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The rising incidence of cancer worldwide is causing an increase in the workload in pathology departments. This, coupled with advanced analysis methodologies, supports a developing need for techniques that could identify the presence of cancer cells in cytology and tissue samples in an objective, fast, and automated way. Fourier transform infrared (FT-IR) microspectroscopy can identify cancer cells in such samples objectively. Thus, it has the potential to become another tool to help pathologists in their daily work. However, one of the main drawbacks is the use of glass substrates by pathologists. Glass absorbs IR radiation, removing important mid-IR spectral data in the fingerprint region (1800 cm−1 to 900 cm−1). In this work, we hypothesized that, using glass coverslips of differing compositions, some regions within the fingerprint area could still be analyzed. We studied three different types of cells (peripheral blood mononuclear cells, a leukemia cell line, and a lung cancer cell line) and lymph node tissue placed on four different types of glass coverslips. The data presented here show that depending of the type of glass substrate used, information within the fingerprint region down to 1350 cm−1 can be obtained. Furthermore, using principal component analysis, separation between the different cell lines was possible using both the lipid region and the fingerprint region between 1800 cm−1 and 1350 cm−1. This work represents a further step towards the application of FT-IR microspectroscopy in histopathology departments. [ABSTRACT FROM AUTHOR]