Abstract
New innovations in single-molecule localization microscopy (SMLM) have revolutionized optical imaging, enabling the characterization of biological structures and interactions with unprecedented detail and resolution. However, multi-color or hyperspectral SMLM can pose particular challenges which affect image quality and data interpretation, such as unequal photophysical performance of fluorophores and non-linear image registration issues, which arise as two emission channels travel along different optical paths to reach the detector. In addition, using evanescent-wave based approaches (Total Internal Reflection Fluorescence: TIRF) where beam shape, decay depth, and power density are important, different illumination wavelengths can lead to unequal imaging depth across multiple channels on the same sample. A potential useful approach would be to use a single excitation wavelength to perform hyperspectral localization imaging. We report herein on the use of a variable angle tunable thin-film filter to spectrally isolate far-red emitting fluorophores. This solution was integrated into a commercial microscope platform using an open-source hardware design, enabling the rapid acquisition of SMLM images arising from fluorescence emission captured within ∼15 nm to 20 nm spectral windows (or detection bands). By characterizing intensity distributions, average intensities, and localization frequency through a range of spectral windows, we investigated several far-red emitting fluorophores and identified an optimal fluorophore pair for two-color SMLM using this method. Fluorophore crosstalk between the different spectral windows was assessed by examining the effect of varying the photon output thresholds on the localization frequency and fraction of data recovered. The utility of this approach was demonstrated by hyper-spectral super-resolution imaging of the interaction between the mitochondrial protein, TOM20, and the peroxisomal protein, PMP70.
Highlights
With the advent of single-molecule localization microscopy (SMLM), new limits of optical resolution have been realized, and the range of biological questions that can be addressed with fluorescence imaging has increased dramatically
To assess how localization is affected by intensity, superresolution images acquired for both fluorophores were analyzed at center wavelengths of 670 nm (CW 670) and CW 700 over a range of photon output thresholds (POTs) [Figs. 5(a) and 5(b)]
Scale bars = 5 μm. (c) A plot of localization frequency vs photon output threshold for all four fluorophore-spectral window combinations (RSE = 1.93%–3.5% and N = 3). (d) A plot of the fraction of localizations recovered vs threshold for all four fluorophore-spectral window combinations (RSE = 2.2%–3.8%, N = 3). (e) A bar graph of theoretical crosstalk at a series of thresholds for spectral windows CW 670 and CW 700
Summary
With the advent of single-molecule localization microscopy (SMLM), new limits of optical resolution have been realized, and the range of biological questions that can be addressed with fluorescence imaging has increased dramatically. Several of the far-red emitting photoswitchable fluorescent molecules commonly used in dSTORM imaging have been shown scitation.org/journal/rsi experimentally to exhibit favorable and reliable photophysical properties in a buffered reducing environment.12,14 These include an inducible dark state transition, high duty cycle, moderate to high photon output, and sensitivity to 405 nm excitation.. Multi-channel SMLM data are very sensitive to chromatic aberrations, which can become the limiting factor in determining the extent of colocalization uncertainty between structures imaged in two separate spectral channels For these reasons, several groups have recently focused on developing methods for spectrally resolving probes with overlapping far-red emission spectra. We examined the performance of a TTF in distinguishing between a pair of farred SMLM fluorophores with overlapping emission spectra using a single excitation laser line and full-field hyperspectral imaging on a single EMCCD camera
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