We evaluated 26 organic dyes spanning the visible and near infrared (NIR) spectral range. These dyes include: blue-absorbing (Atto 488, Alexa 488, Atto 520, fluorescein, FITC, Cy2), yellow-absorbing (Cy3B, Alexa 568, TAMRA, Cy3, Cy3.5, Atto 565), red-absorbing (Alexa 647, Cy5, Atto 647, Atto 647N, Dyomics 654, Atto 655, Atto 680, Cy5.5), and NIR-absorbing (DyLight 750, Cy7, Alexa 750, Atto 740, Alexa 790, IRDye 800CW) fluorophores. They can be categorized structurally as cyanines (Cy2, Cy3, Cy3B, Cy3.5, Cy5, Alexa 647, Dyomics 654, Cy5.5, Cy7, DyLight 750, Alexa 750, Alexa 790, IRDye 800CW), rhodamines (Alexa 488, Atto 488, Atto 520, TAMRA, Atto 565, Alexa 568), oxazines (Atto 655, Atto 680), carbopyronines (Atto 647N, Atto 647, Atto 740), and fluoresceins (FITC, fluorescein).
Previously, STORM imaging has been performed either with activator-reporter dye pairs3,6,12,14,40,41
, in which "activator" molecules are used to enhance the activation rate of the photoswitchable "reporter" molecules, or photoswitchable reporters alone8,10,11,14–16
. Since the intrinsic properties of the photoswitchable reporters limit the STORM imaging performance in both cases in a similar manner, we characterized these properties in the reporter-alone configuration.
Method of photoswitchable probe characterization
We characterized the switching properties using dye-labeled proteins that were adsorbed to coverglass and illuminated with light at 488 nm, 561 nm, 647 nm, or 752 nm, to excite blue, yellow, red, or NIR dyes, respectively. Under these conditions, many fluorophores reversibly transition between on and off states with a characteristic duty cycle and number of emitted photons per switching event. Although it is often convenient to use an additional “activation laser” to increase the on-switching rate and hence speed up the imaging process, the on/off duty cycle determined in the absence of the activation laser represents the lowest achievable duty cycle for a dye and thus sets the resolution limit based on the Nyquist criterion. The number of detected photons per switching event is typically the same independent of whether an additional activation light is used.
For each dye, we determined four switching properties: (i) number of photons detected per switching event; (ii) on/off duty cycle; (iii) survival fraction; and (iv) number of switching cycles. We recorded the number of photons detected in each switching event for many molecules, from which we constructed a photon number histogram and determined the mean value. The on/off duty cycle was measured as the fraction of time spent in the on state averaged over many molecules. Because the molecules were originally in the on state and a certain illumination time is required for them to reach a quasi-equilibrium between the on and off states, we used a sliding window of 100 sec to monitor changes in the on/off duty cycle as a function of exposure time. The duty cycle value during the time window 400–600 sec was used to represent the characteristic, equilibrium duty cycle because the dyes typically achieved quasi-equilibrium between on and off states by this point. Since some dyes switched for a limited number of cycles before photobleaching, a substantial fraction of molecules may be photobleached by the time the on/off equilibrium is reached. We therefore also characterized the survival fraction of unbleached molecules as a function of illumination time. This quantity can be used together with the time-dependent duty cycle value to determine what degree of photobleaching has occurred prior to achieving a sufficiently low density to resolve single molecules. Finally, we also determined for each molecule the number of switching cycles during the data acquisition time, which represents a lower bound value given that some molecules did not photobleach before the end of the observation period.
We correlated the above properties with the quality of STORM images for each dye by recording images of different cellular structures, including microtubules which have a linear (or cylindrical) morphology, and clathrin-coated pits (CCPs) which have a spherical morphology. Because the switching performance and photostability of many dyes are enhanced by the presence of a primary thiol and/or a low oxygen environment, we performed experiments in the presence of an enzymatic oxygen scavenging system and a primary thiol, either β-mercaptoethanol (βME) or mercaptoethylamine (MEA), unless otherwise indicated.
illustrates our method of dye characterization, using Alexa 647, Atto 655, and Cy5.5 as representative examples. We show examples of single-molecule fluorescence time traces (, Supplementary Fig. 1
), histograms of photon number distributions (), and duty cycle plots along with the corresponding survival fractions (). The dyes exhibited three distinct switching behaviors: high photon yield per switching event and low duty cycle (Alexa 647); low photon yield and low duty cycle (Atto 655); and high photon yield and high duty cycle (Cy5.5).
Photon number and duty cycle
To demonstrate the importance of photon number and duty cycle, we recorded three-dimensional (3D) STORM images of CCPs, which provided an excellent metric for assessing a dye’s performance because of their small size (~100–200 nm diameter) and the requirement for high spatial resolution to reveal their hollow, spherical shell-like structure. The 3D STORM images were taken using astigmatism imaging, the most easily implemented 3D super-resolution imaging approach40
. When imaging Alexa 647-labeled CCPs, the hollow shape of the pit was clearly resolved () due to the high photon yield of individual switching events (~5,000 photons, ) and low on/off duty cycle (~0.001, ). The xy
- and xz
cross-sections taken from the midplanes of an example CCP demonstrate the expected cup-like shape of the pit (). The superposition of xy
cross-sections of multiple CCPs showed the expected hollow, ring-like structure ().
In contrast to Alexa 647, the low photon yield of Atto 655 (~660 photons per switching event, ), and the resulting low localization precision, blurred the CCP image substantially such that its hollowness was no longer apparent (), despite the fact that the duty cycle of Atto 655 (~0.001, ) was comparable to that of Alexa 647. In a different manner, the high duty cycle of Cy5.5 (~0.007, ) also negatively impacted the quality of the CCP images (). Because of the large fraction of time each dye spent in the fluorescent state, a considerable reduction in labeling density (or photobleaching of molecules prior to imaging) was necessary to ensure single-molecule detection and localization. As a result, the sparse localization pattern no longer resolved the hollow morphology of the CCP, despite the high photon yield of Cy5.5 (~6,000 photons per switching event, ). These examples demonstrate that both high photon number and low duty cycle are essential to achieving high-resolution STORM images. In Supplementary Figures 2–27
and , we show the wide range of photon number and duty cycle values of the 26 dyes.
Summary of switching properties of the 26 dyes tested in this study
Photostability and number of switching cycles
The dyes also exhibited large variations in their survival fraction and number of switching cycles before photobleaching (Supplementary Figs. 2–27
, ). Although the three dyes in showed comparably high survival fractions after the molecules reached an on/off equilibrium state (), some dyes photobleached much more rapidly. For example, Cy2 showed little recovery to the on state after the initial switching off event, and by the time the duty cycle reached equilibrium, only a very small fraction of molecules survived. Consequently, we were unable to reconstruct a STORM image using Cy2 (Supplementary Fig. 7
). An interesting example is Atto 565 (Supplementary Fig. 13
). Despite having the highest photon yield per switching cycle among all the dyes screened, its relatively low survival fraction upon reaching on/off equilibrium led to overall lower quality STORM images compared to many other dyes with lower photon yields but higher survival fractions.
Related to the survival fraction is the number of switching cycles afforded by a fluorophore. While it is desirable to have a single switching cycle for some applications, such as for the purpose of counting molecules, in many cases, a large number of switching events is advantageous. Specifically, the detection of multiple switching events from the same fluorophore reduces the stochasticity of the localization error, and in the limit of many cycles, the mean localization positions converge with the true positions of the fluorophores. (Supplementary Fig. 28a
). This effect directly impacts the STORM image quality in that a dye with a low number of switching cycles often results in an image with more jagged and ill-defined spatial features due to localization errors, whereas a dye with a large number of switching cycles results in a smoother and more continuous image due to repetitive sampling of the same structure and thus lower noise in the final image (Supplementary Fig. 28b
). For instance, dyes with relatively few switching cycles, such as Cy3, produced STORM images with sparse localization densities and overall poor quality, despite its high photon number and low on/off duty cycle (Supplementary Fig. 11
). In contrast, fluorophores that exhibited a high number of switching cycles, such as Atto 488 (Supplementary Fig. 2
), resulted in more continuous and better-defined microtubule or CCP structures, in spite of a relatively low photon yield.
Sensitivity of fluorophores to violet light activation
During acquisition of a STORM movie, the number of molecules present in a given frame decreases as the fluorophores are photobleached. This reduction, in turn, decreases the information content in a single frame and increases the number of frames required to construct an image. The use of an “activation” laser of a different wavelength from the excitation laser can increase the activation rate and thus reduce the image acquisition time. In the case of activator-reporter pairs, the activator molecules allow the reporter to be activated by using visible light of different colors6
. When a photoswitchable reporter alone is used for STORM imaging, the activation rate is typically controlled by an ultraviolet or violet light10,20,39
. We tested how the dyes responded to short wavelength activation by switching a field of molecules to a stable dark state and then measuring the fraction of molecules that recovered following a pulse of violet (405 nm) light. Several red and NIR dyes showed appreciable recovery after 405 nm illumination, while even the most sensitive blue and yellow dyes showed modest recovery ().
Sensitivity to violet photoactivation for the 26 dyes tested in this study
Dependence of switching properties on buffer composition
The switching properties of dyes can depend strongly on buffer composition. We tested the switching performance of all dyes under four different buffer conditions: (i) our standard buffers containing both an oxygen scavenging system (glucose oxidase with catalase, or GLOX) and a primary thiol (βME or MEA) — "GLOX + thiol"; (ii) a buffer with GLOX but no thiol — "GLOX only"; (iii) a buffer with a thiol (MEA) but no GLOX — "Thiol only", and (iv) a buffer with neither GLOX nor thiol — "No GLOX or thiol". We found that nearly all of the tested dyes photobleached rapidly under the "No GLOX or thiol" or "Thiol only" conditions, while several dyes switched reasonably well in the "GLOX only" condition (Atto 488, Alexa 488, Atto 520, Cy3B, and Atto 680; see and Supplementary Figs. 29–30
). All 26 dyes achieved equal or better imaging results when using buffers containing both GLOX and thiol (, Supplementary Figs. 29–30
Evaluation of the effect of buffer composition on the switching properties for the 26 dyes tested in this study
To more concretely illustrate the effect of thiol or oxygen on dye switching, we quantitatively characterized the switching properties of Alexa 647, Atto 488, and Atto 655 under the four buffer conditions stated above (Supplementary Fig. 29
). Consistent with previous observations, Alexa 647 required thiol for photoswitching and the photon yield per switching cycle, photostability, and number of switching cycles further increased when oxygen was removed by GLOX42–44
. In contrast, Atto 488 switched well in the absence of thiol, but performed best in buffer containing GLOX. In particular, the photostability of Atto 488 increased in the order of “No GLOX or thiol” or "Thiol only" < "GLOX only" < "GLOX + thiol", and this order correlated well with STORM image quality. In the case of Atto 655, which has been previously proposed to switch well in the presence of a thiol under ambient (high) oxygen concentrations without the need of an oxygen scavenger system15
, we observed substantially better performance in the "GLOX + thiol" condition and found that even the "GLOX only" condition yielded better photostability and image quality than the "Thiol only" condition.
Different thiols can be used to facilitate switching of dyes. We quantitatively characterized all 26 dyes in both MEA (10 mM) and βME (140 mM), where the chosen thiol concentrations were in the range of typical usage reported previously6,12,39
. In some cases, the switching behavior was rather sensitive to the thiol concentration, as illustrated by more detailed investigations of Atto 488, Cy3B, Alexa 647, Atto 655, and DyLight 750. We found that low to moderate concentrations of MEA enhanced the dye’s switching performance and STORM image quality (, Supplementary Fig. 29
), but high concentrations (100 mM) of MEA diminished either the number of switching cycles (Cy3B), or the number of photons per switching cycle (Atto 488 and Alexa 647), or both (DyLight 750 and Atto 655) (Supplementary Fig. 31
). Optimal performance was achieved under relatively low MEA concentrations for these five dyes (1–10 mM, Supplementary Fig. 31
), in contrast to the 50–200 mM concentrations previously recommended12,39
Some dyes also behaved differently in the presence of different thiols. For instance, the duty cycles of Atto 488 and Alexa 647 were substantially lower in the presence of MEA than in βME (Supplementary Figs. 2, 14
, w h ile DyLight 750 showed the opposite trend (Supplementary Fig. 22
). More dramatically, STORM images of good quality could be acquired with Atto 647N and Alexa 750 in the presence of βME, whereas the survival fractions for both dyes were reduced to a nearly unusable level when using MEA (Supplementary Fig. 17, 24
These results emphasize the importance of buffer formulation in STORM imaging. We therefore recommend that practitioners optimize buffer conditions for a given probe.
Dependence of switching properties on light intensity
The imaging laser intensity is another adjustable parameter during STORM imaging. Often, we found the number of photons per switching cycle to be largely independent of laser intensity because both the photon emission rate and off-switching rate tend to depend linearly on the excitation intensity (Supplementary Fig. 32
). Some exceptions were observed, such as the case of Cy3B where the number of photons per switching cycle increased with the excitation intensity (Supplementary Fig. 32
). The equilibrium duty-cycle values also did not vary substantially with the laser intensity, as expected when both off- and on-switching rates depend linearly on the excitation intensity. On the other hand, we observed a decrease in the survival fraction and the total number of switching cycles with increased excitation intensity for some dyes, such as Atto 655 (Supplementary Fig. 32
). Therefore, while strong excitation intensity is often used to increase the switching rate and thus imaging speed, excessive laser intensity could reduce the image quality.
Summary of characterization for 26 dyes
summarizes all four important properties that affect the STORM image quality, photon number, duty cycle, survival fraction, and number of switching cycles. Full details are included in Supplementary Figures 2–27
for each of the 26 fluorophores, including dye structures (when available), fluorescence excitation and emission spectra, representative single-molecule fluorescence traces, photons per switching event, duty cycle, survival fraction, as well as 2D STORM images of microtubules and CCPs. summarizes the sensitivity of the dyes to activation by violet (405 nm) light, and summarizes the on/off switching performance under different buffer conditions.
Overall, we observed large variability in the switching properties of the different dyes. The number of detected photons per switching event ranged from a few hundred to several thousand (). This wide range led to wide variability in the localization precision and hence STORM image resolution. An even broader range of duty cycles was observed, spanning two orders of magnitude from 0.0001 to 0.04 (). While the duty cycle can be adjusted to some extent by changing buffer conditions, in particular by the use of different thiols12,39
, for some dyes, this parameter is simply too high to obtain adequate image quality despite the broad range of conditions tested.
Accordingly, the STORM image qualities afforded by these dyes also showed large variability. Dyes with high photon numbers, low duty cycles, high survival fractions, and many switching cycles in general performed well and produced high quality images (discussed below), while dyes with low photon numbers, high duty cycles, low survival fractions, and/or few switching cycles did not (e.g., fluoresein, FITC, Cy2, Cy3, Atto 565, Cy5.5, Atto 740, Alexa 790, IRDye 800CW).
Several dyes within our screen showed good overall properties, resulting in high-quality super-resolution images. Among these dyes, Alexa 647 (along with its structural analog Cy5) and Dyomics 654 emerged as top choices for their overall excellent properties. When used to image microtubules, we observed that Alexa 647 and Dyomics 654 were able to resolve the hollowness of immunostained microtubule filaments (). The transverse profile of localizations () shows two resolved peaks separated by 36–38 nm, as expected for the projection of a 25 nm diameter cylinder that has been broadened by primary antibodies prior to staining with dye-labeled secondary antibodies.
Alexa 647 and Dyomics 654 resolve the hollow structure of immunostained microtubules
Multi-color STORM imaging with spectrally distinct dyes
From our screen we identified dyes with good performance in each of four distinct spectral ranges (blue: 480–540 nm, yellow: 545–600 nm, red: 640–700 nm, and NIR: 740–805 nm) to demonstrate four-color STORM imaging. These dyes were Atto 488 in the blue range, Cy3B in the yellow range, Alexa 647, Cy5, and Dyomics 654 in the red range, and DyLight 750, Cy7, and Alexa 750 in the NIR range. Several of these dyes (Atto 488, Dyomics 654, DyLight 750, and Cy7) were used for STORM imaging for the first time. It should be noted that the red dyes Alexa 647, Cy5, and Dyomics 654 performed considerably better than even the best-performing dyes in other spectral regions. In comparison, Atto 488, Cy3B, DyLight 750, Cy7, and Alexa 750 were substantially dimmer, and Cy3B additionally exhibited a lower number of switching cycles and a wider distribution of photons per switching cycle. Nevertheless, when compared to other dyes within each corresponding spectral band, these dyes exhibited the best overall performance in terms of photon output, duty cycle, survival fraction, number of switching cycles, and quality of super-resolution images.
To demonstrate four-color imaging, we first imaged a model sample of in vitro
prepared microtubules labeled with Atto 488, Cy3B, Alexa 647 (or Dyomics 654), and DyLight 750 (, Supplementary Fig. 33a
). The microtubules were labeled separately with each of these dyes and then mixed prior to imaging. The excellent spectral separation afforded by the probes (, Supplementary Fig. 33b
) allowed us to achieve low crosstalk between the four channels (, Supplementary Fig. 33c–f
). We observed 0–3% crosstalk between different channels, except for Cy3B which exhibited higher crosstalk (~8%) into the Atto 488 and Alexa 647 (or Dyomics 654) channels (, Supplementary Fig. 33g
). Quantitative analysis of single, repetitively switching fluorophores allowed us to determine the localization precisions to be 29 nm for Atto 488, 22 nm for Cy3B, 17 nm for Alexa 647 (or Dyomics 654), and 30 nm for DyLight 750 (measured as the full-width at half maximum of the localization distribution of individual probes, Supplementary Fig. 34
). The variation corresponded well with photons per switching event for each dye.
Four-color STORM imaging of in vitro assembled microtubule filaments and crosstalk analysis
Four-color STORM imaging of cellular structures
Finally, we demonstrated the utility of these probes in multicolor imaging of four different cellular targets. For this, we immunostained cells for tubulin, Tom20 (marker for mitochondrial outer membrane), ATL1 (marker for endoplasmic reticulum (ER)), and acetylated tubulin using Atto 488, Cy3B, Alexa 647, and DyLight 750, respectively. shows STORM images for each of the four individual channels over the same field of view within a single cell. The excellent spectral separation allowed for easy color distinction of each cellular structure. shows the magnified, overlaid images of the ER with mitochondria and microtubules with acetylated tubulin, respectively. In many instances, mitochondria nestled with extensive contacts with the ER network (). As expected, only a subset of microtubules are acetylated (). Similar results were obtained when replacing Alexa 647 with Dyomics 654 (Supplementary Fig. 35