| Autor: |
Fernando SI, Martineau JT, Hobson RJ, Vu TN, Baker B, Mueller BD, Menon R, Jorgensen EM, Gerton JM |
| Jazyk: |
angličtina |
| Zdroj: |
Optics express [Opt Express] 2023 Sep 25; Vol. 31 (20), pp. 33565-33581. |
| DOI: |
10.1364/OE.499929 |
| Abstrakt: |
By engineering the point-spread function (PSF) of single molecules, different fluorophore species can be imaged simultaneously and distinguished by their unique PSF patterns. Here, we insert a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to produce distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrate that the resulting PSFs can be localized spatially and spectrally using a maximum-likelihood estimation algorithm and can be utilized for hyper-spectral super-resolution microscopy of biological samples. We produced superresolution images of fixed U2OS cells using X-PSFs for dSTORM imaging with simultaneous illumination of up to three fluorophore species. The species were distinguished only by the PSF pattern. We achieved ∼21-nm lateral localization precision (FWHM) and ∼17-nm axial precision (FWHM) with an average of 1,800 - 3,500 photons per PSF and a background as high as 130 - 400 photons per pixel. The modified PSF distinguished fluorescent probes with ∼80 nm separation between spectral peaks. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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