Abstract
Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution.
Highlights
Fluorescence microscopy is a widely used imaging tool for cell and molecular biology, and one of the strengths of this technique is the variety of fluorescent probes which may be used to label the specimen
Multicolor super-resolution fluorescence microscopy has been demonstrated by several means, such as employing fluorophores with different fluorescence activation wavelengths,[12,13,14,15] fluorophores with well-separated emission spectra,[9,16,17,18,19] by ratiometric imaging of fluorophores with overlapping emission spectra,[9,20,21] or by taking advantage of other spectral properties, such as fluorescence lifetime.[22]
When 1n approaches one per diffraction-limited volume, the images of individual activated fluorophores begin to overlap substantially, inhibiting their precise localization. In this manner the equilibrium blinking fraction sets an upper bound for the density of fluorophores which may be present on the sample before blinking interferes with stochastic optical reconstruction microscopy (STORM) data acquisition.[4,38,39]
Summary
Fluorescence microscopy is a widely used imaging tool for cell and molecular biology, and one of the strengths of this technique is the variety of fluorescent probes which may be used to label the specimen. To demonstrate that the reactivaphores, Alexa 750 and Alexa 647 (dye structures and fluores- tion wavelength for Alexa 750 can be adjusted into the visible cence spectra are shown in Figure S1 in the Supporting Infor- range by pairing it with an activator fluorophore having the mation), and use these dyes to obtain two-color STORM appropriate absorption spectrum, we labeled antibodies images of biological samples Based on these data, we com- with both Alexa 750 and Cy3. Ure 2 g reveals spatial organization at sub-diffraction-limit length scales (Figure S6 in the Supporting Information) Since both of the photo-switchable dyes used may be switched for multiple cycles, it is possible to directly quantify the localization precision from the experimental data by analyzing the distribution of positions obtained for repeated localizations of a single fluorophore within the sample. The lower localization precision obtained for Alexa 750 compared to Alexa 647 was expected, resulting from the lower number of photons obtained for this dye (Figure 1)
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