After the cleaning process to remove media, a drop of 10 ll with
approximately 485 parasites was deposited on a calcium fluoride
(CaF2) window. The sample was dried by an Eppendorf Concentrator,
which allows the thin film formation of the unanalyzed Leishmaniasis.
The IR spectra was collected by a microscope (Spotlight
Perkin Elmer 400, USA) coupled to FT-IR spectrophotometer (Spectrum
400), which was controlled by a computer with Spotlight 400
software, version 3.6.2.
The sample was analyzed in four random regions, totaling 22
points. The spectra were recorded in regions with approximately
the same thickness. The band centered at 1650 cm1 had the highest
value of absorbance around 0.9 to 1.0 for all spectra. Each spectrum
was obtained from an average of 64 spectra per point with
spectral resolution of 4 cm1. The spectral range was from 4000
to 750 cm1.
These spectra were analyzed by cluster analysis and deconvolution
procedure base on spectra second derivatives. The multivariate
statistical analysis was performed by Cluster Analysis [29]
using OPUS software version 4.2 with the following parameters:
second derivative, smoothing 9 points, Ward’s algorithm, and Scaling
to 1st range method. Cluster analysis classifies objects into
groups which show similarities [30] and is commonly used to rapidly
differentiate/classify spectra of microorganisms [31].
Gaussians curve fitting was used in the observed band deconvolutions,
thereby quantifying the contribution of specific biochemical
species in each region of the spectrum [32]. The biochemical
changes, which underlie the separation obtained by cluster analysis,
can then be elucidated.