Fig 1.
Spectral properties of chosen dyes.
Normalized excitation (filled) and emission (lines) spectra of F18 (green), MitoTracker Red (magenta), ATTO647N (red), Tb(III) (black), and Eu(III) (blue).
Table 1.
Excitation parameters and filters used for microscopy.
Fig 2.
Areas used for imaging one zeolite using the four excitation wavelengths.
Fig 3.
Schematic presentation of the model system.
The model system is based on Ln(III)-doped zeolites and stained polyvinyl alcohol and is used to experimentally determine noise and fluorescent signals. Ln(III)-doped zeolites can be used as fix points for locating the region of interest and for instrument benchmarking.
Table 2.
Photophysical properties of the chosen dyes and lanthanide ions.
Fig 4.
Model system for the scanning confocal fluorescence microscopy.
Total intensity spectra and confocal fluorescence microscopy images of Eu(III)@LTA with ATTO467N in the PVA film following 465 nm excitation and Eu(III)@LTA with F18, MitoTracker Red, and ATTO467N in the PVA film following excitation at 465 nm or at 488 nm monitored through a green bandpass filter (539–556 nm) or a red bandpass filter (604–644 nm) of an Edmund Optics filter kit. The profiles of the diagonal lines (dotted) through the images are plotted beneath showing the pixel intensities.
Fig 5.
The brightness and measured intensity of the dyes in the specific conditions used.
a) Brightness is the product of the quantum yield and the molar absorptivity of the dye. The brightnesses of Eu(III) and Tb(III) are calculated from the quantum yield of their DOTA-complexes and the absorptivities of their acetate salts (our own measurements). b) The measured intensities correspond to the fluorescent signals arising from each fluorescent probe (fully integrated spectra) upon different excitation wavelengths.
Fig 6.
Fluorescent signal of each dye at their optimal excitation wavelength.
Each emission window is simulated by integrating the acquired emission spectra only in the wavelength area determined by the commercial optical emission bandpass filters. The Edmund Optics filter kits are a standard set of filters for 1) FITC, 2) Texas Red, and 3) Cy5, with wavelength ranges of the emission bandpass filters at 539–556 nm, 604–644 nm, and 672–712 nm, respectively. The Chroma laser bandpass filter sets for 4) FITC, 5) mCherry, and 6) ATTO647N had wavelength ranges of 539–550 nm, 593–667 nm, and 669–741 nm, respectively. The background from PVA-sample without dyes was subtracted. Error bars illustrate the standard deviation in the observed signals of two separate pixels in the images.
Fig 7.
Emission spectra of F18, MitoTracker Red, and ATTO647N upon excitation at 465 nm, 488 nm, 560 nm, and 633 nm.
Fig 8.
Fluorescent signal of dyes in simulated emission windows upon different excitation wavelengths.
Each emission window is imitated by integrating the acquired emission spectra only in the wavelength area determined by the commercial optical bandpass filters. The Edmund Optics filter kits are a standard set of filters for 1) FITC, 2) Texas Red, and 3) Cy5, with transmission wavelength ranges of the emission bandpass filters at 539–556 nm, 604–644 nm, and 672–712 nm, respectively. The Chroma laser bandpass filter sets for 4) FITC, 5) mCherry, and 6) ATTO647N had wavelength ranges of 539–550 nm, 593–667 nm, and 669–741 nm, respectively. The background from PVA-sample without dyes was subtracted. The dashed lines are guides for the eye at intensity of 5000 a.u. Error bars represent the standard deviation of two separate pixels in each image.
Fig 9.
Bleaching of F18, MitoTracker Red and ATTO647N.
The spectra were recorded in 1 s intervals for 100 s. For F18 the whole spectrum was integrated, for MitoTracker Red only the wavelength range of 578–817 nm was integrated, and for ATTO647N the wavelength range of 567–817 nm was integrated. The integrated intensities were normalized to the range of 0–1. The excitation powers can be found in the methods section.