Hadamard-Transform Fluorescence Excitation-Emission-Matrix Spectroscopy

Abstract

We present a fluorescence excitation-emission-matrix spectrometer with superior data acquisition rates over previous instruments. Light from a white light emitting diode (LED) source is dispersed onto a digital micromirror array (DMA) and encoded using binary n-size Walsh functions ("barcodes"). The encoded excitation light is used to irradiate the liquid sample and its fluorescence is dispersed and detected using a conventional array spectrometer. After exposure to excitation light encoded in n different ways, the 2-dimensional excitation-emission-matrix (EEM) spectrum is obtained by inverse Hadamard transformation. Using this technique we examined the kinetics of the fluorescence of rhodamine B as a function of temperature and the acid-driven demetalation of chlorophyll-a into pheophytin-a. For these experiments, EEM spectra with 31 excitation channels and 2048 emission channels were recorded every 15 s. In total, data from over 3000 EEM spectra were included in this report. It is shown that the increase in data acquisition rate can be as high as [{n(n + 1)}/2]-fold over conventional EEM spectrometers. Spectral acquisition rates of more than two spectra per second were demonstrated.

HPLC-Detector Based on Hadamard-Transform Fluorescence Excitation-Emission-Matrix Spectroscopy

“…Note that the multiplex advantage of HT-EEM scales with ( n + 1)/2 compared to a sequential excitation with a single wavelength and simultaneous collection of the entire emission spectrum. When comparing to a conventional EEM spectrometer which acquires each (λ exc ,λ em ) data pair sequentially, this advantage increases to approximately n 2 /2 …”

“…The multiplexed HT-spectra were demodulated after the HPLC separations were complete as described before . The decomposition of the multivariate matrices into component matrices can be performed with a variety of computational analysis tools.…”

“…Recently, we introduced a much faster EEM spectrometer achieving for the first time an acquisition rate of higher than 30 spectra per minute. − This EEM instrument uses a fully programmable 31-channel light source and a conventional 2048-channel fluorescence photodiode array spectrometer. The programmable light source produces 31 different combinations of 31 wavelength channels based on Hadamard-matrix multiplexing .…”

“…The programmable light source produces 31 different combinations of 31 wavelength channels based on Hadamard-matrix multiplexing . A fluorescence spectrum is recorded for each combination (“bar code”) of excitation wavelength channels, and the resulting data cube is demultiplexed using the inverse Hadamard transform yielding the EEM. , …”

“…Our first Hadamard-transform EEM (HT-EEM) instrument was based on a programmable light source that used a digital micromirror array (DMA) as a polychromator to select discrete excitation wavelengths. 14 Unfortunately, the DMA design prevented the use of the DMA with UV light, limiting the light source to the visible region (λ exc = 400−750 nm).…”