**8. Recent trends and future perspectives**

Though basic research has been extensively carried out including pilot scale experimentation of clinical trials in using FTIR measurements for diseases diagnosis, the potential has not been practically exploited due to inability of FTIR based diagnosis to be an independent technique for classification of diseased tissues. The role of a pathologist has been indispensable and pivotal in the preliminary process of sample selection. Advances made through development of FPA based techniques partially overcome this requirement where automated identification of diseased regions in tissues using programs like cluster analysis or ANN/PNN provide pseudocolored images depicting tissue morphology. The rapidity of the technique is however compromised in these cases. Moreover, while a pathologist could quickly look at areas of interest, the automation mandatorily examines the entire section, making it a time consuming affair. In case of complex material like melanoma

Chemometrics of Cells and Tissues Using

**0**

for assigning the worst prognosis.

**0.5**

**1**

**1.5**

**Area(Middle)/Area(Top)**

**2**

**2.5**

IR Spectroscopy – Relevance in Biomedical Research 305

colonoscopes, colposcopes or other endosocopic tools maybe used to detect malignant or abnormal tissues in body cavities. While the optical systems could help to pin point locations , the FTIR system could provide a quantitative evaluation of the abnormal tissue in vivo , without the requirement of the pathologist and help the surgeon make real time decisions. This is especially important as histologically normal regions can be diseased when evaluated by FTIR spectroscopy (Argov et al 2004, Sahu et al 2004, 2010). For example, IR probes mounted on colposcopes may not only determine the areas of malignancy in the cervix but also possibly be able to determine the stage of the CIN. Similarly, examination of body fluids or other material for microbial infections without requiring long culture time can be rapidly done if detection systems are developed where the type of changes associated with different kinds of microbes are clearly defined and established (Bombalska et al 2011, Maquelin et al 2003). The ability of FTIR to detect biochemical changes in organs other than the one affected to possibly use these as indicator of the health status of patients has been another growing area of interest, mainly because these use materials like hairs and nails that are dispensable or blood samples that are easily obtained compared to biopsies (Lyman et al 2005, Khanmohammadi et al 2007). This type of FTIR based diagnosis would help decrease the cost through early intervention. FTIR based diagnosis of diseases, microbes, and healthy and unhealthy conditions is thus a future possibility that is less destructive and harmless compared to conventional methods (Toubas et al 2006). Similar approach can be used to

> **1234 Patient**

Fig. 6. Evaluation of polyposis in biopsies using the biomarkers derived from crypts. The Crypts from patients 1 and 2 had a polyposis while those of patients 3 and 4 did not. The values in the y axis are derived from the ratios of the integrated absorbance in the region 900-1185 cm-1 obtained at the middle and top of the crypt. The color codes are independent and used only to distinguish crypts from one another. Crypts with a value less than 1 are indicative of a propensity to have polyposis. At least 2 such abnormal crypts are required

Another clinically relevant application of FTIR spectroscopy related to the , in vivo diagnosis of diseases in human organs like colon, rectum, oesophagus, cervix, alimentary tract, nasopharynx and other areas deals with fiber optic based IR probes that can be

diagnose microbial presence in blood samples (Maquelin et al 2003).

and different grades of nevi, the process still needs supporting techniques like immuno histochemistry/histology for verifications before an FTIR measurement is made. The complex dynamics of epithelial tissues that are most prone to carcinogenesis, vary from one organ to another. Thus, clear knowledge of the type of metabolic pattern variation in these tissues is a prerequisite for both acquisition and interpretation of the spectral data. This makes the technique interdisciplinary and involves people with extra specializations requiring special training. However owing to the simplicity in data acquisition and automation with most post spectral processing, these limitations may be overcome easily.

An objective and quantitative method like FTIR spectroscopy becomes crucial when metabolomics of cells and tissues are desired without requirement of different chemical analyses, as it simultaneously can monitor different metabolites. It has the ability to distinguish between microorganisms depending on their biochemical variation and this has been demonstrated in both bacteria and fungi (Sahu et al 2006b, Salman et al 2011). Similarly, minute changes can occur between the different stages of cancer or malignancy which are difficult to detect even with histochemical analyses. Under these circumstances, FTIR becomes handy as it distinguishes among different grades of premalignant and malignant tissues based on markers like carbohydrates which may not be easily stained in normal laboratory practices (Mark et al 2005). Observation of metabolite variation along tissues reflecting the dynamic nature through FTIR spectroscopy has been one of the most important findings (Chiriboga et al 1998a, Salman et al 2004, Sahu et al 2010) that has implications for cancer diagnosis. For example abnormal crypt proliferation can detect in colonic tissues to re-evaluate resection margins (Sahu et al 2010) consequently decreasing the number of surgical interventions. The technique becomes an inexpensive alternative method to follow disease progression or regression quantitatively over a period of time (Zelig et al 2011). Similarly, the growth patterns of microbes are being studied using FTIR spectroscopy. The technique has also become a method of choice to monitor drug effects on cells and tissues as it can easily monitor changes occurring due to onset of processes like apoptosis and necrosis (Zelig et al 2009).

In addition to these abilities, the availability of a qualitative evaluation along with quantization adds in determining the status of the tissues. For example, as shown in figure 6, the values of the ratio of integrated absorbance of several crypts in colonic biopsies shows two different patterns. The biopsies of patients diagnosed with polyposis show a low ratio of 1 or less for many crypts while biopsies without any polyposis have most crypts displaying a the ratio greater than 1. While conventional statistical analysis would look at values like the average for determining the probability of the biopsy being normal or abnormal, the present system allows the pathologist to examine each biopsy with the privilege of being able to designate the biopsy as abnormal based on the values of more than one crypt going with the medical practice of assigning the worst prognosis. This is an easier approach where spectral data from the biopsy cannot be analyzed using more complicated methods like DCF analysis. Thus, the spectral analysis can be tailor made to suit the availability of qualified personnel.

The fiber optic based IR probes are being increasingly examined for utilization as *in vivo* detection systems as these are non toxic. Several reports on fiber-optic based IR sensors for diagnosis of malignancies reflect the trend of incorporation of this technique in future clinical practices (Mackanos and Contag 2010). This in combination with light based

and different grades of nevi, the process still needs supporting techniques like immuno histochemistry/histology for verifications before an FTIR measurement is made. The complex dynamics of epithelial tissues that are most prone to carcinogenesis, vary from one organ to another. Thus, clear knowledge of the type of metabolic pattern variation in these tissues is a prerequisite for both acquisition and interpretation of the spectral data. This makes the technique interdisciplinary and involves people with extra specializations requiring special training. However owing to the simplicity in data acquisition and automation with most post spectral processing, these limitations may be overcome easily. An objective and quantitative method like FTIR spectroscopy becomes crucial when metabolomics of cells and tissues are desired without requirement of different chemical analyses, as it simultaneously can monitor different metabolites. It has the ability to distinguish between microorganisms depending on their biochemical variation and this has been demonstrated in both bacteria and fungi (Sahu et al 2006b, Salman et al 2011). Similarly, minute changes can occur between the different stages of cancer or malignancy which are difficult to detect even with histochemical analyses. Under these circumstances, FTIR becomes handy as it distinguishes among different grades of premalignant and malignant tissues based on markers like carbohydrates which may not be easily stained in normal laboratory practices (Mark et al 2005). Observation of metabolite variation along tissues reflecting the dynamic nature through FTIR spectroscopy has been one of the most important findings (Chiriboga et al 1998a, Salman et al 2004, Sahu et al 2010) that has implications for cancer diagnosis. For example abnormal crypt proliferation can detect in colonic tissues to re-evaluate resection margins (Sahu et al 2010) consequently decreasing the number of surgical interventions. The technique becomes an inexpensive alternative method to follow disease progression or regression quantitatively over a period of time (Zelig et al 2011). Similarly, the growth patterns of microbes are being studied using FTIR spectroscopy. The technique has also become a method of choice to monitor drug effects on cells and tissues as it can easily monitor changes occurring due to onset of processes like

In addition to these abilities, the availability of a qualitative evaluation along with quantization adds in determining the status of the tissues. For example, as shown in figure 6, the values of the ratio of integrated absorbance of several crypts in colonic biopsies shows two different patterns. The biopsies of patients diagnosed with polyposis show a low ratio of 1 or less for many crypts while biopsies without any polyposis have most crypts displaying a the ratio greater than 1. While conventional statistical analysis would look at values like the average for determining the probability of the biopsy being normal or abnormal, the present system allows the pathologist to examine each biopsy with the privilege of being able to designate the biopsy as abnormal based on the values of more than one crypt going with the medical practice of assigning the worst prognosis. This is an easier approach where spectral data from the biopsy cannot be analyzed using more complicated methods like DCF analysis. Thus, the spectral analysis can be tailor made to suit the

The fiber optic based IR probes are being increasingly examined for utilization as *in vivo* detection systems as these are non toxic. Several reports on fiber-optic based IR sensors for diagnosis of malignancies reflect the trend of incorporation of this technique in future clinical practices (Mackanos and Contag 2010). This in combination with light based

apoptosis and necrosis (Zelig et al 2009).

availability of qualified personnel.

colonoscopes, colposcopes or other endosocopic tools maybe used to detect malignant or abnormal tissues in body cavities. While the optical systems could help to pin point locations , the FTIR system could provide a quantitative evaluation of the abnormal tissue in vivo , without the requirement of the pathologist and help the surgeon make real time decisions. This is especially important as histologically normal regions can be diseased when evaluated by FTIR spectroscopy (Argov et al 2004, Sahu et al 2004, 2010). For example, IR probes mounted on colposcopes may not only determine the areas of malignancy in the cervix but also possibly be able to determine the stage of the CIN. Similarly, examination of body fluids or other material for microbial infections without requiring long culture time can be rapidly done if detection systems are developed where the type of changes associated with different kinds of microbes are clearly defined and established (Bombalska et al 2011, Maquelin et al 2003). The ability of FTIR to detect biochemical changes in organs other than the one affected to possibly use these as indicator of the health status of patients has been another growing area of interest, mainly because these use materials like hairs and nails that are dispensable or blood samples that are easily obtained compared to biopsies (Lyman et al 2005, Khanmohammadi et al 2007). This type of FTIR based diagnosis would help decrease the cost through early intervention. FTIR based diagnosis of diseases, microbes, and healthy and unhealthy conditions is thus a future possibility that is less destructive and harmless compared to conventional methods (Toubas et al 2006). Similar approach can be used to diagnose microbial presence in blood samples (Maquelin et al 2003).

Fig. 6. Evaluation of polyposis in biopsies using the biomarkers derived from crypts. The Crypts from patients 1 and 2 had a polyposis while those of patients 3 and 4 did not. The values in the y axis are derived from the ratios of the integrated absorbance in the region 900-1185 cm-1 obtained at the middle and top of the crypt. The color codes are independent and used only to distinguish crypts from one another. Crypts with a value less than 1 are indicative of a propensity to have polyposis. At least 2 such abnormal crypts are required for assigning the worst prognosis.

Another clinically relevant application of FTIR spectroscopy related to the , in vivo diagnosis of diseases in human organs like colon, rectum, oesophagus, cervix, alimentary tract, nasopharynx and other areas deals with fiber optic based IR probes that can be

Chemometrics of Cells and Tissues Using

of having large amounts of spectra for analyses.

Mar;21(3):297-301.

2002.Apr;7(2):248-54.

*Opt.* 2002 Jan;7(1):100-8.

Sep;46(9):4344-53.

*Spectrosc.* 1997. June; 51 (6):792–797.

2005).

**10. Conclusion** 

**11. References** 

IR Spectroscopy – Relevance in Biomedical Research 307

extending Fiberoptic detection systems to different organs is a future potential (Lucas et al

The technique of FTIR spectroscopy is now being used for different purposes like identification of organisms and their classification, determination of status of tissues, monitoring the effects of drugs on cell lines, monitoring treatment regimen and studying interactions between biological systems in the arena of biomedical research. Owing to its simplicity of data acquisition compared to more sophisticated methods like NMR and development of computational methods for rapid data processing, its relevance in biomedical research is bound to increase over the coming years, in spite of the initial block

Ali, K.; Lu, Y.; Christensen, C.; May, T.; Hyett, C.; Griebel, R.; Fourney, D.; Meguro, K.;

Argov, S.; Ramesh, J.; Salman, A.; Sinelnikov, I.; Goldstein, J.; Guterman, H. & Mordechai, S.

Argov, S.; Sahu, R.K.; Bernshtain, E.; Salman, A.; Shohat, G.; Zelig, U. & Mordechai, S. (2004.

a FTIR-microspectroscopy approach*. Biopolymers.* 2004 Dec 5;75(5):384-92. Benedetti, E.; Bramanti, E.; Papineschi, F.; Rossi, I. & Benedetti. E. (1997). Determination of

Bindig, U.;Winter, H.; Wäsche, W.; Zelianeos, K. & Müller, G. (2002).Fiber-optical and

Bogomolny, E.; Huleihel, M.; Suproun, Y.; Sahu, R.K. & Mordechai S. (2007). Early spectral

Bombalska, A.; Mularczyk-Oliwa, M.; Kwaśny, M.; Włodarski, M.; Kaliszewski, M.;

Bourassa, P.; Dubeau, S.; Maharvi, G.M.; Fauq, A.H.; Thomas, T.J. & Tajmir-Riahi, H.A.

microspectroscopy. *J Biomed Opt.* 2007 Mar-Apr;12(2):024003.

*Spectrochim Acta A Mol Biomol Spectrosc*. 2011 Apr;78(4):1221-6.

Resch, L. & Sharma, R.K. (2008). Fourier transform infrared spectromicroscopy and hierarchical cluster analysis of human meningiomas. Int J Mol Med. 2008

(2002). Diagnostic potential of Fourier-transform infrared microspectroscopy and advanced computational methods in colon cancer patients*. J Biomed Opt.*

Inflammatory bowel diseases as an intermediate stage between normal and cancer:

the relative amount of nucleic acids and proteins in leukemic and normal lymphocytes by means of fourier transform infrared microspectroscopy. *Appl.* 

microscopic detection of malignant tissue by use of infrared spectrometry. *J Biomed* 

changes of cellular malignant transformation using Fourier transform infrared

Kopczyński, K.; Szpakowska, M. & Trafny, E .A. (2010). Classification of the biological material with use of FTIR spectroscopy and statistical analysis.

(2011). Locating the binding sites of anticancer tamoxifen and its metabolites 4 hydroxytamoxifen and endoxifen on bovine serum albumin. *Eur J Med Chem*. 2011

inserted to obtain spectra of the surface layers. Studies pertaining to these objectives have utilized fiber-optic sensors for diagnosis of malignancies (Li et al 2005, Katkuri et al 2010) and under conditions of aqueous interference (Bindig et al 2003). Presently malignant tissues are excised during colonoscopy and send for evaluation. With the development of suitable probes, it is envisoned that other than the area with the symptoms, adjoining areas can be evaluated to determine the extent of spread of disease and redefine the resection margin. Another potential use would be to differentiate diseases with similar symptoms like IBD and cancer using such probes. This would help in early intervention and prevent recurrent surgeries resulting in less patient discomfort and less expenses. Thus, the field of FTIR based disease diagnosis can be utilized in various levels such as the simple diagnosis using IR microscopes to advanced in-vivo methods using fiberoptic sensors.

#### **9. Discussion**

The diagnostic potential of FTIR spectroscopy relies upon its ability to detect and monitor changes in the biochemical fingerprint of abnormal cells, tissues and molecules as compared to the normal conditions along with its ability to monitor minute spectral differences among closely related samples. Thus, establishing reference set of spectra of every biological entity that is being studied is an integral part as with other spectroscopic methods, often the references data base built using model compounds, whose exact spectral contribution is known (Benedetti et al 1996). Biological samples are procured after many different types of procedures and thus, each type may demand different approach for spectral acquisition and analysis. In addition, the availability of the instrument set up and resources would play an important role in deciding which types of samples can be analyzed in a particular clinical set up. For example, when both FTIR microscopy and normal spectroscopy is available, blood components can be examined by the normal method while tissues and biopsies can utilize the microscopy. Similarly, the tissue architect dictates the type of measurement to be followed. While tissues like cervical epithelium where distinct zones permit the spectral acquisition at several locations within a uniform region (Figure 3) more complex tissues like melanoma and nevus make it imperative that the location is carefully selected to not exclude desired region and not include regions that do not meet the criterion of either normal or diseased conditions (Hammody et al 2005). Complicated tissues like colonic epithelium present two possibilities of data acquisition, along the cross section (Argov et al 2002,2004) or longitudinal section (Sahu et al 2004b, 2010) of the crypts and inevitably the availability of microscopic or FPA facilities. Understanding the spectral differences due to instrumentation has been undertaken with the objective of tailoring the techniques to suit set ups and also verifying the universal applicability of certain parameters compared to others (Hammody et al 2007).

The biogenesis of each cell or tissue needs to be studied before a suitable methodology is designed to obtain spectral data for the specific purpose it is intended to serve. The various protocols are also required to be suitably modified depending upon whether a sample is fresh, frozen or paraffin embedded to exclude interfering substances (Sahu et al 2005). The spectroscopy of microorganisms on the other hand may be simpler (Sahu et al 2006b).Both FTIR and Raman spectroscopy use vibrational spectroscopy and can be used independently or in combination with each other as a means of spectroscopic evaluation of biological samples(Mourant et al 2003b, Krishna et al 2005, Oust et al 2006). The prospective of extending Fiberoptic detection systems to different organs is a future potential (Lucas et al 2005).
