Fig 1.
Definition of terms and description of data treatment methods I and II.
Top: Definitions of symbols describing data arrays, content of arrays, and the method of determination. Bottom: schematic description of data processing leading to the resolved signals.
Fig 2.
Description of the different signals used in the presented data treatment methods.
Spectra created from the sum of the pixel-by-pixel treated spectra, total intensity images, and intensity profile plots corresponding to the blue dashed line in the total intensity images. The plotted signals arise from treatment of data recorded following excitation of a PVA thin film stained with F18, MitoTracker Red, ATTO647N, and LTA zeolites doped with europium(III) ions. Assignment of signals: Sā, raw data. S, smoothed raw data; X, channel-by-channel background subtracted data with a built-in function; L, resolved data; Bexp, experimentally determined background signal from another area of the sample.
Fig 3.
Walkthrough of methods I and II for resolving sharp emission bands in spectrally resolved data.
In method I, the smoothed raw data (S) is differentiated, and the smoothed gradient (Ī) is used to calculate the signal L(I) as the absolute value of the gradient. In method II, the smoothed gradient (Ī) is compared with the threshold T1 to provide the first set of feature onset and end anchors. Using the thresholds T2, and T3 the final anchor points are determined and the baseline (B) is defined. Subtracting the baseline (B) from the raw data resolves the fluorescent signal L(II) as the sharp emission bands. This baseline is identical to the background signal. The integration time of the spectrum shown is 1 second. All signals are extracted from a single bright pixel in the image shown in Fig 2.
Fig 4.
Smoothed spectra S and resolved spectra L(II) from individual pixels.
Each spectrum corresponds to an individual pixel in an image of PVA thin film stained with F18, Mito Tracker Red, ATTO647N and LTA zeolites doped with europium(III) ions following 465 nm excitation and PVA thin film stained with F18, Mito Tracker Red and LTA zeolites doped with terbium(III) ions following 488 nm excitation.
Fig 5.
Images of multilabeled samples where method II is used to resolve the fluorescent signal from sharp emission bands and the background.
Smoothed raw data S (integrated for all the pixels in the image) and RGB images where green color indicates the background signal B from F18, MitoTracker Red, and ATTO647N and red or blue color indicate the fluorescence L(II) from Eu(III) or Tb(III), respectively. The signal of the lanthanides L(II) is integrated inside the spectrally filtered band indicated for each image, whereas the signal B is integrated over the whole spectral region. In the case of Eu(III) the spectral region in gray color was not integrated, since the Eu(III) band in that region was not sharp enough to be resolved by method II. The R, G, and B images that form the merged RGB image are individually normalized in order to resolve the features detected in the background signal and from the lanthanide centered emission. In the bottom row, the resolved spectra L show that several lanthanide(III) ions can be imaged simultaneously if the fluorescent signals are integrated within a specific lanthanide band as illustrated by the dashed lines.
Fig 6.
Comparison of signal and background of raw data and different background subtraction methods.
Total fluorescent signal along the diagonal lines (Fig 2 for Eu, data for Tb not shown) and signal vs. background in images of PVA thin film stained with F18, Mito Tracker Red, ATTO647N and LTA zeolites doped with europium(III) ions following 465 nm excitation and Tb@LTA with F18 and MT following 488 nm excitation. The signal or background level corresponds to the maximum or minimum of the intensity profile, respectively.