⁰

On the other hand, electron transfer reactions can be used to control the lifetime of the dark state of fluorophores.²⁷ The idea behind this is that, dependent on the redox properties of fluorophores, their triplet states can be depopulated by electron transfer reactions with suitable reducing and oxidizing agents to form long-lived radical ions (nonfluorescent states) and to repopulate the singlet state (fluorescent state). This method offers the possibility to engineer the lifetime of the dark state of fluorophores by addition of different concentrations of reducing and oxidizing reagents provided that oxygen is carefully removed from the solvent. Thus, the method enables the use of a variety of fluorophores for photoswitching microscopy with high optical resolution using only a single continuous wave laser line.²¹

Alternatively, one might reduce the triplet state of fluorophores using a suited reducing agent and repopulate the singlet ground-state using molecular oxygen as oxidizing agent naturally present in aqueous buffer at ∼250 μM concentrations.²⁴ Therefore, fluorophores have to be selected that are comparably easy reduced by electron donors, e.g. oxazine and rhodamine derivatives.^28–31,27 On the other hand, the fluorophores should exhibit a low energy of the first reduced state to be insensitive to oxidation by oxygen. Taking these considerations into account, a reducing agent has to be selected that selectively reduces excited fluorophores once they enter the triplet state but remains almost ineffective as long as the fluorophore resides in the excited singlet state (Fig. 1A). Reducing agents with the desired mild reducing properties are thiol-containing compounds such as β-mercaptoethylamine (MEA) or glutathione. Following excitation of the fluorophore intersystem crossing to a triplet state can occur with the rate k_isc. Dependent on the reduction potential of the fluorophore 10–100 mM of the reducing agent have to be added to efficiently deplete the triplet state by the formation of a radical anion with the rate k_red. The first excited singlet state, S₁, however, should not be efficiently quenched by the thiol-containing reducing compound to ensure high fluorescence brightness.

Fig. 1 (A) Underlying photophysical processes of photoswitching microscopy with ATTO520, ATTO565, ATTO655, ATTO680, and ATTO700 with subdiffraction optical resolution. Following excitation of the fluorophores (k_ex) to their first excited singlet state, S₁, the excited state energy is either released via fluorescence emission with rate k_F (dependent on the fluorescence quantum yield), or the triplet state is occupied via intersystem crossing (k_isc). The triplet state is depopulated either by intersystem crossing (k_isc′) or by an electron transfer reaction. Dependent on the triplet lifetime of the fluorophore and its reduction potential 10–100 mM of β-mercaptoethylamine have to be added to efficiently reduce the fluorophore with rate k_red. The generated radical anions of the selected ATTO-dyes exhibit a relatively high thermal stability. Because oxidation of the radical anion by oxygen, i.e. the repopulation of the singlet ground state (k_ox), is very inefficient the lifetime of the radical anions can easily outlive more than several 100 milliseconds. (B) Fluorescence time trace of a single ATTO520 labeled goat anti-mouse F(ab′)₂ fragment adsorbed non-specifically on a glass coverslide recorded under continuous illumination with a frame rate of 10 Hz in the presence of 100 mM MEA. (C) PSF of a single ATTO520 labeled goat anti-mouse F(ab′)₂ fragment (black dots) approximated by a Gaussian function (black line), yielding a FWHM of ∼350 nm. The localization accuracy, determined from 77 individual localizations of the same ATTO520 fluorophore, was determined to 20 nm (red curve). Measurements were performed in PBS, pH 7.4 at a laser power of 30 mW at 514 nm using TIRF microscopy.

Since oxidation of the radical anion by oxygen, i.e. the repopulation of the singlet ground state (k_ox), is very inefficient the dark state exhibits a relatively high thermal stability with lifetimes of several hundred milliseconds. However, the lifetime of the radical anion state can be easily increased further by decreasing the oxygen concentration (e.g. oxygen removal) thus supporting the critical role of oxygen as oxidizing agent.³²

The lifetime of the fluorescent state is almost exclusively controlled by the excitation power as expected for photoinduced intersystem crossing in the presence of high concentrations of triplet quencher (10–100 mM) and can be adjusted between tens to hundreds of milliseconds dependent on the fluorophore properties, respectively. On the other hand, the lifetime of the nonfluorescent dark state, i.e. the lifetime of the radical anion state, is mainly controlled by the oxygen concentration, the oxygen accessibility of individual fluorophores, and their redox properties.

In aqueous buffer in the presence of 100 mM MEA several standard fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, and ATTO700 volunteer for photoswitching microscopy with subdiffraction optical resolution. As exemplified in Fig. 1B for an individual ATTO520 labeled F(ab′)₂ fragment adsorbed non-specifically on a glass coverslide reversible transitions of the fluorophore between a fluorescent and nonfluorescent state are observed under continuous laser illumination. Typically, 1000 to 2000 photons emitted by the fluorophore were used for each localization, which suggests a theoretically achievable localization accuracy of less than 10 nm.²³ On the other hand, the localization accuracy, determined from 77 individual localizations of the same ATTO520 fluorophore, was determined to ∼20 nm (Fig. 1C).

The difference of experimental and theoretical values is caused most probably by mechanical instabilities of the microscope system. Similar photoswitching properties as illustrated for ATTO520 in Fig. 1 were found for a number of other standard fluorophores, such as ATTO565, ATTO655, ATTO680 and ATTO700.

To demonstrate the potential of photoswitching microscopy for subdiffraction-resolution fluorescence imaging of cellular structures we used fixed COS-7 cells and stained the microtubule network applying immunocytochemistry and antibody fragments labeled with ATTO520 and ATTO655 (Fig. 2). The experiments were carried out in PBS buffer, in the presence of 100 mM MEA. As can be easily seen in Fig. 2A and 2B photoswitching images show superior resolution as compared to conventional wide-field images of the microtubule network. Frame stacks of up to 16000 images were recorded with a rate of 10 Hz to demonstrate the evolution of a high-resolution fluorescence image with time (Fig. 2C–2F). These experiments demonstrate that high quality subdiffraction-resolution images can be reconstructed already from 4000–8000 images.

Fig. 2 Photoswitching microscopy performed with the two standard fluorophores ATTO655 and ATTO520. The upper right (A) and lower parts (B) of the images are conventional immuno-fluorescence images of microtubules in COS-7 cells labeled with a primary antibody and ATTO655 (A) and ATTO520 (B) labeled F(ab′)₂ fragments. Photoswitching microscopy images with subdiffraction resolution (scale bar 1 μm) are superimposed in the lower left (A) and upper part (B) of the images to visualize resolution improvement. Measurements were performed in PBS, pH 7.4 in the presence of 100 mM MEA at a laser power of 30 mW at 647 nm (A) and 514 nm (B) with a frame rate of 10 Hz. (C–F) Evolution of a superresolution image with ATTO520 as fluorophore (1000–16000 frames corresponding to measurement times of 100–1600 s).

Dual-color photoswitching microscopy achieving subdiffraction resolution is demonstrated in Fig. 3. The microtubule network of COS-7 cells was labeled with a 1 : 1 mixture of F(ab′)₂ fragments labeled with ATTO520 and ATTO655. Measurements for ATTO520 and ATTO655 were recorded sequentially, with a filter change in between. The excellent spatial resolution achieved for both fluorophores is immediately visible because both fluorophores were attached to the same structure. The dual-color photoswitching image (Fig. 3B) easily reveals structural details that are hidden in the conventional wide-field image (Fig. 3A). In accordance with previously published work,⁸ small chromatic aberrations in the range of 50 to 100 nm were observed between the two color channels which was corrected manually by superimposing the final images. These data evidence that multicolor photoswitching microscopy can be readily used to study protein colocalization in cells with subdiffraction resolution.

Fig. 3 Dual-color photoswitching microscopy with ATTO520 and ATTO655 labeled microtubules in COS-7 cells. The reconstructed photoswitching image is shown in (B) and compared to the corresponding conventional wide-field image (A). Measurements were performed sequentially in PBS, pH 7.4 in the presence of 100 mM MEA at laser powers of 30 mW at 647 nm and 514 nm with a frame rate of 20 Hz. 16000 images were measured from the two spectrally different fluorophores (scale bar 5 μm).

Moreover, multicolor photoswitching microscopy can be used for molecular quantification of proteins localized in specific subcellular compartments.¹⁸ In Fig. 4 ATTO520 labeled Fab-fragments and ATTO655 labeled antibodies were used to label the microtubule network and cytochrome c oxidase in the inner mitochondrial membrane of COS-7 cells, respectively. The high-resolution fluorescence image (Fig. 4B) was obtained from a series of ∼10000 individual localizations of individual single antibodies and clearly shows much more structural details. By further processing of the subdiffraction-resolution fluorescence image using a threshold algorithm that identifies sites of multiple localizations the total number of proteins (corresponding to localizations) located in a specific subcellular area can be obtained.¹⁸

Fig. 4 Dual-color photoswitching microscopy with ATTO520 labeled microtubules and ATTO655 labeled cytochrome c oxidase localized in the inner mitochondrial membrane of COS-7 cells. The reconstructed dual-color photoswitching image (expanded section) is shown in (B) and compared to the corresponding conventional wide-field image (A). Measurements were performed subsequently in PBS, pH 7.4 in the presence of 100 mM MEA at laser powers of 30 mW at 647 nm and 514 nm with a frame rate of 20 Hz. 16000 images were measured from the two spectrally different fluorophores (scale bar 5 μm).

Conclusion

We demonstrate photoswitching microscopy with an optical resolution of ∼20 nm using standard organic fluorophores out of different spectral regions. Especially rhodamine and oxazine derivatives whose triplet states can be depopulated in the presence of 10–100 mM thiol-containing reducing compounds such as β-mercaptoethylamine to form radical anions are suited to achieve subdiffraction resolution. Due to the low reactivity of the radical anion states of these fluorophores with oxygen they are thermally stable under aqueous buffer conditions. The lifetime of the fluorescent and nonfluorescent state can be controlled by the excitation intensity, the concentration of the reducing agent, and the oxygen concentration, and accessibility of the fluorophore and can be adjusted between tens to hundreds of milliseconds. Thus, commercially available standard fluorophores and fluorescent probes, e.g. labeled antibodies or Fab-fragments can be directly used as molecular photoswitches at the single molecule level. The fact that molecular oxygen does not have to be removed simplifies the method and suggests the compatibility of some of these standard fluorophores for photoswitching microscopy even in living cells. Living cells contain besides many thiol-containing proteins, glutathione in the lower millimolar concentration range (i.e. up to 10 mM). The tripeptide glutathione is a major intracellular non-protein thiol compound with reducing properties and is essential for the optimum activity of some enzymes and other cellular macromolecules as well as for detoxicitation.^33–35 As recently demonstrated,²² some fluorophores can in fact be switched reversibly between a fluorescent and nonfluorescent state in the presence of 10 mM glutathione. Therefore, we believe that our new method might be also applicable for in vivo multicolor photoswitching microscopy with subdiffraction resolution using selected standard fluorophores. In addition, one should bear in mind that reversibly photoswitchable fluorophores are useful for molecular quantification experiments in cells because the number of active (fluorescent) fluorophores can be easily adjusted via the excitation intensity.¹⁸

Acknowledgements

This work was supported by the Biophotonics and the Systems Biology Initiative (FORSYS) of the German Ministry of Research and Education (BMBF, grants 13N9234 and 0315262).

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