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Updated: Sep 28, 2025

AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells
Published on: June 23, 2023
High-precision background correction and artifact suppression for ultrafast spectroscopy by quasi-simultaneous
E Deniz1, J G Löffler1, A Kondratiev1
1Institute of Biophysics, Goethe University Frankfurt am Main, Frankfurt am Main 60438, Germany.
Abstract:
Alternating acquisition of background and sample spectra is often employed in conventional Fourier-transform infrared spectroscopy or ultraviolet-visible spectroscopy for accurate background subtraction. For example, for solvent background correction, typically a spectrum of a cuvette with solvent is measured and subtracted from a spectrum of a cuvette with solvent and solute. Ultrafast spectroscopies, though, come with many peculiarities that make the collection of well-matched, subtractable background and sample spectra challenging. Here, we present a demountable split-sample cell in combination with a modified Lissajous scanner to overcome these challenges. It allows for quasi-simultaneous measurements of background and sample spectra, mitigating the effects of drifts of the setup and maintaining the beam and sample geometry when swapping between background and sample measurements. The cell is moving between subsequent laser shots to refresh the excited sample volume. With less than 45 μl of solution for 150 μm optical thickness, sample usage is economical. Cell assembly is a key step and covered in an illustrated protocol.

