Optimization and validation of cryostat temperature conditions for trans-reflectance mode FTIR microspectroscopic imaging of biological tissues

Abstract

In Fourier transform infrared (FTIR) microspectrocopy, the tissue preparation method is crucial, especially how the tissue is cryo-sectioned prior to the imaging requires special consideration. Having a temperature difference between the cutting blade and the specimen holder of the cryostat greatly affects the quality of the sections. Therefore, we have developed an optimal protocol for cryo-sectioning of biological tissues by varying the temperature of both the cutting blade and the specimen holder. Using this protocol, we successfully cryo-sectioned four different difficult-to-section tissues including white adipose tissue (WAT), brown adipose tissue (BAT), lung, and liver. The optimal temperatures that required to be maintained at the cutting blade and the specimen holder for the cryo-sectioning of WAT, BAT, lung, and liver are (-25, -20 °C), (-25, -20 °C), (-17, -13 °C) and (-15, -5 °C), respectively. The optimized protocol developed in this study produced high quality cryo-sections with sample thickness of 8-10 μm, as well as high quality trans-reflectance mode FTIR microspectroscopic images for the tissue sections. •Use of cryostat technique to make thin sections of biological samples for FTIR microspectroscopy imaging.•Optimized cryostat temperature conditions by varying the temperatures at the cutting blade and specimen holder to obtain high quality sections of difficult-to-handle tissues.•FTIR imaging is used to obtain chemical information from cryo-sectioned samples with no interference of the conventional paraffin-embedding agent and chemicals.

Keywords: Cryo-sectioning and FTIR microspectroscopic imaging of biological tissues; Cryostat; FTIR; Microspectroscopic imaging; Tissue sectioning.

Graphical abstract

Fig. 1

Schematic representation of the overall experimental workflow for the tissue imaging. For each tissue sample (A) cryomold was half-filled with OCT, (B,C) tissue sample was placed in the cryomold and covered with OCT, (D) cryomold was placed inside the cryostat for 3 min until completely frozen, (E) temperature at the cutting blade and specimen holders was adjusted to optimal values (F,G,H) specimen block was removed from the cryomold and placed on the specialized metal grid that fit onto the specimen holder, (I,J) sections were cut in the cryostat, (K) sections were transferred to a low-e microscopic slide, (L) before imaging, sections were kept in a desiccator and dried under vacuum pump, (M,N) IR spectra were recorded on the tissue sections and (O) respective spectral data were obtained.

Fig. 2

FTIR visual images and the corrosponding average absorbance images of (a) WAT, (b) BAT, (c) Liver and (d) Lung, recpectively. The red color represents areas of the highest absorbance while the blue color represents areas with the lowest absorbance.

Fig. 3

Average trans-reflectance FTIR spectra of WAT (1), BAT (2), Lung (3) and Liver (4) tissue sections (10 μm thickness) in the 3300–2800 and 1800–750 cm⁻¹ regions revealing the distinct spectral regions for lipids, proteins, nucleic acids, and carbohydrates. ν = stretching vibrations, δ = bending vibrations, s = symmetric vibrations and as = asymmetric vibrations.

Fig. 4

Second derivative FTIR spectra generated from Fig. 2 of liver, lung, BAT and WAT tissue sections (10 μm thickness) in the regions of (a) 310–2800 cm⁻¹ and (b) 1800–875 cm⁻¹.