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Importance of tissue preparation methods in FTIR micro-spectroscopical analysis of biological tissues: 'traps for new users'.

Zohdi V, Whelan DR, Wood BR, Pearson JT, Bambery KR, Black MJ - PLoS ONE (2015)

Bottom Line: We have systematically examined the spectra for any biochemical changes to the native state of the tissue caused by the three methods of preparation and have detected changes in infrared (IR) absorption band intensities and peak positions.In particular, the position and profile of the amide I, key in assigning protein secondary structure, changes depending on preparation method and the lipid absorptions lose intensity drastically when these tissues are hydrated with ethanol.It is therefore imperative to consider tissue preparative effects when preparing, measuring, and analyzing samples using FTIR spectroscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy & Developmental Biology, Monash University, Clayton, Victoria 3800, Australia.

ABSTRACT
Fourier Transform Infrared (FTIR) micro-spectroscopy is an emerging technique for the biochemical analysis of tissues and cellular materials. It provides objective information on the holistic biochemistry of a cell or tissue sample and has been applied in many areas of medical research. However, it has become apparent that how the tissue is handled prior to FTIR micro-spectroscopic imaging requires special consideration, particularly with regards to methods for preservation of the samples. We have performed FTIR micro-spectroscopy on rodent heart and liver tissue sections (two spectroscopically very different biological tissues) that were prepared by desiccation drying, ethanol substitution and formalin fixation and have compared the resulting spectra with that of fully hydrated freshly excised tissues. We have systematically examined the spectra for any biochemical changes to the native state of the tissue caused by the three methods of preparation and have detected changes in infrared (IR) absorption band intensities and peak positions. In particular, the position and profile of the amide I, key in assigning protein secondary structure, changes depending on preparation method and the lipid absorptions lose intensity drastically when these tissues are hydrated with ethanol. Indeed, we demonstrate that preserving samples through desiccation drying, ethanol substitution or formalin fixation significantly alters the biochemical information detected using spectroscopic methods when compared to spectra of fresh hydrated tissue. It is therefore imperative to consider tissue preparative effects when preparing, measuring, and analyzing samples using FTIR spectroscopy.

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Related in: MedlinePlus

Average FTIR spectra from liver tissue: hydrated tissue ATR spectrum (blue), formalin fixed transmission spectrum (black), desiccator dried transmission spectrum (pink) and ethanol dehydrated transmission spectrum (green).
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pone.0116491.g003: Average FTIR spectra from liver tissue: hydrated tissue ATR spectrum (blue), formalin fixed transmission spectrum (black), desiccator dried transmission spectrum (pink) and ethanol dehydrated transmission spectrum (green).

Mentions: Fig. 1 represents the average FTIR spectra for the heart tissues prepared by the four different techniques. Fig. 2 shows the second derivative spectra of the heart tissues spectra in Fig. 1. Figs. 3 and 4 present the average FTIR spectra and the corresponding second derivative spectra from the liver tissues, respectively. From the spectra obtained for the four different preparation methods it was apparent, especially in the second derivative spectra, that there were many bands exhibiting significant shifts of the absorption band positions compared to the fresh / hydrated tissue. Tables 1 and 2 summarize the peak wavenumber values and their assignments for the important features for the heart and liver tissues, respectively. By considering, in turn, the groups of characteristic bands associated with the key macromolecules the relative effectiveness of the three methods for dehydrating the samples while preserving the biochemistry can be determined. Here the average ATR-FTIR spectrum of the fresh hydrated tissues (Figs. 1 and 3 blue trace) can be considered as the gold standard for preservation of biochemistry. However, it is important to note that because of the strong, broad absorptions of the water located at 1625 and 3200 cm−1, there is some degree of overlap, particularly of the amide I, and a loss of intensity in many absorption bands. Because of this, accurate measurement of peak wavenumber values and intensities are not always possible. The hydrated tissue spectra also exhibited marked differences in the relative strengths of various bands when compared with the dried tissue spectra. It has recently been reported that the extinction coefficients for the bands associated with the nucleic acids are significantly changed when these molecules are taken from hydrated to dehydrated states [11]. It is not unreasonable to assume that the same could be true for other biological macromolecules.


Importance of tissue preparation methods in FTIR micro-spectroscopical analysis of biological tissues: 'traps for new users'.

Zohdi V, Whelan DR, Wood BR, Pearson JT, Bambery KR, Black MJ - PLoS ONE (2015)

Average FTIR spectra from liver tissue: hydrated tissue ATR spectrum (blue), formalin fixed transmission spectrum (black), desiccator dried transmission spectrum (pink) and ethanol dehydrated transmission spectrum (green).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4339720&req=5

pone.0116491.g003: Average FTIR spectra from liver tissue: hydrated tissue ATR spectrum (blue), formalin fixed transmission spectrum (black), desiccator dried transmission spectrum (pink) and ethanol dehydrated transmission spectrum (green).
Mentions: Fig. 1 represents the average FTIR spectra for the heart tissues prepared by the four different techniques. Fig. 2 shows the second derivative spectra of the heart tissues spectra in Fig. 1. Figs. 3 and 4 present the average FTIR spectra and the corresponding second derivative spectra from the liver tissues, respectively. From the spectra obtained for the four different preparation methods it was apparent, especially in the second derivative spectra, that there were many bands exhibiting significant shifts of the absorption band positions compared to the fresh / hydrated tissue. Tables 1 and 2 summarize the peak wavenumber values and their assignments for the important features for the heart and liver tissues, respectively. By considering, in turn, the groups of characteristic bands associated with the key macromolecules the relative effectiveness of the three methods for dehydrating the samples while preserving the biochemistry can be determined. Here the average ATR-FTIR spectrum of the fresh hydrated tissues (Figs. 1 and 3 blue trace) can be considered as the gold standard for preservation of biochemistry. However, it is important to note that because of the strong, broad absorptions of the water located at 1625 and 3200 cm−1, there is some degree of overlap, particularly of the amide I, and a loss of intensity in many absorption bands. Because of this, accurate measurement of peak wavenumber values and intensities are not always possible. The hydrated tissue spectra also exhibited marked differences in the relative strengths of various bands when compared with the dried tissue spectra. It has recently been reported that the extinction coefficients for the bands associated with the nucleic acids are significantly changed when these molecules are taken from hydrated to dehydrated states [11]. It is not unreasonable to assume that the same could be true for other biological macromolecules.

Bottom Line: We have systematically examined the spectra for any biochemical changes to the native state of the tissue caused by the three methods of preparation and have detected changes in infrared (IR) absorption band intensities and peak positions.In particular, the position and profile of the amide I, key in assigning protein secondary structure, changes depending on preparation method and the lipid absorptions lose intensity drastically when these tissues are hydrated with ethanol.It is therefore imperative to consider tissue preparative effects when preparing, measuring, and analyzing samples using FTIR spectroscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy & Developmental Biology, Monash University, Clayton, Victoria 3800, Australia.

ABSTRACT
Fourier Transform Infrared (FTIR) micro-spectroscopy is an emerging technique for the biochemical analysis of tissues and cellular materials. It provides objective information on the holistic biochemistry of a cell or tissue sample and has been applied in many areas of medical research. However, it has become apparent that how the tissue is handled prior to FTIR micro-spectroscopic imaging requires special consideration, particularly with regards to methods for preservation of the samples. We have performed FTIR micro-spectroscopy on rodent heart and liver tissue sections (two spectroscopically very different biological tissues) that were prepared by desiccation drying, ethanol substitution and formalin fixation and have compared the resulting spectra with that of fully hydrated freshly excised tissues. We have systematically examined the spectra for any biochemical changes to the native state of the tissue caused by the three methods of preparation and have detected changes in infrared (IR) absorption band intensities and peak positions. In particular, the position and profile of the amide I, key in assigning protein secondary structure, changes depending on preparation method and the lipid absorptions lose intensity drastically when these tissues are hydrated with ethanol. Indeed, we demonstrate that preserving samples through desiccation drying, ethanol substitution or formalin fixation significantly alters the biochemical information detected using spectroscopic methods when compared to spectra of fresh hydrated tissue. It is therefore imperative to consider tissue preparative effects when preparing, measuring, and analyzing samples using FTIR spectroscopy.

Show MeSH
Related in: MedlinePlus