FT-IR and Raman vibrational microspectroscopies used for spectral biodiagnosis of human tissues

Fourier transform infrared (FT-IR) and Raman vibrational microspectroscopies used for biomedical diagnosis of human tissues are reviewed from basic principle to biological applications. The advantages and disadvantages of both vibrational microspectroscopies are compared to highlight their efficiency and adaptability for noninvasively investigating the chemical compositions of ultrastructual human tissues at different disease states. Biochemical fingerprints applied to the biological samples by using FT-IR and Raman microspectroscopies are illustrated. The spectral biodiagnoses of several diseased human tissues such as ophthalmic disorders (risk factors-induced cataractous lens capsules and lens, lens and corneal calcifications, opacification and contamination of intraocular lens, vitreous asteroid bodies), alcohol-disordered human gastric mucosa, skin disorders (cancer and calcification), brain tumors (pituitary adenomas and astrocytomas), genetic hair roots disorder (glucose-6-phosphate dehydrogenase deficiency, phenylketonuria and congenital hypothyroidism), benign prostatic hyperplasia, and interstitial cystitis investigated by both vibrational microspectroscopies in our laboratory are introduced.


Introduction
Infrared (IR) and Raman spectroscopies have been widely applied to detect the vibrational characteristics of chemical functional groups in diverse materials, including polymers, semiconductors, pharmaceuticals, and others [1][2][3][4].Both techniques not only provide the chemical information for all materials but also offer chemical fingerprinting services for different analytical results, particularly after the Fourier transform (FT) system has been introduced into both techniques.A FT infrared (FT-IR) spectrometer collects an interferogram of a sample signal from an interferometer to obtain the IR spectrum of sample, while FT-Raman spectroscopy uses a laser beam to illuminate the sample and collects Raman scattering from a sample [5].These FT instruments equipped with a computer system enable weak signals to be measured faster and more sensitive than the older dispersive instruments.The computerized FT instrument may save the IR or Raman spectrum of sample for different data processings.Thus, it is interesting for many researchers in the fields of biochemistry, biomedicine, biophysics and biomaterials to use both vibrational spectroscopies to investigate structural components of biosamples [6][7][8].
Although IR and Raman spectroscopies measure the vibrational energies of molecules, both methods are dependent on different selection rules, i.e., an absorption process and a scattering effect (Fig. 1).IR spectroscopy is based on the absorption of electromagnetic radiation, whereas Raman spectroscopy relies upon inelastic scattering of electromagnetic radiation.The sample is radiated by IR light in IR spectroscopy, and the vibrations induced by electrical dipole moment are detected.In Raman spectroscopy, the sample is illuminated by a monochromatic visible or near IR light from a laser source and its vibrations during the electrical polarisability changes are determined [5,8,9].Thus both vibrational techniques may provide complementary information, since the shapes and positions of both spectral bands on the energy scale are closely located for the specific functional groups of the molecule (Fig. 2).Many vibrations are observed in both spectra to serve as a valuable confirmatory evidence of samples.Therefore, the combination of using IR and Raman spectroscopies can offer a more potential approach for analyzing intact sample and provides more detailed chemical information.

FT-IR or Raman microscopy and imaging
Recently, FT-IR or Raman spectroscopy is coupled with a microscope and a computer system, capable of microanalysis of minute samples by using a dedicated detector, mercury cadmium telluride (MCT).The resultant FT-IR or Raman vibrational microspectroscopy can provide molecular information of samples with a high spatial resolution at microscopic level.Samples with microscopic size can be nondestructively analyzed by both vibrational microspectroscopies, particularly in the application of biomedical sciences [10][11][12][13].Thus, the use of both vibrational microspectroscopies have extensively become a great potential over other diagnostic techniques for noninvasively investigating the chemical components of ultrastructual tissues at various disease states, due to the reagentless procedure and no dyes or labels addition for spectral determination [6,14,15].As compared with conventional histopathologic diagnosis, the microspectroscopic chemical information is admitted to be more objective and rapid.Both microspectroscopic techniques not only provide more detailed spectral information of tissue (lipids, proteins, RNA/DNA, carbohydrates) for physicians, but also earlier predict the diagnostic results of diseases.In the current biomedical diagnosis, many investigations have been focused on discriminating normal and malignant tissues at different sites such as oral, stomach, cervical, breast, and skin, colon, etc. [10,12,14].Several excellent review papers to introduce the biomedical applications of FT-IR and Raman microspectroscopies have been reported [6][7][8][11][12][13][14][16][17][18].
More recently, FT-IR and/or Raman microspectroscopic imaging systems have also been developed for applying to biosciences [19][20][21][22][23].The imaging system is a powerful tool to map the spatial compositions distributed in the heterogeneous biological tissues.With the development of new software and instrument, functional group mapping or chemical imaging has become accessible.In common, the spectroscopic images are obtained by mapping with a standard microscope equipped with an XY stage and a computer-controlled motorized stage, but this process is time consuming even to map a few square millimeters in size.FT-IR or Raman spectra may be simultaneously collected at a time in a stepwise manner from different areas of a sample.The absorbance of a band corresponding to a specific chemical component may be plotted as a map.A new imaging capability has been established not only to image heterogeneous regions of tissue and simultaneously to provide spectroscopic and spatial information, but also to show visually the concentrations of components and to highlight their effect from the threedimensional plot.The application of microscopic FT-IR imaging system to the intestinal section of SD rats after feeding either cholesterol alone (A) or cholesterol and the Ginkgo biloba L. extracts (B) is shown in Fig. 3, in which the absorbance images represent the area under the IR peaks at 2917 cm −1 (lipid), 1655 cm −1 (protein) and 1081 cm −1 (phosphate and glycogen) of a 5 μm-thick SD rat intestinal section.The changes in color contour may show the content and distribution of lipid, protein, and phosphate/glycoprotein constituents in tissues [24].Recently, new instruments by using a CCD array will make area mapping much less time consuming.The high performance of CCD focal-plane array (FPA) detector has further enabled the simultaneous collection of several thousand IR spectra to form images within a few minutes [25,26].

Biochemical fingerprints of FT-IR or Raman (micro)spectroscopy
Undoubtedly, a better diagnostic result obtained from FT-IR spectroscopy strongly depends on the quality of the IR spectra determined.According to the standard operation of IR determination, the sample will absorb the IR radiation and exhibits different vibrations such as stretching, bending, deformation or their combined vibrations.These vibrations are just directly correlated to the molecular structure of biosamples.For most studies of spectral biodiagnosis of biosamples, the mid-IR range of spectrum within 4000-600 cm −1 seems to be more optimal than the near-IR range (14 000-4000 cm −1 ) due to an existence of overtone.In the spectral region of 4000-1500 cm −1 several specific bonds are characterized by various stretching modes of functional groups of molecules.Peaks below 1500 cm −1 are significant assignments for deformation, bending and ring vibrations, and are commonly referred as the fingerprint region of the spectrum.Both microspectroscopies are well known as attractive analytical technique, and their applications to the biological and biomedical fields have rapidly increased.The major reason is that many biomolecules, such as proteins, lipids, nucleic acids, and carbohydrates have characteristic and specific vibrational fingerprints [6][7][8]10].Several predominant IR bands in biosamples are amide I (1600-1700 cm −1 : 80% C=O stretching mode), amide II (1500-1600 cm −1 : 60% N-H bending and 40% C-N stretching modes), amide III (1200-1330 cm −1 : 40% C-N stretching and 30% N-H bending modes), asymmetric or symmetric phosphate (near 1225 or 1080 cm −1 ), and glycogen (near 1030 cm −1 ), respectively.Subtle changes in peak shape, peak position, peak intensity and/or area of IR absorbance bands represent different structural conformations of biomolecules.The ratio of IR peak intensity or peak area, 1030 cm −1 /1080 cm −1 (glycogen/phosphate ratio) or 1121 cm −1 /1020 cm −1 (RNA/DNA ratio), may be used as a potential biomarker to predict the cell proliferation in the normal or malignant tissue [27,28].In addition, the mineralization occurred in the tissues may also be monitored by the ratio of 900-1200 cm −1 (ν 1 , ν 3 phosphate stretching mode) and amide I contours (mineral : matrix ratio), or the ratio of 850-900 cm −1 (total carbonate)/900-1200 cm −1 (ν 1 , ν 3 phosphate) [29].Moreover, the mineral crystallinity or maturity may also be deduced from the IR peak intensity ratio of 1020 cm −1 (nonstoichiometric apatite)/1030 cm −1 (stoichiometric apatite) [29,30].
Raman spectroscopy also provides similar information concerning the composition and chemical structure to that of FT-IR spectroscopy.Unlike FT-IR determination, Raman measurement can directly detect the hydrated tissue samples since the Raman scattering signal of water molecule is very weak but the fluorescence induced by laser should be avoided [31].Due to the vibrational activity is different between Raman and FT-IR spectroscopies, some modes in both spectroscopies are active, but others are only Raman or FT-IR active.Thus FT-IR and Raman spectroscopies provide similar and complementary detailed information of molecular vibrations [1][2][3][4][5].Although Raman spectroscopy is a complementary method to the FT-IR spectroscopy, it is particularly optimal to nondestructively analyze the dark and opaque samples, corrosion products and minerals, due to its emission technique [32].For example, the carbonate and phosphate vibrational modes of biominerals in different biological tissues are easily determined by Raman spectroscopy.The characteristic and comparative absorbance bands found in biological samples are shown in Table 1 for both FT-IR and Raman spectroscopies [1][2][3][4][5]33].Recently, the development of a confocal Raman microscopy is becoming a potential tool to control the depth of field, eliminate the image degrading out-of-focus information, and collect serial optical sections from thick specimens [35].This confocal microscopy offers several advantages over conventional optical microscopy, resulting in possible application to the measurement of minute sample and cellular components in single cells [36,37].

Advantages/disadvantages of FT-IR and Raman (micro)spectroscopy
Although the Raman scattering spectrum provides the same type of information as the IR absorption spectrum, both vibrational techniques have characteristic advantages and disadvantages in biomedical applications due to their different mechanism and selection rules.The advantage of FT-IR microspectroscopy over conventional FT-IR spectroscopy is that it is not only fast, non-destructive, higher signal/noise ratio, higher spatial resolution but also multiple accessories can be used, particularly in the pathological applications [38].Furthermore, the secondary structure of the proteins may be noninvasively measured in native environment.Although Raman microspectroscopy has less sensitive Raman signal and may also be interfered by laser-induced fluorescence, it has a confocal property, less water interference, and higher spatial resolution.The characteristic advantages and disadvantages of both vibrational microspectroscopies used in different scientific fields are listed in Table 2 [5,6,31,33].
However, two technical problems should be paid more attention by using both spectroscopic techniques in the practical study of biological tissues.The first problem is water having a very intense IRabsorbing bending mode near 1640 cm −1 , which just directly overlapped to the IR absorbance of protein.Fortunately, modern FT-IR instruments and software may overcome this problem to a great extent.The secondary structure of protein in aqueous system can be overcome by a FT-IR spectroscopy with an attenuated total reflection (ATR) measurement [1][2][3]7,9].On the other hand, there is only a feeble interference of water in the Raman determination.Another problem for Raman determination is the heating destruction to the sample after long-term laser irradiation [39].In order to obtain an enhanced Raman spectrum of sample, the prolonged exposure of a specimen to a laser beam is necessary.However, the heat generated by the laser may alter or destroy the biosample texture in the long-term exposure of measurement.To overcome the heating destruction of sample, the shorter irradiation time with a repeated scanning number seems to be optimal.

Current biological applications of vibrational microspectroscopy
Until now, both FT-IR and Raman vibrational microspectroscopies have been extensively applied to the diverse biological researches, such as the molecular compound distribution (lipids, proteins, nucleic acids, carbohydrate) and chemical information to histological structure of a single cell in cell biology

Table 2
The characteristic advantages and disadvantages of FT-IR and Raman microspectroscopies used in the biological applications [5,6,31 study [10][11][12][13][14], rapid and accurate identification of the nature of colony development and biofilm formation for microorganisms and microbial fermentations [40,41], the chemical and functional characteristics of plant tissues and seeds [42,43], the interaction of implants with biological system [31,44,45], and the detection of drug abuse and drug distribution in a forensic study [46,47].Tables 3 and 4 list many examples and applications of different human tissues determined by using FT-IR and/or Raman spectroscopy combined with/without microscopy .

Spectral biodiagnoses of diseased human tissues in our laboratory
Since both vibrational microspectroscopic detections can qualitatively and quantitatively distinguish the differences from the spectral characteristics of many molecules, thus the molecular and submolecular spectral profiles may be used to define and differentiate the diseased and healthy human tissues.Here, we will review several biomedical diagnoses of disordered human tissues investigated by using FT-IR and/or Raman microspectroscopy in our laboratory.All the studies were approved by the Institutional Review Board of our hospital and all procedures adhered to the Declaration of Helsinki.The schematic diagram of different disordered organs in the human body diagnosed in our laboratory is shown in Fig. 4.

Diagnosis of human ophthalmic disorders 6.1.1. Cataractous lens capsule [120-125]
The lens capsule mainly constructed by type IV collagen is an acellular basement membrane of eye to maintain the shape of the lens.It also protects the interior of the lens from ultraviolet radiation.Although the morphology, physico-chemical properties, functional and conformational structure of type IV collagen have been reported to be easily influenced by many events such as ages, diseases or other foreign stress factors, little attention has been paid to clarify the role of human lens capsule in cataract formation.Our groups had first used a FT-IR microspectroscopy with second-derivative analysis and curve-fitting program to differentiate the grading and maturity of cataracts based on the progressive changes in protein secondary conformation of cataractous human lens capsules (Fig. 5).Moreover, the effect of risk factors (diabetes, glaucoma, systemic hypertension, myopia or heredity) of cataract formation on the protein secondary structures and its compositions of cataractous lens capsules in patients have been investigated.Our studies found that the duration of diabetes was a major influencing factor to alter the protein secondary conformation of the cataractous lens capsules in diabetic patients not only by decreasing the triple helix content but also by markedly increasing the random coil structure.The compositions of both random coil and β-structure (β-sheet and β-turn) in cataractous human lens capsules were increasingly induced by systemic hypertension, myopia or glaucoma, but α-helix content clearly decreased.A possible pathway of cataract formation exacerbated by systemic hypertension or glaucoma was proposed.
According to the results, we proposed that the risk factors might change the protein conformational structure of lens capsule, then cause the alteration of membrane transport and permeability for ions, and finally increase the intraocular pressure to exacerbate cataract formation (Fig. 6).The content of aspartic acid had also been reduced significantly in the lens capsules of hereditary cataractous patients.Our conclusion suggested that the cataract formation might be possibly associated with the age-related alteration of protein secondary structure in the lens capsule and exacerbated by several diseases-related risk factors.

Cataractous lens [126]
Two normal lenses, 10 immature cataractous lenses without any complication and 4 immature cataractous lenses with glaucoma were used after surgical operation for investigating the changes in the hu-Fig.6. Speculative scheme for exacerbating cataract formation by systemic hypertension or glaucoma [124].
man lens lipid and protein structures.The ATR/FT-IR results indicated that the compositional ratio of 2965/2930 cm −1 , due to the methyl groups of proteins and methylene groups of phospholipids, for normal lens was about 0.702, but the ratio for cataractous lenses without glaucoma was 0.382 and for glaucomatous lenses was 0.377.The maximum IR peak position in amide I band of normal lens, immature cataractous lenses without complications or with glaucoma was respectively found at 1632, 1630 or 1622 cm −1 , suggesting that the β-sheet structure was a predominate component in it.The peak intensity ratio of amide I/amide II (1632 cm −1 /1545 cm −1 ) for normal lenses was in the range of 2.20-2.33,whereas this ratio for immature cataractous lenses without glaucoma (1630 cm −1 /1545 cm −1 ) was 1.28-1.41but was 1.04-1.13for glaucomatous lens (1622 cm −1 /1545 cm −1 ).The decreased amount of α-helix and random coil structures but the enhanced content of β-sheet structure in the immature cataractous human lens induced by glaucoma might be due to an intermolecular hydrogen-bonded formation in lenses.The results revealed that the alternations in lens lipid and protein structures played an important role to induce cataract formation.The glaucomatous lens was more pronounced.[127] The chemical composition of a small calcified plaque on the surface of a senile cataractous lens isolated from a male patient was determined by using FT-IR and confocal Raman microspectroscopies.The noncalcified area of the same lens and hydroxyapatite (HA) were selected as a control.Several unique IR absorption bands at 960, 1034 and 1090 cm −1 assigned to the ν 1 and ν 3 phosphate stretching modes and at 875 cm −1 attributed to carbonate band were clearly displayed in the IR spectra of calcified plaque.A peak at 961 cm −1 due to the ν 1 phosphate stretching mode was also evidenced in the Raman spectra of calcified plaque and was near to 958 cm −1 for HA (Fig. 7).In fully mature minerals, the carbonate ions may occupy two anionic sites of the apatite structure or may be in surface site locations [128].The CO 2− 3 ions occupying the OH − sites are generally defined as type-A carbonate apatites, but the CO 2− 3 ions occupying the PO 3− 4 sites are defined as type-B carbonate apatites.The IR spectra of minerals exhibited three components of these carbonate bands in the spectral region of 860-890 cm −1 : type-A carbonate (>878 cm −1 ), type-B carbonate (>871 cm −1 ) and an unstable carbonate location (near 866 cm −1 ), respectively.In this study, the calcified plaque formed within the cataractous lens was found to mainly consist of a mature HA, in which the content of type-A carbonate apatites (11.4%), type-B carbonate  apatites (55.6%) and liable surface carbonate ions (33.0%) were close to the result of HA sample after applying with curve-fitting program (Fig. 8).A higher content of the liable carbonate implied that the calcification in this calcified lens was incomplete and still in progress.[129] FT-IR microspectroscopy combining with ATR microsampling technique and micro-Raman confocal spectrophotometer were used to detect the component of opaque deposited materials on the surface of explanted acrylic hydrogel intraocular lens (IOL).The brand-new IOL exhibited a very smooth, transparent and featureless surface by observing with a confocal laser scanning microscope, but the explanted IOL had an irregular cerebriform-like opaque appearance.Both ATR/FT-IR and Raman microspectroscopic analyses showed that the deposit on the surface of explanted IOL was consisted of octacalcium phosphate and type-B carbonate apatites, leading to the opalescence of IOL.It has been reported that the early stages of immature poorly crystalline HA having peaks at 960, 985, 1020, 1038, 1055, and 1075 cm −1 might shift to the mature HA with peaks at 960, 982, 999, 1030, 1055, 1075, 1096, and 1116 cm −1 .The former belonged to the nonstoichiometric apatites, whereas the latter was stoichiometric apatites.In the present study, the second-derivative IR spectra of HA (A) and explanted IOL (B) also exhibited similar peaks to the above positions, as shown in Fig. 9. Obviously, the HA sample used in this study was close to the stoichiometric and mature crystalline HA.However, the calcified deposits on the explanted IOL was similar to the freshly precipitated, immature poorly crystalline HA.Both vibrational microspectroscopic examinations also confirmed the mineralization still in progress on the surface of IOL even ocular implantation for 2 years.

Identification of oily-like material on IOL [130]
A small oily-like hump on the tempo-upper quadrant of anterior surface of IOL was found under a slit-lamp microscope for an old man after IOL implantation.The surgeon failed to remove the hump with Nd:YAG laser.One year after implantation, his vision decreased to 20/400 because this oily-like hump extended to the central part of IOL surface.The oily-like material deposited on the explanted IOL surface after IOL exchange was examined by FT-IR and confocal Raman microspectroscopies.The IR bands near 2955, 2920, and 2850 cm −1 for oily-like material were assigned to the asymmetric and symmetric CH stretching modes, the peaks at 1463 and 1377 cm −1 were the result of the CH 2 and CH 3 bending vibrations.The FT-IR spectrum of oily-like material were just the same as that of the fatty acyl chains of ointment base for garamycin ophthalmic ointment.This implied that the oily-like material should be the oily base of ointment.Raman bands at 2931, 2887, and 2856 cm −1 (asymmetric and symmetric CH stretching modes) and at 1444 and 1303 cm −1 (CH 2 and CH 3 bending vibrations) also confirmed the results of FT-IR spectrum.This result identified a long-term retained garamycin ophthalmic ointment on an IOL after sutureless cataract surgery.

Corneal calcification [131]
The chemical composition of the excised corneal calcified opaque deposit on the surface of an excised cornea from a male patient was also quickly and quantitatively investigated by microscopic ATR/FT-IR and confocal Raman spectroscopies.
Microscopic observation indicated that a white-grayish opaque plaque-like deposit was seen (Fig. 10).A peak at 1020 cm −1 assigned to the stretching vibration of phosphate of the immature poorly crystalline and nonstoichiometric HA was observed from the IR spectrum of corneal calcified deposit, as compared with the peak at 1031 cm −1 due to the mature, crystalline and stoichiometric HA [132].In addition, higher contents of two IR spectral peaks at 871 cm −1 (type-B carbonate apatite) and at 866 cm −1 (a labile carbonate) after curve-fitting were also evidenced in the corneal calcified deposit.The dominate peak at 959 cm −1 due to its stretching mode of phosphate was also found in the Raman spectrum of corneal calcified deposit.The results indicated that the corneal calcified deposit was consisted of an immature poorly crystalline HA with higher content of the type-B carbonate apatite within the corneal collagen matrix.[133] Asteroid hyalosis (AH) is a degenerative disease of the vitreous that consists of multiple discrete yellowish-white minute spherical particles, termed asteroid bodies (ABs), suspended within the vitreous humor.The origin, composition, and formation of ABs in the vitreous are still incompletely elu-Fig.11.Representative photograph of one ABs specimen suspended within the vitreous humor and Raman spectra of two ABs specimens from both AH patients, and β-carotene as well as lutein [133].cidated, although several techniques had been attempted to investigate their structure and composition [134][135][136].Until now, the structural compositions of ABs had been proposed to consist of calcium hydroxyapatite, sphingomyelin, triglyceride, cholesterol, ceramide dihexoside, cerebroside sulfate, proteoglycans, and glycosaminoglycan in the vitreous humor.In this study, the morphology and components of the minute AB specimens excised from two AH patients were observed by optical microscopy and determined by using confocal Raman microspectroscopy.The high performance liquid chromatography (HPLC) was also used to verify the component.Figure 11 showed the representative photograph of one AB specimen suspended within the vitreous humor.A lot of particles entrapped in the vitreous gel were clearly observed in the AB aggregates.The particle size of ABs ranged approximately between 20 and 50 μm.Both AB specimens displayed similar appearances.Two unique peaks at 1528 and 1157 cm −1 were found in the Raman spectrum of the AB specimen for patient 1 alone, which were in close agreement with that of the Raman peaks at 1525 and 1158 cm −1 for β-carotene and/or lutein [137].However, HPLC analytical data clearly verified that the retention time for the extracted sample from the AB specimen of patient 1 was observed at 13.68 min and just identical to that of β-carotene at 13.75 min rather than lutein at 2.97 min.Furthermore, the lack of any peak in the HPLC profile for the AB specimen of patient 2 also confirmed the absence of Raman peaks at 1525 and 1158 cm −1 .Thus this study strongly suggested that β-carotene as a unique component of ABs might be specifically de-tected from the AB specimen of one AH patient by using confocal Raman microspectroscopy and HPLC analysis.

Alcohol-induced structural alteration of human gastric mucosa
Gastric mucous gel that lines the surface of gastric mucosa is crucial for epithelial protection.In order to simulate the effect of ethanol in the alcoholic drinks on the human gastric mucosa, the ATR/FT-IR microspectroscopy was used to investigate the protein secondary structure of human gastric mucosa (isolated from human gastric wall) after treatment with ethanol, in vitro.The effect of concentration and exposure time of ethanol on the structural changes of this gastric mucosa was also studied.The peak intensity and position of IR spectra for gastric mucosa was changed significantly with the increases of ethanol concentration and length of exposure [138,139].The IR peak intensity due to the β-sheet and/or β-turn conformation in amide I and II bands of gastric mucosa was clearly increased after treatment with ethanol.Moreover, the peak at 1635 cm −1 shifted to 1630 cm −1 after treatment with 40% ethanol for 3 h, or 80% ethanol for 1 h, and a distinct shoulder also appeared at 1643 cm −1 .This shift was occurred more rapidly and was more pronounced after exposure of mucosa to 80% ethanol.Ethanol treatment might also transform the secondary structure of amide III in gastric mucosa from α-helix to random coil with extensive unfolding.The absorption between 1180 and 980 cm −1 assigned to glycoprotein structure was also reduced after treatment with ethanol.This strongly revealed that ethanol markedly influenced the conformational structure of proteins and carbohydrates of gastric mucus gel.The dehydration and interference of hydrophobic interactions in the isolated mucus gel after pretreatment with ethanol might be responsible for this conformational change.[140] Pilomatrixoma (PMX) is a benign cutaneous epithelial tumor that occurs in the dermis or subcutaneous tissues.PMX may be diagnosed from sebaceous cysts by noninvasive physical imaging examinations, which is only capable of providing morphological information.In this study, confocal Raman microspectroscopy was used to qualitatively identify and distinguish the chemical components of the PXM tissues from that of normal skin tissue.The normal skin dermis, type I collagen and HA were used as control.The excised specimens from two patients were diagnosed as a typical PMX, in which one specimen was a soft mass but the other was a hard mass.The Raman spectrum of normal skin dermis was found to be similar to the Raman spectrum of type I collagen.The major differences of Raman peak intensity between normal skin dermis and soft or hard PMX mass were obvious at 1622, 1558, 1400-1230, 1128, 1000-850, 749 and 509 cm −1 (Fig. 12).In particular, the peak at 1665 cm −1 assigned to amide I band shifted to 1655 cm −1 and the peak at 1246 cm −1 corresponding to amide III band was reduced in its intensity in hard PMX mass.The significant changes in collagen content and its structural conformation, the higher content of tryptophan, and disulfide formation in PMX masses were markedly evidenced.In addition, the shoulder and weak peak at 960 cm −1 assigned to the stretching vibration of PO 3−  4 of HA also appeared respectively in the Raman spectra of soft and hard PMX masses, suggesting the occurrence of calcification of HA in the PMX tissue, particularly in the hard PMX mass.

Skin calcification [141]
The clinical diagnosis of calcinosis cutis has been established by time-consuming histopathological or immunohistochemical evaluation.In this study, a young Chinese lady had undergone surgery for a calcinosis cutis (Fig. 13).The skin lesion was totally excised and cut into two equal parts to send one for  H&E stain and the other for FT-IR and confocal Raman microspectroscopic studies.Our result indicated that the whole skin calcified deposit in the skin lesion was found to be a well developed, mature and hard mass.Several FT-IR absorption bands at 870, 961, 1031, 1547 and 1658 cm −1 were detected in the IR spectrum of deposit.The Raman spectral bands at 1665 and 1450 cm −1 (collagen); 1519 and 1156 cm −1 (β-carotene); and 1072 and 958 cm −1 (HA) were also obtained.In this study, we first described that the skin calcified deposit of a calcinosis cutis was composed of collagen, β-carotene and type-B carbonate HA using FT-IR and Raman microspectroscopic investigations.

Identification of brain tumors
Human pituitary adenomas are the most frequent intracranial neoplasms originating from gland cells of adenohypophysis, which can be responsible for different endocrine and tumoral symptoms.According to the basis of the endocrinological potential of the adenoma, it can be classified into clinically silent and active adenomas [142].We had used a reflectance FT-IR microspectroscopy to determine the secondary structure and composition of the different human pituitary adenomas.The silent pituitary adenomas exhibited similar protein secondary structure and conformational composition, but active pituitary adenomas revealed different behavior.The reflectance IR spectrum of the silent pituitary adenoma revealed a maximum peak at 1651 cm −1 in the amide I band, whereas the maximum peak was located at 1653 or 1657 cm −1 for active pituitary adenoma.The differences in secondary structure for different human pituitary adenomas might possibly be due to the different protein conformations of the proliferated adenoma tissues and various hormones shared [143].
The benign and malignant astrocytomas were also determined by reflectance FT-IR microspectroscopy [144].The IR peak maximum in the amide I band of the tissues from recurrent malignant astrocytoma markedly appeared at higher frequency (1655 or 1663 cm −1 ), which was significantly different from that of the tissues from benign astrocytoma at 1651 cm −1 and tissues from malignant astrocytoma at 1652 cm −1 .Malignant astrocytoma indicated slightly different compositions in the protein secondary structure from benign astrocytoma.A significant increase in β-turn structure (1660, 1668, 1682 and 1691 cm −1 ) but a marked decrease in β-sheet (1628 and 1618 cm −1 ) and in random coil (1645 cm −1 ) structures were observed in the protein secondary structure of the recurrent malignant astrocytoma.The phenomenon was more pronounced in recurrent malignant astrocytoma pretreated with radiation and chemotherapy.The rapid cell proliferation and cell differentiation of malignant astrocytoma with or without recurrence might be the possible explanations for the different compositions of protein conformational structures.6.5.Diagnosis of genetic disorders from scalp hair roots 6.5.1.Glucose-6-phosphate dehydrogenase deficiency [145] Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an important hereditary metabolic defect that leads to acute hemolytic anemia on ingestion of certain drugs and foods [146].The clinical symptoms of this genetic disease can be prevented if it is detected early; thus neonatal diagnosis is important.We had demonstrated that FT-IR microspectroscopy could be a rapid and effective diagnostic method to differentiate the scalp hair roots of normal neonates (n = 67) into the anagen, catagen or telogen phases of the hair growth cycle (Fig. 14), using IR peak area ratio of 2854 cm −1 /2873 cm −1 (A) or 1084 cm −1 /amide II band (B) (p < 0.001).Moreover, G6PD-deficient neonates (n = 39) could be accurately diagnosed from telogen phase hair roots owing to significant differences in IR peak area ratios Fig. 14.Peak area ratio of the 2854 cm −1 /2873 cm −1 (A) or 1084 cm −1 /amide II band (B) against the number of normal neonates in three different phases of hair growth cycle: ", anagen phase; !, catagen phase; 2, telogen phase [145].The shaded area represented the normal range. of 2854 cm −1 /2873 cm −1 or 1084 cm −1 /amide II band, compared to normal values in healthy neonates.Hair root analysis seems to be a useful complement to serum and urine analysis in the diagnosis of genetic diseases.[147] Classic phenylketonuria (PKU) is an autosomal recessive disorder caused by failure of hydroxylation of phenylalanine (PA) to tyrosine.When high levels of PA are accumulated, it may cause severe mental retardation [148].Eight normal newborn infants (PA level <2 mg/dl) and 2 classic PKU infants before and after dietary control with a low PA diet were selected for study.The FT-IR spectra of these hair roots were determined by using FT-IR microspectroscopy.Our findings strongly illustrated that two unique peaks at 1339 and 1169 cm −1 was observed in the hair root of PKU patients before dietary control.Both unique peaks were also verified in the IR spectrum of L-cysteinesulfinic acid.It has been reported that L-cysteinesulfinic acid, as one of the products in the sulfur metabolism of cysteine, can convert to sulfate and being excreted from the urine via transamination [149], although the reason is still unclear.This study reported on the marked difference in FT-IR spectra of hair roots between normal infants and PKU patients before and after dietary control.The evidence of L-cysteinesulfinic acid in hair roots of PKU patients might disappear with dietary control, as shown in Fig. 15.[150] Congenital hypothyroidism (CH) is a common preventable disease mainly caused by thyroid dysgenesis or homonogenesis defects.CH is characterized by low levels of serum triiodothyronine (T3) and thyroxine (T4), and elevated levels of thyroid-stimulating hormone (TSH) [151].In the present study, we used FT-IR microspectroscopy to detect the biophysical properties of anagen scalp hair roots of neonates with CH.The serum levels of T3, T4, free T4 (fT4) and TSH for normal (n = 20) and thyroid hormone deficient (n = 2, serum TSH level >10 mU/l) newborns were also determined by radioimmunoassay.The screening results indicated that the serum levels of T3, T4, fT4, and TSH for normal Fig. 15.FT-IR peak intensity ratio of the scalp hair roots for normal and PKU infants [147].Key: PKU infants 1 and 2: infants before dietary control.PKU infant 1A: infant 1 after dietary control for 3 hrs.PKU infant 2A: infant 2 after dietary control for 1 week.*Plasma PA level.newborns were all within the normal limit.However, patients 1 and 2 had lower T4 and fT4 levels but the highest TSH level (>60-fold to normal value), and were clinically diagnosed to be the typical severe neonatal hypothyroidism.The FT-IR results indicated that the lower composition near 1053 cm −1 (also assigned to the aromatic iodide stretching band) in the IR spectra of the hair roots for CH patients was directly associated with the lower serum level of T4 and fT4, and the elevated TSH levels.

Congenital hypothyroidism
6.6.Detection of benign prostatic hyperplasia 6.6.1.Epithelial and stromal growth in the human BPH [152] Benign prostatic hyperplasia (BPH) is one of the most common diseases in elderly men.It describes an overgrowth of the epithelium and stroma in the human prostate, in which both tissues proliferate at different rates [153].We utilized a reflectance FT-IR microspectroscopy to pathologically image and examine the structural changes of the molecular constituents in BPH tissues.Two successive thin tissue slices were obtained by cutting the tissue with a microtome: one unstained slice was used for FT-IR microspectroscopic analysis and the other stained slice was used for position referencing.The result showed that the IR maximum peak in the amide I band for BPH epithelial tissue was located at 1638 cm −1 assigned to the contribution of random coil and β-sheet structures.However, an intense IR absorption peak in amide I band was at 1630 cm −1 for BPH stromal tissue due to the predominant β-sheet structure.The higher peak intensity of several IR bands at 1337, 1281, 1238, 1206, 1055 and 1034 cm −1 , assigned to the C-O stretching mode of collagen, was observed in BPH stromal tissue (Fig. 16).This suggested that the higher content of collagen was observed in the stromal tissue than in the epithelial tissue of the proliferous BPH.[154,155] The thermotherapy has been successfully used to treat the BPH patients by using a transurethral prostatic heating device.We had used a novel system by combining the microscopic FT-IR spectroscopy and differential scanning calorimetry (DSC) to non-isothermally (from 25 to 60 • C) or isothermally (47 • C, 3 h) simulate the clinical BPH thermotherapy, in vitro.The protein conformational structure of the epithelium and stroma of BPH tissue during both thermal treatments were also investigated.The results revealed that the isolated epithelium and stroma of BPH tissue could slightly change their secondary structure from α-helix to β-structure in the temperature range of 25-60 • C, like the other thermal-sensitive protein.However, this thermal-induced conversion behavior in the stroma was less sensitive than that in the epithelium during thermal treatment.The different thermal response between the epithelium and stroma of BPH might be due to the different constitutions in the epithelium and stroma of BPH.On the other hand, no significant change was evidenced in the secondary structure for each sample either before or after isothermal study (Fig. 17), suggesting the integrality and safety of BPH thermotherapy in a 47 • C for 3 h treatment course.

S.-Y. Lin et al. / FT-IR and Raman vibrational microspectroscopies used for spectral biodiagnosis of human tissues
Fig. 17.Three-dimensional plots of FT-IR spectra of epithelium (A) and stroma (B) of BPH with respect to heating time [155].[156] Transurethral resection of the prostate (TURP) is one of the gold-standard surgical treatments for BPH to alleviate the symptoms and signs of urinary outflow obstruction and to improve quality of life.In the process of TURP, the higher electrosurgical energy via cutting loop was used to remove the prostatic tissue piece.Here, the ATR/FT-IR microspectroscopy was applied to study the effect of the higher electric cutting heat of TURP treatment on the protein conformation of stroma and epithelium in BPH tissue.The tissue obtained by a non-heating process of prostatic needle biopsy was used as a control.The results indicated a predominantly higher proportion of β-sheet conformation of the epithelium or stroma in BPH tissue at 1633 or 1630 cm −1 in the amide I band, but after treatment with TURP both IR maximum peaks shifted to 1644 or 1646 cm −1 due to random coil structure, respectively.Our study showed that TURP might convert the protein conformational structure of both epithelium and stroma in BPH tissue from β-sheet to random coil due to the higher temperature used.This conformational interconversion might possibly relate to the occurrence of complications or the alteration of functional integrity of BPH.

Diagnosis of interstitial cystitis after PST
Interstitial cystitis (IC) is a chronic bladder disorder occurring predominantly in women.There is lack of a uniform, definitive and worldwide standard technique for clinical diagnosis of IC [157].Twenty-two participants were screened by clinical features, history, potassium sensitivity test (PST) and urodynamic evaluations.The freeze-dried water samples voided from all the participants after PST were directly determined by using a confocal micro-Raman spectroscopy to investigate their chemical compositions.Participants with or without IC symptom were separated into control and clinical groups, according to the above examinations.The participants in the clinical group were further divided into mild and severe subgroups by PST.A significant increase in urinary frequency but a marked reduction in bladder capacity, maximum cystometric capacity and maximum voiding flow rate were obtained for clinical group of IC participants, as compared with the result of control group (p < 0.05).The symptom of urinary pain and urgency was also significant difference between the mild and severe subgroups (p < 0.05).The Raman result indicated that the Raman band near 1003-1005 cm −1 assigned to phenylalanine was observed for control groups and mild subgroups, but the Raman band at 1010 cm −1 due to tryptophan was found for severe subgroups (Fig. 18).This study first suggested a possible application of Raman microspectroscopy to certify again the results of PST for IC diagnosis [158].Phenylalanine or tryptophan seemed to act as a biomarker to assist the diagnosis of IC after PST.Particularly, the appearance of tryptophan might be used to discriminate the severity of IC symptom.

Conclusions and future prospects
FT-IR and Raman vibrational spectroscopic techniques have been extensively used to diagnose and differentiate the chemical differences between diseased and normal tissues based on the IR absorption or Raman scattering spectra via the molecular vibrations of biosamples.Recently, several new improvements are now being developed.Both vibrational microspectroscopies coupling with the modern fiberoptic probes have been designed to directly reach less assessable organs and in vivo measure the spectra of various levels of cells, tissues and organs in their most natural state in quite a few seconds, in which the tissue without the need of biopsy can be easily detected.The detection of atherosclerosis and cervical cancers has been successfully performed [159,160].Another new design for vibrational spectroscopic techniques is a combination of FT-IR and Raman system.This FT-IR/Raman combination microprobe system enables a simultaneous examination of one fixed sample area by both techniques, without the need for any sample positioning or instrument adjustment.This is a single microscope simultaneously coupling to an IR interferometer and a Raman spectrograph, in which the IR beams and laser are coincident at the sample and provide the spectral information of the same material site [161,162].
Although there are several advantages for both vibrational microspectroscopies, whether the spectral information being able to satisfy clinical diagnostic requirements and assist the physicians' diagnosis and treatment plan for patients is not yet available.Thus further investigations and improvements of FT-IR and/or Raman vibrational microspectroscopy are necessary to make these approaches in vivo applicable to routinely use in clinical analysis at a much earlier disease stage [18,[163][164][165][166].

Fig. 3 .
Fig. 3.The application of microscopic FT-IR imaging to the intestinal section of SD rats after feeding cholesterol (A) or cholesterol and the Ginkgo biloba L. extracts (B).

Fig. 4 .
Fig. 4. The schematic diagram of different disordered organs in the human body diagnosed in our laboratory.

Fig. 8 .
Fig.8.The assignment and composition of the curve-fitted original FT-IR spectrum of HA reference sample (A) and the difference spectrum between non-calcified area and calcified plaque (B)[127].

17 Fig. 12 .
Fig. 12. Raman spectra of the normal skin dermis, type I collagen, HA, soft and hard PMX masses by normalizing the peak intensity within the region of 1460-1448 cm −1[140].Key: The decreasing ( ) and increasing ( ) tendencies of specific Raman specks.

Fig. 16 .
Fig. 16.Optical micrograph of a stained BPH tissue biopsy and the corresponding reflectance FT-IR spectra of the unstained same tissue at five different sites [152].

Fig. 18 .
Fig.18.Raman spectra and Raman shift of different samples[158].Key: a, intact phenylalanine; b, intact tryptophan; c, freeze-dried water sample of control groups; d, freeze-dried water sample of mild subgroups; e, freeze-dried water sample of severe subgroups.

Table 3
Examples for detection and identification of human tissues by FT-IR microspectroscopy