**5. Protocols for sample preparation**

As the sources of samples for FTIR spectroscopy can be different, they need special preparations adopting different kinds of procedures depending on the samples.

### **5.1 FTIR spectroscopy of homogeneous materials**

#### **5.1.1 Drug macromolecules interactions**

Studies of drug interactions by FTIR spectroscopy are easily carried out in systems using D2O. Various approaches are possible. The drug and protein/nucleic acid interaction is carried out under solution conditions at predetermined stoichiometry and the mixture is then added to a film from which spectral data are obtained. The individual components are also used as references or controls. The difference spectra between the control and with the ligands is sued to quantify the amount of bound ligands or study the changes in the protein secondary structure by monitoring the shifts in peak intensities or variation in peak intensities of different functional groups (Bourasssa et al 2011). FTIR-ATR system can also be used for studying the interactions using a dialysis system.(Krasteva et al 2006, Kumar & Barth (2011).

#### **5.1.2 Microbial cultures**

Bacterial cultures growing in the log phase are fixed with formaldehyde (final concentration 0.25%) and washed three times with saline containing 0.25% formaldehyde and once with saline only. The pellet is resuspended with saline to an OD450 = 0.3 (about 20 ml). One microliter of the suspension is spotted on Zinc-Selenium slides and air dried for 6 hours to remove any water in the sample under a laminar air flow chamber. Microscopic FTIR measurements are made in transmission mode using the FTIR microscope IRscope II with a mercury-cadmium-telluride (MCT) detector, coupled to the FTIR spectrometer (Bruker Equinox model 55/S, OPUS software). Absorbance is measured from an area of 100 nm diameter by setting the slit to 100 microns. Regions of thickness of about 10 microns (as seen from the ADC values) are selected and 128 scans co added for each spectrum. For each sample at least five spots are measured and the average spectra calculated. Similar procedure can be adopted for yeast cells. In case of fungi which have a tendency to grow as filaments (hyphae), the filaments are harvested and briefly tweezed or torn to get uniform untangled mass as observed under the microscope. These are then washed in water or saline before mounting on the slides. These can also be measured using ATR systems as they are highly adherent and a spread sample is likely to have non uniformity in thickness owing to the clumping tendency of the filaments (Salman et al 2011).

#### **5.1.3 Cell lines and transformed cultures**

Cell lines are cultured under suitable conditions (e.g. 37°C in RPMI medium supplemented with 10% of newborn calf serum (NBCS) and the antibiotics penicillin, streptomycin) and

Chemometrics of Cells and Tissues Using

**5.3 Cellular transformations** 

malignant transformation.

IR Spectroscopy – Relevance in Biomedical Research 297

contain regions where the crypts are seen in a transverse or longitudinal section. The measurements can be made on either type of section. However, the transverse sections are usually measured for conventional diagnosis and grading for cancer or premalignancy (Argov et al 2002, Argov et al 2004). Measurements along the crypt height have been employed to understand metabolic activity in terms of spectral changes (Salman et al 2004) or to define abnormal proliferation in the epithelium (Sahu et al 2004a, Sahu et al 2010). In these cases, a region of the slide free from contaminants is used for the background. The histology of the tissue is evaluated on the complementary slide which is stained with H&E. Regions are selected that indicate the required stage of disease and then measured by locating the identical spots on the ZnSe slide. At least five measurements on each sample are carried out. The morphology of the colonic epithelium is well defined and it is easy to locate the crypts even while viewing under the optical microscope. Thus, it is easier to locate the crypts and measure them. Usually in these tissues, measurements are made by adjusting the slit to 120 microns that results in an area of 100 micron diameter. As most formalin fixed tissues are paraffin embedded and processed, the spectroscopy in the higher region can also be used to monitor contaminants like blood and paraffin or any residual cleansing reagent like xylol (Sahu et al 2005). Both the transverse sections of the crypt, where the entire crypt would fit into the area of measurement or several locations along the crypt in the longitudinal section can be measured based on the requirement of the studies (Argov et al 2002, Argov et al 2004, Sahu et al 2004a, Salman et al 2004). In case of measurement along the length of the crypt the replication is carried out by measuring several crypts at similar distance form the base or apex unlike the cross section where several crypt circles are measured. For more complex tissues such as skin, where different kinds of abnormalities occur (both benign and malignant) the exact location of the sample measurement area requires the involvement of an expert dermatologist/pathologist to pin point and mark the areas on the complementary slides (Hammody et al 2007). Advancement in technologies have made it possible to automatically measure entire regions of the biopsy using the FPA detector systems and the spectra corresponding to every pixel of the measured area can be obtained. This type of measurement is slow though automated and helps to map entire regions. However the study on a complementary slide to demarcate the exact location of histological entities is still essential to precisely correlate the spectra. The processing is similar to microscopy but owing to a large

amount of acquired data, automated computational methods become necessary.

Cellular transformations are used to understand spectral changes happening due to biological changes in cells and tissues, often inducing controlled changes in cell lines using genes or viruses. Cells grown in tissue culture plates are treated with polybrene (a cationic polymer required for neutralizing the negative charge of the cell membrane) for 24 h before infection with the virus. Free polybrene is removed and incubated with the high titer infecting virus stock at 37°C for 2 h. The unabsorbed virus particles are removed by washing the cells in fresh warm medium and fresh medium is added. Several wells are used in a study including a control group passed through similar conditions without the infecting virus. After various stages of progression the cells are examined for the appearance of malignant transformation and cells from sets of wells harvested at different time intervals used for spectra acquisition. Cells can also be grown on soft agar or stained to confirm their

passed to obtain confluent cells . Adherent cells are harvested using a cell lifter while cells in suspension are taken directly. The cell suspensions is centrifuged for 5 minutes at 3000 rpm and the pellet washed in phosphate buffered saline several times ( 3-5 times). After the washing, the cells are washed at least two times in normal saline and resuspended in saline such that the cells density is about 1x106/ml. One microlitre of this suspension is spotted on a ZnSe window and allowed to air dry in a laminar air flow chamber for several hours. A microlitre of the last supernatant before suspension may also be spotted to be used as a reference for any unwanted materials. Spectra from this sample should ideally be similar to the background spectra. Cells and microbes like fungus may also be grown on IR transparent matrices when they are analyzed by ATR or in reflectance mode. Precautions are however required to avoid artifacts due to very thin layers of drying of cells (Mourant et al 2003a).

#### **5.1.4 Samples from body fluids such as blood or urine**

Body fluids usually contain cells and non cellular components, tissue debris and a fluid or matrix. When the cells are the material of interest, they are isolated into pure or homogeneous forms using various separation techniques that utilize the different sedimentation coefficient of the cells and including Fluorescence activated cell sorter (FACS) when required to isolate a pure population based on their cell surface markers. In case of blood samples, the collection is made in heparin or EDTA to prevent the clotting. Then they are separated by a histopaque or Ficoll gradient. The cells are then washed in PBS followed by normal saline as for cultured cells. For spectra of other components such as plasma from blood, the plasma or sera obtained from blood is directly spotted on the ZnSe windows without further processing. However, if the liquid is very viscous, it can lead to super saturated spectra. To avoid this, the fluid is spread by dragging the drop in several directions with the tip and spreading it. Study of other body fluids like urine may require filtration of the liquid to remove debris and its concentration to reduce the amount of water before it is loaded on matrices. In case of study of the cellular components, a prior separation of different cell components maybe required.

#### **5.2 FTIR spectroscopy of tissue samples**

Most of the diagnostic potential of FTIR spectroscopy has been evaluated in tissues that relate to carcinogenesis with an objective to diagnose malignancy or premalignancy. Thus, conventionally the material available was formalin fixed, paraffin embedded tissues. In most of these cases, the tissue is sliced to a suitable thickness( usually 10 microns), processed to remove the embedding material and mounted on a slide ( usually a ZnSe slide) before it is used for measurements. Such slides are stored in histology frames dipped in 70% ethanol to prevent contamination. A consecutive section that is congruent to the section being measured is usually used for H&E staining. Thus, both the optical as well as the FTIR measuring capabilties of the microscope are used to pin point the exact location of measurement. For example, in tissues like cervical epithelium, where there is a decrease in glycogen level from the superficial layer to the basal layer ( Chiriboga et al 1998a), and also the reduction of glycogen is an important marker for CIN or cervical cancer, it becomes important to measure at defined regions, namely the intermediate layers of the tissue to accurately be able to classify the stage of CIN (Figure 3a)(Mark et al 2004). In case of colonic epithelium, the tissue can

passed to obtain confluent cells . Adherent cells are harvested using a cell lifter while cells in suspension are taken directly. The cell suspensions is centrifuged for 5 minutes at 3000 rpm and the pellet washed in phosphate buffered saline several times ( 3-5 times). After the washing, the cells are washed at least two times in normal saline and resuspended in saline such that the cells density is about 1x106/ml. One microlitre of this suspension is spotted on a ZnSe window and allowed to air dry in a laminar air flow chamber for several hours. A microlitre of the last supernatant before suspension may also be spotted to be used as a reference for any unwanted materials. Spectra from this sample should ideally be similar to the background spectra. Cells and microbes like fungus may also be grown on IR transparent matrices when they are analyzed by ATR or in reflectance mode. Precautions are however required to avoid artifacts due to very thin layers of drying of cells (Mourant et

Body fluids usually contain cells and non cellular components, tissue debris and a fluid or matrix. When the cells are the material of interest, they are isolated into pure or homogeneous forms using various separation techniques that utilize the different sedimentation coefficient of the cells and including Fluorescence activated cell sorter (FACS) when required to isolate a pure population based on their cell surface markers. In case of blood samples, the collection is made in heparin or EDTA to prevent the clotting. Then they are separated by a histopaque or Ficoll gradient. The cells are then washed in PBS followed by normal saline as for cultured cells. For spectra of other components such as plasma from blood, the plasma or sera obtained from blood is directly spotted on the ZnSe windows without further processing. However, if the liquid is very viscous, it can lead to super saturated spectra. To avoid this, the fluid is spread by dragging the drop in several directions with the tip and spreading it. Study of other body fluids like urine may require filtration of the liquid to remove debris and its concentration to reduce the amount of water before it is loaded on matrices. In case of study of the cellular components, a prior

Most of the diagnostic potential of FTIR spectroscopy has been evaluated in tissues that relate to carcinogenesis with an objective to diagnose malignancy or premalignancy. Thus, conventionally the material available was formalin fixed, paraffin embedded tissues. In most of these cases, the tissue is sliced to a suitable thickness( usually 10 microns), processed to remove the embedding material and mounted on a slide ( usually a ZnSe slide) before it is used for measurements. Such slides are stored in histology frames dipped in 70% ethanol to prevent contamination. A consecutive section that is congruent to the section being measured is usually used for H&E staining. Thus, both the optical as well as the FTIR measuring capabilties of the microscope are used to pin point the exact location of measurement. For example, in tissues like cervical epithelium, where there is a decrease in glycogen level from the superficial layer to the basal layer ( Chiriboga et al 1998a), and also the reduction of glycogen is an important marker for CIN or cervical cancer, it becomes important to measure at defined regions, namely the intermediate layers of the tissue to accurately be able to classify the stage of CIN (Figure 3a)(Mark et al 2004). In case of colonic epithelium, the tissue can

**5.1.4 Samples from body fluids such as blood or urine** 

separation of different cell components maybe required.

**5.2 FTIR spectroscopy of tissue samples** 

al 2003a).

contain regions where the crypts are seen in a transverse or longitudinal section. The measurements can be made on either type of section. However, the transverse sections are usually measured for conventional diagnosis and grading for cancer or premalignancy (Argov et al 2002, Argov et al 2004). Measurements along the crypt height have been employed to understand metabolic activity in terms of spectral changes (Salman et al 2004) or to define abnormal proliferation in the epithelium (Sahu et al 2004a, Sahu et al 2010). In these cases, a region of the slide free from contaminants is used for the background. The histology of the tissue is evaluated on the complementary slide which is stained with H&E. Regions are selected that indicate the required stage of disease and then measured by locating the identical spots on the ZnSe slide. At least five measurements on each sample are carried out. The morphology of the colonic epithelium is well defined and it is easy to locate the crypts even while viewing under the optical microscope. Thus, it is easier to locate the crypts and measure them. Usually in these tissues, measurements are made by adjusting the slit to 120 microns that results in an area of 100 micron diameter. As most formalin fixed tissues are paraffin embedded and processed, the spectroscopy in the higher region can also be used to monitor contaminants like blood and paraffin or any residual cleansing reagent like xylol (Sahu et al 2005). Both the transverse sections of the crypt, where the entire crypt would fit into the area of measurement or several locations along the crypt in the longitudinal section can be measured based on the requirement of the studies (Argov et al 2002, Argov et al 2004, Sahu et al 2004a, Salman et al 2004). In case of measurement along the length of the crypt the replication is carried out by measuring several crypts at similar distance form the base or apex unlike the cross section where several crypt circles are measured. For more complex tissues such as skin, where different kinds of abnormalities occur (both benign and malignant) the exact location of the sample measurement area requires the involvement of an expert dermatologist/pathologist to pin point and mark the areas on the complementary slides (Hammody et al 2007). Advancement in technologies have made it possible to automatically measure entire regions of the biopsy using the FPA detector systems and the spectra corresponding to every pixel of the measured area can be obtained. This type of measurement is slow though automated and helps to map entire regions. However the study on a complementary slide to demarcate the exact location of histological entities is still essential to precisely correlate the spectra. The processing is similar to microscopy but owing to a large amount of acquired data, automated computational methods become necessary.

### **5.3 Cellular transformations**

Cellular transformations are used to understand spectral changes happening due to biological changes in cells and tissues, often inducing controlled changes in cell lines using genes or viruses. Cells grown in tissue culture plates are treated with polybrene (a cationic polymer required for neutralizing the negative charge of the cell membrane) for 24 h before infection with the virus. Free polybrene is removed and incubated with the high titer infecting virus stock at 37°C for 2 h. The unabsorbed virus particles are removed by washing the cells in fresh warm medium and fresh medium is added. Several wells are used in a study including a control group passed through similar conditions without the infecting virus. After various stages of progression the cells are examined for the appearance of malignant transformation and cells from sets of wells harvested at different time intervals used for spectra acquisition. Cells can also be grown on soft agar or stained to confirm their malignant transformation.

Chemometrics of Cells and Tissues Using

Figure 2C) or peak areas after deconvolution (Figure 2D).

taken to indicate the intensity at the particular wavenumber (Fig 2F).

the ADC rates are empirically chosen between 4000-5000 counts/sec.

**6.1 Spectral acquisition using ATR/FEWS systems** 

**7. Advances in data analysis** 

spotted sample.

(Sahu et al 2010).

IR Spectroscopy – Relevance in Biomedical Research 299

background spectra when further measurements are made. The reference samples if any are then measured. The thickness of the sample is usually reflected in the ADC rates. A ADC value of less than 3000 usually denotes a thickness of less than 10 microns and often leads to noisy spectra. On the other hand regions with a very high ADC value of (>6000) can give rise to supersaturated spectra. Once a set of reference spectra are obtained and they display required characteristics, the samples are measured. Ideally 3-5 spots are measured on each

The background corrected spectra is then baseline corrected for the entire region using a rubber band baseline function (Figure 2A). Selected spectral regions of interest are then separately cut from the entire spectrum and a second baseline correction is made (e.g region between 800-1800 cm-1). This is followed by a normalization of the spectra (Fig.2B,C). The different spectra are then averaged and the average spectra used to represent a sample. Intensities at different wavenumbers obtained form this processed spectra are the inputs for various mathematical analyses. For example, the ratio of intensities at 1545 and 1045 cm-1 (

Usually a region with normal morphology is taken as a reference or control while identifying the malignancy in a tissue to avoid any heterogeneity due to individual variations while comparing normal and abnormal tissues (Argov et al 2002). With recognition of the fact that normal tissues may still display abnormal FTIR spectra it becomes important to precisely define a normal tissue or biological entity which in itself could lead to better understand dynamics of spectral variation during disease progression

Second derivatives and higher order derivatives of spectra are used to avoid the errors creeping in due to bias originating from baseline selection methods (Figure 2E). In this case when the intensities are measured, the minima correspond to the maxima (peak intensity) in the original spectra. Sum of the value at the minima with the value at the nearest maxima is

ATR measurements can be done using Bruker FT-IR Tensor 27 Spectrometer equipped with a liquid nitrogen cooled mercury-cadmium-telluride (MCT) detector and coupled with Horizontal Attenuated Total Reflectance Accessory (HATR (Horizontal Attenuated Total Reflectance), PIKE technologies Inc,) systems (Bogomonly et al 2009). The accessory is connected with a nitrogen reservoir, which enables to preserve dryness of the sample and maintaining an inert atmosphere. The design employs a pair of transfer optics to direct the infrared beam to one end of IR transmitting crystal. A similar pair of flat mirrors directs the beam from the other end of the ATR crystal to the spectrometer detector. The ATR crystal made up of ZnSe is of a trapezoid shape with its thickness suitable to produce optimum performance. To reduce variance each sample is measured several times (at least five) and

Unique spectral fingerprints of biological entities in the mid IR region are the manifestation of several components absorbing at different wavenumbers, with overlap. Thus, both the

#### **6. Spectra acquisition and data processing**

Spectra can be obtained either in transmission or reflective mode or both from a FTIR microscope (Argov et al 2002, Chang et al 2003). Samples loaded on a mounting material such as a ZnSe slide are observed under the microscope for their uniformity and thickness and representative regions are selected. Prior to measuring the samples a background measurement is made in a region free of any samples or reference material. This spectrum is saved as a background (Figure 2a). The data processor automatically subtracts these

A. Initial absorbance spectrum collected in transmission mode (a), background (b) and background subtracted and rubber band baseline corrected spectrum (c). B. Spectra in the region 800-1800 cm-1 after cutting from the entire spectrum, baseline correction and normalization to amide I peak (a) or amide II peak (b). C. Area normalization of the spectrum after cutting the region between 800-1800 cm-1 preceded by baseline correction and followed by offset correction. The vertical lines denote intensities at 1545 and 1045 cm-1 which can be used for quantification of carbohydrates. D. Deconvolution of the spectra to obtain intensity at various peaks using Gaussian fit of the spectra. (E.) Second derivative of the spectra. (F) Selected region in the second derivative spectra where the intensity at a selected wavenumber is calculated by taking the intesities at the minimum (a) and the adjacent maximum (b).

Fig. 2. Spectral acquisition and processing.

Spectra can be obtained either in transmission or reflective mode or both from a FTIR microscope (Argov et al 2002, Chang et al 2003). Samples loaded on a mounting material such as a ZnSe slide are observed under the microscope for their uniformity and thickness and representative regions are selected. Prior to measuring the samples a background measurement is made in a region free of any samples or reference material. This spectrum is saved as a background (Figure 2a). The data processor automatically subtracts these

> **0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5**

> > **0.0 0.5 1.0 1.5 2.0**

**) 900 950 1000 1050 1100 1150 1200 1250 1300 -0.0015**

Absorbance (AU)

**-0.0010 -0.0005 0.0000 0.0005 0.0010 0.0015**

**Second Derivative (AU)**

A. Initial absorbance spectrum collected in transmission mode (a), background (b) and background subtracted and rubber band baseline corrected spectrum (c). B. Spectra in the region 800-1800 cm-1 after cutting from the entire spectrum, baseline correction and normalization to amide I peak (a) or amide II peak (b). C. Area normalization of the spectrum after cutting the region between 800-1800 cm-1 preceded by baseline correction and followed by offset correction. The vertical lines denote intensities at 1545 and 1045 cm-1 which can be used for quantification of carbohydrates. D. Deconvolution of the spectra to obtain intensity at various peaks using Gaussian fit of the spectra. (E.) Second derivative of the spectra. (F) Selected region in the second derivative spectra where the intensity at a selected wavenumber is

E F

C D

**Absorbance (AU)**

A B

**1800 1600 1400 1200 1000 800**

**Wavenumber (cm-1**

**1800 1600 1400 1200 1000 800**

**Wavenumber cm-1**

**Wavenumber (cm-1**

**)**

**a b**

**)**

**b a**

**6. Spectra acquisition and data processing** 

**4000 3500 3000 2500 2000 1500 1000**

**c**

**b**

**a**

**Wavenumber (cm-1**

**1800 1600 1400 1200 1000 800**

**Wavenumber (cm-1**

**1800 1600 1400 1200 1000 800**

**Wavenumber (cm-1**

Carbohydrate

**)**

calculated by taking the intesities at the minimum (a) and the adjacent maximum (b).

**)**

**0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7**

> **0.00 0.05 0.10 0.15 0.20**

**-0.004 -0.002 0.000 0.002 0.004**

Fig. 2. Spectral acquisition and processing.

**Second Derivative (AU)**

**Absorbance (AU)**

AmideII

**Absorbance (AU)**

background spectra when further measurements are made. The reference samples if any are then measured. The thickness of the sample is usually reflected in the ADC rates. A ADC value of less than 3000 usually denotes a thickness of less than 10 microns and often leads to noisy spectra. On the other hand regions with a very high ADC value of (>6000) can give rise to supersaturated spectra. Once a set of reference spectra are obtained and they display required characteristics, the samples are measured. Ideally 3-5 spots are measured on each spotted sample.

The background corrected spectra is then baseline corrected for the entire region using a rubber band baseline function (Figure 2A). Selected spectral regions of interest are then separately cut from the entire spectrum and a second baseline correction is made (e.g region between 800-1800 cm-1). This is followed by a normalization of the spectra (Fig.2B,C). The different spectra are then averaged and the average spectra used to represent a sample. Intensities at different wavenumbers obtained form this processed spectra are the inputs for various mathematical analyses. For example, the ratio of intensities at 1545 and 1045 cm-1 ( Figure 2C) or peak areas after deconvolution (Figure 2D).

Usually a region with normal morphology is taken as a reference or control while identifying the malignancy in a tissue to avoid any heterogeneity due to individual variations while comparing normal and abnormal tissues (Argov et al 2002). With recognition of the fact that normal tissues may still display abnormal FTIR spectra it becomes important to precisely define a normal tissue or biological entity which in itself could lead to better understand dynamics of spectral variation during disease progression (Sahu et al 2010).

Second derivatives and higher order derivatives of spectra are used to avoid the errors creeping in due to bias originating from baseline selection methods (Figure 2E). In this case when the intensities are measured, the minima correspond to the maxima (peak intensity) in the original spectra. Sum of the value at the minima with the value at the nearest maxima is taken to indicate the intensity at the particular wavenumber (Fig 2F).

#### **6.1 Spectral acquisition using ATR/FEWS systems**

ATR measurements can be done using Bruker FT-IR Tensor 27 Spectrometer equipped with a liquid nitrogen cooled mercury-cadmium-telluride (MCT) detector and coupled with Horizontal Attenuated Total Reflectance Accessory (HATR (Horizontal Attenuated Total Reflectance), PIKE technologies Inc,) systems (Bogomonly et al 2009). The accessory is connected with a nitrogen reservoir, which enables to preserve dryness of the sample and maintaining an inert atmosphere. The design employs a pair of transfer optics to direct the infrared beam to one end of IR transmitting crystal. A similar pair of flat mirrors directs the beam from the other end of the ATR crystal to the spectrometer detector. The ATR crystal made up of ZnSe is of a trapezoid shape with its thickness suitable to produce optimum performance. To reduce variance each sample is measured several times (at least five) and the ADC rates are empirically chosen between 4000-5000 counts/sec.
