Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations

Abstract

Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample.

Fig 1

Spectral-FPALM experimental setup (a). Filters (F) blocked reflected laser light and cellular auto-fluorescence. Emitted fluorescence was split by a 50:50 beamsplitter (BS). The reflected path was redirected to the camera and spread into its spectrum with a dispersive prism (P) creating the Spectral Channel (b, right). Light transmitted through the BS formed the Spatial Channel (b, left). Two additional mirrors (M2 and M3) ensured that the same focal planes were imaged. Three different colors of fluorescent beads (yellow-green, orange, and red) were simultaneously imaged (b). Colored boxes represent spatial locations (b, Spatial Channel) and corresponding spectral images and spectral distributions (b, Spectral Channel). The red bead (c) was taken as the reference bead since there was no noticeable shift between the vertical positions in the spatial and spectral channels. The orange bead (d) and yellow-green bead (e) show downward shifts of the spectral distribution of a few pixels (d) to approximately 12 pixels (e), indicating different fluorescent spectra. Calibration enabled measurement of the spectral profile (f, solid lines) of each of the beads in (c, d, and e). The dip in spectral intensity ~560 nm (gray box) was due to notch filters. Measurements with a spectrometer (f, dashed lines) confirmed the spectral measurements of Spectral-FPALM. Scale bar is 2 μm in (b) and 1 μm in (c, d, and e).

Fig 2. Spectral-FPALM measurements of spectral wandering and identification of multiple fluorescent species.

Three examples of photoactivatable fluorophores undergoing spectral wandering are shown. Individual molecules spectrally wandered before photobleaching; (a) PAmKate, from mean emission wavelength ~600 nm to ~625 nm and back again; (b) CAGE 590 from ~620 nm to ~645 nm before photobleaching, and (c) Dendra2 from ~590nm to ~620nm before photobleaching. Error bars shown in (a-c) are due to shot noise from the number of detected photons. Single and multi- color images of NIH-3T3 cells were recorded using Spectral-FPALM. Single molecules were localized and identified based on the criteria shown in Fig B in S1 File. Each panel represents a different sample; Dendra2-HA (d), PAmCherry-cofilin (e), and PAmKate-TfR (f) and all three labels (g). The fraction of molecules identified as each fluorescent species is displayed at the bottom right of the single color cell panels (d-f). Misidentifications of Dendra2 (d) and PAmCherry (e) are less than 5%. Misidentification of PAmKate as PAmCherry is ~12%, largely due to fewer numbers of molecules in the PAmKate sample and large fraction of spectral wanderings of PAmKate (Table A in S1 File). Scale bars are 2 μm.

Fig 3. Super-resolution maps of spectral wandering and emission wavelength differences for localized individual molecules.

(a) Super-resolution render of wavelength-dependence of emission spectra of individual localized molecules shows spatial gradients in emission wavelength of Dendra2-HA. Cyan points: molecules below the median wavelength. Magenta points: molecules above the median wavelength. Molecules which emitted for more than one consecutive frame are plotted using their position in each frame. Scale bar 2 μm. (b) Super-resolution render of spatial distribution of molecules with and without spectral wandering. Green points: molecules not exhibiting spectral wandering. Yellow points: molecules exhibiting spectral wandering. Molecules which emitted for more than one consecutive frame are each localized to an averaged single position. Scale bar 2 μm.