Mie scatter corrections in single cell infrared microspectroscopy.

Area of Science:

• Spectroscopy
• Microscopy
• Biophysics

Background:

• Mie scattering significantly complicates chemical interpretation of infrared microscopy spectra from single cells and tissues.
• Existing Mie scatter correction algorithms for infrared spectroscopy often require extensive computation or lack accuracy.

Purpose of the Study:

• To critically review existing Mie scattering correction algorithms.
• To develop and validate an improved iterative algorithm for accurate retrieval of pure absorbance spectra from distorted infrared microscopy data of single cells.

Main Methods:

• Developed an iterative Extended Multiplicative Scatter Correction (EMSC) algorithm utilizing the van de Hulst approximation for extinction efficiency.
• Reduced the number of independent parameters in the EMSC meta-model from three to two, decreasing calculation time by a factor of 10.
• Incorporated the Hilbert transform and FFT algorithm for Kramers-Kronig relations, further accelerating the process by a factor of 100.

Main Results:

• The novel iterative EMSC algorithm successfully retrieves pure absorbance spectra from simulated and measured distorted infrared spectra.
• The algorithm demonstrates a 10x speed improvement due to parameter reduction and an additional 100x speed improvement from Hilbert transform integration.
• Validated the algorithm using simulated spectra based on exact scattering theory and measured spectra from single lung cancer cells.

Conclusions:

• The developed iterative EMSC algorithm offers a significantly faster and more accurate method for correcting Mie scattering in infrared microscopy of single cells.
• This advancement improves the chemical interpretation and multivariate analysis of cellular infrared spectra, crucial for biological and medical research.
• The optimized algorithm provides a valuable tool for researchers analyzing complex biological samples with infrared microscopy.