application of bio medical engg

8/3/2019 Application of Bio Medical Engg

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IOMEDICAL APPLICATIONS OF LASERSP.K.Gupta

Biomedical Applications Section, Centre for Advanced TechnologyIndore, 452 013, India

Ab,"ract

The ose of la,ser,s jar medical diagnosLe, and jar therapy initialed by photo-excitalian


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healing, "eatmem of pulmona'}' nrheulmis andtcearmem of paio of various etiologies, Themechanisms for these thetapeuric effeCtS arc nOtvc'}' well undmtood. The dinid pmemial ofthis cather simple and inexpeosive thmpeuticmodality is motivating considerable work in thisditeetion and sobstamial pmgcess is beingmade",Realizing the profoond influence opri,,1diagnosis and photntherapy can have on theqoali'}' of health eare, work in these arca> w"initiared ar CAT in 1992-93. Several Stodies havebeen made to investigate the photO-biologic-,1effem of Iasec icradiation on liviog ocgaoisn1'and to explore the me of laver inducedtlooreseence speCtroscopy fot cancer diagnosis,Following stodies on sampb reswed at surgeryor binpsy from patietm wirh cancer of difTeretltarga",. systems suirable foc clinical use have beendevelnped at CAT. One unit has been i",ralledat a cancer screeuing center for screening patientswith neopl"m of urerine eervix. The other unith" been used to Cat'}' OUI a pilot study on 25patients wirh histopatbologically confirmedcanw of oral eaviry and eocouraging multsobrained.Work has also been initiated ,It CAT in someoth" promising areas, One of these is thedevelopmem of teehni,]ues for optical in"gingthrough turbid media. Thesc have rhe porentialro pmvide sob-millimeter resolotion imagingwithom rhe need for ionizing radiation andassociated risks". The orher aetivi'}' taken op isdevdopment ofbser trap and mierobeam vcr up,These set ops "'c finding widesp


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onsidenble pwmi;e af this appmach fa.iagnosis of .hc canee. of va.ious mgans like"ioc cmix, csoplugus, lung, b,'east, and onl


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PATIENTFig.2. &a""pl"'fl"h, in"g.,."djlua",nin"miryfl'N, /&"""i"d 'po'" afpai"dcan"'maud",",,1 a,,1Un""i",ftam12.parim""kmd","udam.P"im' numb"1" 4badcau",afal",lm (A);5" 8hadca"",afbuoalm"a" (BM)aud9" 12badcan",af"ng.u,(T).Cancer of oral cavil]Oral cane is one of the most common cancersin India and several other Sourh Asian counrries.Laser induced fluorescence technique isparticularly wdl suited for early detection of oralcancers due to the easy accessibility of this organ.Studies on 337 nm excited autofluorescencespectra from oral tissues (alveolus. buccal mucosaand the tongue tissue samples obtained frompatients with the cancer of oral cavity) revealedsignificant differences in the total. specrrallyintegrated fluorescence intensity


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ORAL:'c'3. S",," plo,ft' ,bt 'p"",/ly imwa"d fI~mtm" in"mi,i" Jivm tan"rom IORAL-q and ~,,"a' IORAL-N)';", nf,}" nml ,auiry,ht ",,' >amp/'iu in""iga,,d. Th, b", ,how mtan "',, I "andad ,",iaNnn.

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PATIENT NUMBERFig. 4. &m" plotft, tht 'P"aI/y in"f7a"dfl."""'mtt imtmiry nfpaim! ,"'urow and adjoining nanna/Im..t tinut>ampktJivm'"parim..

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dimiminating cancerous from notmal oralrissues were -90% 0= the sample sizeinvesrigated". Use of a stepwise mulri-varialinear regression (MVLR) analysis with ten inputpmmetetS led to only a muginal improvementin the discriminarion suits". It is pertinent tonore rhat having a discrimination parameterbased only on L,I,().) h", the advantage of amuch simpler experimental =gemcnt, sinceno spectral resolution is required. It can howeverbe used as a good discrimination parameter onlywhcn the diflerence in intensity values formalignant and normal sites is much more thanrhe variations possible due to the variouscxperimental factOrs. Applicability to in-vivostudics therefore needs to be investigated. TheUF based approach is expected to lead to anearly diagnosis and may allow detection of pre-malignant alterations for which ptesently nodlective non-in=ive method exists.

Breast cancer

Autofluorescence spectroscopy of bre"'t tissuewith N, laser excitation sbowed thar rhecancerous tissue was considerably morefluorescent comp=d to the normal and thebenign tumor tissue""'. A scatret plot for theintegrated fluorescence intensity from pairedcancerous sires and adjoining normal breasttissue sires from 10 patients is shown in Fig. 4.The ratio of the mean fluorescence intensity forcancerous sites to that from normal and bcnigntumor sites were .2.82 and 3.23 respectively inthe study involving 63 patients, 28 with ductalcarcinoma and 35 with fibroadenoma". With300 nm and 488 nm excitation the canccroussites were more fluorescent rhan the normal.However, no statistically significant diffetencewas observed in the fluorescence intensity ofcancerous and benign tumor sites. Therefore,while cancerous tissue could be discriminatcdfrom normal with good sensitivity and

specificity, the discrimination results were poorfor discriminating cancerous from benign tumortissue. With 337 nm excitation, the use offluorescence intensity as a discriminationparameter could howevcr, discriminate canceroustissue from, both normal and benign tumorrissue, with sensitivity and specificity values of>99% ".Excitation-emission spectroscopy" and time-resolved m=utements. carried. out on breasttissue autofluorescence have revealed a significantvariation in the concentration of fluorophores inmalignant, benign tumor and normal tissuetypes. Some of rhe predictions of thespectroscopic analysis have been confirmed bybiochemical estimation". This variation in theconcentration of the fluorophores explained whythe discrimination resultS obtained with 337 nmexcitation we" much better compared to thatobtained with the use of orber excitationwavelengrhs by other researchers".Although b,,"'t cancer is not a superficialdisease, the UF based diagnosis of brmt canCercan be conveniendy done during needlc biopsy.This is of interest because X-ray mammography,the best available means of detecting br=tcancer at present, has two important drawbacks.First it leads to a vety large number of falsepositives, i.e., a vety large ptOportion (60-90%)of mammograpbically abnormal detection turnout to be benign upon invasive br=t biopsy,leading to avoidable trauma and psychologicalStress to patients n. Secondly, frequent exposureto ionizing x-ray tadiation during mammographyhas potential hazards, howsoever remote. Laserscreeuing can be used without the adverse effects",sociated with the use of ionizing radiation.Furtbet, the tesultS of the in-vitro studies suggestthat the UF technique may offer much imptovedspecificity.

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rine cancer

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In-vivo studiesA schematic of the UF based system developedat CAT for in-vivo elinical studies is shown inFig. 5. It consists of a sealed-off N, laser (7 ns,100 I'}, 10 Hz), an optical fiber probe, and agareable iotensified CCO detector. Thediagnostic probe, developed in-house is a fIberbundle, whieh has tWOlegs; one contains a singlequartz tibet (NA 0.22, core diame


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discrimination algorithm developed coulddifferentiare rhe squamous cell carciuoma of rheoral caviry from normal "Iuamo", ti"ue wirh asemiriviry and specificiry, towards cancer, of86% and 63%, respectively, The resultS areshown in Fig. 7. The reason for rhe relativelylower specificiry values appears ro be rhe fact rhatmost of rhe parienes who panicipated in rhisstudy were at an advanced stage of malignancies.Some of rhe visually uninvolved sires assumed tobe nmmal may therefo nOt be truly normal.This follows because the normal appearing

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region surrounding rhe cancero", rurnm mayhave biochemical changes due to rhe field.effectof rhe malignancy. Indeed, when rhediscriminarion analysis was carried our on rhebasis of the spectra averaged over all canceroussites and the spectra averaged over all normalsites from a patient, a sensiriviry and specificirytowards cancer of 100% were obrained. Theremarkably good results obrained on sireaveraged spectra suggests rhar a few of rhenormal sires had signarures very different fromthe orher sites of the group.

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Studies on PhotobioactivationThe u,e of light for non-,urgical rherapeuticappli"",io", exploits photochemical reactionsinitiated by pboto-excitation of naturalchromophores or exogenous drugs localized inthe tissue. Some well-known example.. of thetberapeuuc use of light" are its use inthe teeatment of psoriasis, neonatal jaundice, skintuberculo,is, photodynamic therapy of cancer,etc. Several ",;carchers have also reported matinadiation wirh narrow bandwidth light Oaser)can have profound effect on cellular cultures andanimal models' and can alw lead to rherapeuticeffects in humans'."', like, accelerated woundhcaling, treatment of pain nf different origins,etc.. Although the usage of lasm for non-,urgicallaser therapeutic applicatinns is gtowing it hasnot yet become an established clinical modality"because the mechanisms for many of rhe photo-thetapeutic effem ace not vety well undersrood.The clinical porential of mi, tatber simple andinexpensive therapeutic modality is motivatingconsiderable work in this direction andsubstantial progtess is being made". It isperrinent to note rhat for these phoro-therapeuricapplications any conventional light sourcegenerating the appropriate wavelength and withthe desired paramerers (engy, pulse durationetc.) can be used. However, rhe berrer control onlaser light characreristics ofren makC1iphototherapy more convenient with rhe use oflasers.At CAT ,everal studies have been canied out toinvestigate the effect of laser radiarion on cellularculrures and animal models. Our studies" on rheeffecr of N, laser irradiation on the skin ofanimal models (albino rabbirs and mice) showedthat N, laser inadiation at cerrain doses can leadto a proliferation of cells in rhe active epidermallayer of me rabbit/mice skin. Such proliferationof cells can comribute to fuster healing of mewounds reponed in several clinical studies.

Furmer, me fact mat an inhibition of cellproliferation was observed at higher dosehighlights the importance of a carefrd study ofme parametric dependence in order ro elicit medC1iiredclinical response. Since macrophages playan important role in rhe process of woundhealing, effi:cr of light on macrophagC1i bas alsobeen studied and stimularion of macrophagC1iobserved following He-Ne laser inadiation".The studi", on narrow bandwidm lighr


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otOchemical and not intrinsic photosensitiwduced photodynamic action.. A singl~ cell gelectwphmesis '" up was also developed anded to investig"e UVA induced DNA damagemice pedton,,1 macwphages"'.tudies on photodynamic effect ofotnsensidsm on cellulae cultutes and animalve also been initiated" CAT. Studies wece,ied out on the photodynamic inactindon ofillus ,"brill, cells by He-Ne la"e itradi"ion insence of methylene blue (MB) and toludinene (TB). Results show th" outside: the cellth MB and TB lead to photodynamicnemion of singlet oxygen causing a decase inll memb"ne fludity. Howevet, whereas TBnuins outside ehe cell and does nm lead tonecation of feee "dicals, in MB mediatedotodynamk action, supewxide cadica!nerated int"cellulacly is the majoe contributmthe cell damage". Investig"ions have also been,ied out photodynamic effect of MC540 andlA on cell lines of epithelial neoplasm and onlA induced accumulation of pwtopotphydnp) intumm and skin of fibwsaccoma beatingice. Effect of glucose p-treatment andactionated delivery of AlA on Pp accumulaeiontumoe and tumoe to skin mio of Pp was

vestigated. A pilot study on photodynamicecapy of animal tumms has also been weiedr'". This was a collaboeative wack with a gwupRMC to chatacteei" a photosensitisee dcugented jointly by Radiation Medicine CentreMC), Mnmbai, and liT, Mnmbai.

evelopment of Techniques for Optical

ptical imaging has the pmential w pwvide sub-illimme eesolution imaging without the neede ionizing "di"ion and associated eisks. Thendamental pwblem with optical imaging is" in contrast w Hays optical photons aceCongly scattered in tissue, which leads to a

bluccing of ehe image. Sevecal appwaches can beused to pick out the useful image beating lightfwm the backgwund-scatteeed light" Oneappwach is w exploit the fact that the scatteredlight emerges fwm ehe dssue in all dictions andalso takes longer time to emerge as compad wthe component of light which does not undergoany scattering oe is peedominantly foewatdscattered. Therefo, using sp"ial filtecs and byuse of ultta-shott mpmal gates (of picosecond Isub-pico"cond duration) image beatingcomponents can be filted out. Though ehe'patial solution achievable in these techniquesis vel)' high (few tens of micwns), these can notbe used fm imaging thwugh laege dssuethickness since the amount of image beaeinglighe hils off exponentially with the incase inthe thickness of the scatteeing medium. Oneappwach to addss this pwblem is to ',electivelyamplifY the image beacing light using non-lineaeoptical eechniques. Experiments on opticalimaging through eutbid media have been c",iedour ar CAT using a Streak Cameca with tempoca!wlution of - 10 ps and also by urilizingstimulated Raman scattering (SRS) appwach foerime garing. A sparial solution of -300 ~m hasbeen obsecved in rhe present set-up.Development of Laser Optical Trap-Microbeam Ser UpDevelopment of laser trap and micwbeam set uphas also been taken up ar CAT becau" it isfinding widespread applications in biomedicinefor non-contact micwmanipularion ofmicwscopic objects". Laser micwbeam isessentially a pulsed lasee beam coupled to amicwscope. The lacge intensities (of up to 10"W/cm' with UV lasec micwbeam) generated atrhe focal point of the large numerical apetturemicwscope objecrive can be used to Cut,perforate m fuse micwscopic objects with sub-micwmetee accuracy. As these intensities arise

123 I-


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only ac the focal point. it is possible to workwithin the depth of a transpacent object withoutopening it. Optical tWeeze, or laser optical trapuses the light of a CW infrared laser for transpOt(of microscopic objecrs. Here. the gradient forcesarising due to the lacge gradient of light intensityin rhe focussed laser beam and the resultinggradient in the light pressure and the electricfield ace used to trap microscopic objecrs at thefocal point of the laset beam. Unlike mechanicalmicrotools. the optical trap is gentle andabsolurely stetile and can be used to capture,move and position single cells or subcellularpanicles without direct contact or significantdamage. A laset trap set up has recently beenmade operarional ar CAT and used to trappoiystyrene microsphetes and motile bacteria"'""damo"", and E-Coh).

ConclusionsThere exist considerable current interest in thedevelopment oflaser-based techniques for in-,itu.near teal time diagnosis. Sevetal studies havebeen carried our at CAT on laser inducedfluorescence of tissues resected at surgety orbiopsy from pacients suffering from cancer oforal cavity. breast or uterus. These studies haveprovided very encouraging resulrs ondiscrimination of cancerous tissue sites frombenign tUmor tissue Ot normal tissue sites. N,laser based systems have been developed forclinical studies and ace being used for in-vivostudies on the diagnosis of cancer of oral cavityand urerine cervix. A pilot study on 25 patienrswirh histopathologically confirmed squamouscell carcinoma of oral cavity has been completedand satisfactory discrimination results obtained.Photobioactivation using endogenous andexogenous chromophores also holds considerablepromise for a variety of therapeutic applications.However, realizations of this potential requirescareful studies on the effects of narrow

bandwidth light on celiular cultUres and animalmodels so that the mechanisms tesponsible forthe various photo-therapeutic effecrs are betterunderstood. Several studies in this direction havealso been carried out ac CAT.

AcknowledgmentsThe author will like to thank his colleagues atBiomedical Applications Section and LaserApplications and Electronics Division, CAT whohave contributed to the wack described in thepaper. It is also a pleasure to thank Dr. D. D.Bhawalka


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. Karu, T I., "Phorobiology oflow-power laserrherapy", Harwood Aeademic Publishers,London, 1989.

. Ohshirao, T, and Calderhead, R. G., "Lowlevel laser therapy a prKrical inrroducrion",John Wiley and Sons, Chichester, 1988;Ohshirao, T, "Low reacrive level lasertherapy: pracrical applicarions", John Wileyand Sam, Chichester, 1991.

. Hender,"n, B. W., and Dougherty, T J.,(eds.), "Phorodynamic Therapy BasicPrinciples and Applicarions", Ma


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28. Regan, J. D., and Parcish, J. A., (eds.), "Thescience of photomedicine", Plenum PiaPacific Cancer Conference, 12-15December, 1999, Cancer Institute, Chennai.

Abaut the auth"..

(MSr., Lurk",w Uni"mity, 1973) i"iucd the mtwhile La", Sreti", BARC, in,ating thwugh the BARC haining Seh"l. Hi, ""a"h intem", while at BARC,

included n,,-line., aptiealfrequency ,,""minn, CO, I.,m and CO, /a", pumped malew/a, g"Imm. Dr. Gupta war at H"int Watt Uni",ri'y, UK frum N"emb" 1979 to N"emb" 1981" a Camm"wealth Schola"hip Awa,d and late' nn a pnttdnttocal ftllawrhip dueing 1988-89He ubtained hir Ph.D. "'pre fram H"int Watt Univmity in 1981 ft' hit w"k nn 4fttient mid-infra"d genreati" by optical pumping mnlreul., garer and ",n-line., aptieal mixing.Dr. Gupta m"ed tn Cen"eftcAdvanrrd (CAT), Indu", in Ma"h 1990. At CAT,he initiated w"k nn CO, I.", pumpedfir,-infra"d larm and an biamedieal applieati"r aJlmm. He pmently hradr the

Biumedical Applieati'" Seai", at CAT, D,. Gupta h., raclie"eaintd the 1988 NS Satyamucthy Mem"ia/ awaed ofthe Indian PhyrirrA",tiatinn fir, hit "m,ibutia", un mid-infra"d "hennt "U"".

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application of bio medical engg

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