Literature Review
2.4 Emerging Raman techniques and their applications in life scienceslife sciences
Stokes line intensity is proportional to laser intensity, and inversely proportional to excitation wavelength (Istokes 1/ λ4). Choosing a shorter wavelength increases Stokes line intensity, however, fluorescence is also wavelength dependent and typically in-creases with shorter wavelengths. This requires a trade-off between obtaining higher signal intensities at shorter wavelengths versus lower background at longer wavelengths.
Sample concentration also has influence over line intensity. And choice of wavelength can change depending on factors such as sample thickness, phase, required measure-ment depth and the substrate on which the sampling is made. There are a number of emerging Raman setups that can increase line intensity using resonance effects.
For example, Resonance Raman Spectroscopy (RSS) occurs when the excitation fre-quency matches that of an electronic transition of an analyte. The transition increases the magnitude of the oscillating dipole moment. The resonance, when combined with
the freqeuncy of the laser generating the Raman signal, can result in line intensity increases in the order of 106 [McCreery2005]. It is an alternative to increasing con-centration. The method has been used to detect carotenoid concentrations in human skin stratum corneums non invasively [Ermakov et al. 2005]. Carotenoid levels were shown to correlate well with oxidative stress levels in subjects independent of dietary consumption. Haemoglobin oxygen saturation has also been measured in rats with deep violet excitation [Ward et al. 2007] and in single trapped blood cells [Gessner et al. 2004]. Interference from fluorescence is often a problem and may overwhelm the Raman signal making detection of the RRS signals over the background difficult for many samples. Also, the technique works only on spectral regions where the laser matches the appropriate electronic transition wavelength, limiting applicability (E.g.
resonance of carotenoids closely matches 488nm excitation).
Surface Enhanced Raman Scattering (SERS) enhances Raman signals of analytes when they are absorbed onto or near the surface of certain noble metals. Collective oscilla-tions of the conduction electrons (plasmons) move freely within the metal. The surface plasmons resonate upon excitation and are coupled with the energy of the incident radiation if the frequency of the excitation laser matches that of the plasmons. The effect is typically confined to local fields but the raman signals may be amplified by 103 -107. It is an alternative to increasing laser intensity. The technique allows a wide range of inorganic and organic analytes, including single molecules, to be observed with much lower acquisition times and laser powers. It also greatly improves the detection limit of low concentration solutions. Classical Raman setup is able to detect Adenine in solution at down to 10-3 M concentrations, while with SERS the detection limit is increased to concentrations as low as 10-8 M [K¨ammer et al. 2014]. Many groups have combined this technique to make use of the SERS enhancement in microfluidic devices to detect single cells and create rapid cell sorting assays for bacteria [Li et al. 2012].
SERS has also been used to create cellular pH maps within pH limits between 2 to 8, allowing the detection of cell reactions to external stimuli [Wang et al. 2008, Bishnoi et al.2006]. Functionalizing the nanoparticles to detect specific molecules of interest is worth particular mention. The potential for combining substrate particles with various tumour-targeting antibodies is an exciting area gaining rapid interest. Oral cancer cells were found to align to gold nanorods containing antibodies targeting epidermal growth factor [Huang, Ernberg, and Kauffman 2009] and liver cancer cell were targeted with silver nanoparticles containing antibodies HER2 and CD10 [Kim et al. 2006]. The en-hancement effect decays considerably with increasing distance form the metal surface,
which poses a limitation for this technique. Reproducing surfaces with identical states of aggregation and roughness is also difficult and requires highly controllable deposition methods. The typical metals used also photodecompose giving the produced substrate limited operational time.
Raman signals may also be enhanced via nonlinear excitation as seen in coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) that are beyond the scope of this review. Briefly; they involve a pump and Stokes laser that are incident on the sample. When the frequency difference between the pump and Stokes beams matches the Raman transition of a chemical bond, the molecular oscillations are coherently driven. In CARS, a second pump laser further interacts with the now excited chemical bond to give rise to an anti-Stokes signal several orders of magnitude greater than spontaneous Raman signals. In SRS, the enhancement arises from an en-ergy transfer from the pump to the Stokes beam when the frequency difference matches a Raman transition. The techniques are chemically specific as only selected molecular vibrations are amplified. This brings some particular advantages including faster data acquisition (by focusing all energy to track a single bond) and also in cases where two components have Raman peaks in the same position, it may be possible to differentiate each component by shaping the pump pulse to each component. They have been used for video-rate imaging of tissues and to monitor drug delivery into skin [Saar et al.
2010, Belsey et al.2014, Mansfield et al. 2013, Galli et al.2014, Friedrich et al.2015, Evans et al. 2005].
Table2.1:SummaryofRS-basedfindingsfromskincancerpapers AuthorMainfeaturesidentifiedOthersignificantNumberofsamplesSystem [Silveiraet al.2012]800-1000cm-1 and1250-1300cm-1 regions higherforBCCindicatinghigherprotein content.1620cm-1 peakofhaemoglobin lowerforBCC.
ANOVAalgorithmforclassi- ficationwasabledifferentiate BCCfromnormaltissuewith sensitivityandspecificityof 88%and83%respectively.
830nm laser upto 350mW power [Martinet al.2004]1200-1400cm-1 AmideIIIband:1270 cm-1 peakislowerforBCC,1333cm-1 peakishigherforBCC.800-970cm-1 band attributedtocollagen,prolineandhy- droxyproline:showsgeneralreductionin BCC.
Tissuetypediscriminationus- ingPCAwithsensitivityand specificitiesover83%
21xBCCsections, 18xnormaltissue sections
1064nm laser, 300mW power [Nijssenet al.2002]Significantdifferencesinlipidandnucleic acidcontentforBCCcomparedtodermis. SignificantdifferenceinDNAcontentfor BCCcomparedtoepidermis.
Ramanmappingstudy.Per- formingKCAoverthemap clearlyseparatedBCCfrom dermis.Classificationsen- sitivityandspecificitywas 100%,93%forBCCvsder- mis.BCCvsepidermiswas harder.
15xBCCcryosec- tions850nm laser, 100mW [Lieberet al.2008b]1542-1556cm-1 regionfortryptophan, 807-821cm-1 region(unassigned)and758- 772cm-1 trytophanregionswereconsid- erablydifferentbetweennormalandbcc tissue.
Thisstudywasdoneinvivo!21suspectlesions, each825nm laser, 40mW power
Table2.2:SummaryofRS-basedfindingsfromskincancerpaperscontinued AuthorMainspectralfeaturesidentifiedOthersignificantNumberofsamplesSystem [Gniadecka etal.1997]1680-1640cm-1 AmideIband:1650cm-1 peakintensityislowerforBCC,thismight indicatedisturbanceofα-helixstructure. 1300-1230cm-1 AmideIIIband:1270 cm-1 peakislowerforBCC.Ratioof1290- 1220cm-1 vs.1360-1290cm-1 peakareas arefoundtodiscriminateBCCfromnor- mal.1000-800cm-1 region:significantdif- ferencesbetweenBCCandnormal.Ratio of900-830cm-1 vs.990-900cm-1 peakar- easarefoundtodiscriminateBCCfrom normal.Thesepeaksareattributedto proline,valineandpolysaccarides.
ANNmodellingoverthe3500- 400cm-1 regionconfirmedvi- sualobservations.
16xBCCsections, 16xnormaltissue sections
1064nm laser, 300mW power [Choietal. 2005,Baek etal.2006]
1656cm-1 AmideIbandisshiftedto1589 cm-1 forBCC1302cm-1 AmideIIIband isshiftedto1328cm-1 forBCC.1085 cm-1 phospholipidandnucleicacidpeakis shiftedto1048cm-1 forBCC.1441cm-1 lipidandproteinpeakisshiftedto1450 cm-1 forBCC.
Bandshiftsinsteadofintensi- tieswereusedtodiscriminate tissuetypeswithPCA,MAP, fuzzyandSVM.Discrimina- tionaccuracieswereover96%.
10xbiopsies(Spec- traforbothBCC andnormalskinwere obtainedfromeach biopsy) 514.5 nmlaser 20mW power
Table2.3:SummaryofRS-basedfindingsfromskincancerpaperscontinued AuthorMainfeaturesidentifiedOthersignificantNumberofsamplesSystem [Pereiraet al.2004]AmidesIandIIIweresignificantlydif- ferentbetweenSCCandnormalindicat- ingchangestoproteinstructure.860, 939cm-1 peakshigherinnormalandas- signedtocollagen.Peaksassociatedwith nucleicacidshigherinSCC(1555-1560, 1244-1272cm-1 .
-8xintactSCCand normalskinsamples1064nm laser, 200mW power [Philipsen etal.2013]Lookedatratios:I3250/I2930-waterbad higherinPN,BCCandMM.I1660/I1450 -nosignificantdifference.I1250/I1450- higherinnormalcomparedtoall.Ratio differentenoughforPN,BCCandMM toseparateeach.I1250/I1310-PN,MM >BCC.
Thisstudywasdoneinvivo. Alsoincludedclassification. Sensitivityandspecificityfor: MM93.3and96.4%.For BCC88and85.5%.ForPN 87.8and84.2%.
x55normal,x25 BCC,x41Pig- mentednevi,x15 melanoma
1064nm, 120mW [Fendeland Schrader 1998]
Collagenrelatedbandslower,Histone, DNA,RNAbandshigherinkaposisar- coma.Higherlipidcontentineczemasam- ples.
Melaninfluorescencereported veryhighinmelanoma.Intact9xeczema, 1xpsoriaticskin, 4xkaposisarcoma, 4xnormalskin,? melanomasamples.
1064nm, 300mW power [Luietal. 2012]UsedPCA,LDAandPLStosep- aratei.skincancersfrombenignle- sions,ii.melanomasfromnonmelanomas, iii.melanomasfromnonmelanomapig- mentedlesions.Found1055-1800cm-1 re- gionsoptimalforseparating,iii.and500- 1800cm-1 forseparatingcancerandpre- cancersfromnormal.
Sensitivityandspecificityfor i,ii,iiivariedfrom90-99%to 15-68%.
Invivostudyon 518samplesof melanoma,BCC, SCC,AK,neviand SK
785nm
Table2.4:SummaryofRS-basedfindingsfromskincancerpaperscontinued AuthorMainfeaturesidentifiedOthersignificantNumberofsamplesSystem [Gniadecka etal.2004]AmideIflattenedforMM.AmideIand 1450cm-1 proteinbandsshiftedsignifi- cantlyinMM,BCCandSKbutnotin PN.
Neuralnetworkanalysison allsamples.Sensitivityand specificity:ForMM85and 99%,ForBCC97and98%, ForSK96and100%,For PN78and97%.SKmost commonlymisdiagnosedwith BCC.
x22melanoma,x41 PN,x48BCC,x23 SK,x89normal.
1064nm, 300mW power [Larraona- Puyetal. 2009]
788cm-1 peakofDNAconsiderablyhigher forBCC(theintensityofthispeakwas alsofoundtovarybetweenepidermis anddermislowerindermis-.At- tributedtohighercelldensitiesinBCC tissue.Significantdifferencein1093and 1350cm-1 typeIcollagenpeaksforBCC comparedtodermis.
Ramanmapsofthetissuesec- tionsshowedclearseparation ofepidermis,dermis,BCC andhairfollicles.
20microntissuesec- tions785nm laser power unknown [Bodanese etal.2012]1271-1333cm-1 AmideIII/CH3CH2twist- ingband:1271cm-1 peakislowerfor BCC,1333cm-1 peakishigherforBCC. 800-1000cm-1 regionofC-Cvibrationsat- tributedtolipidsandproteins:showsgen- eralreductioninintensityforBCC.
Tissuetypediscrimination poweroftwomethodsiscom- pared.1-PCAdiscrimination withsensitivityandspecifici- tiesover90%.2-Biochemical modelbaseddiscrimination oncollagencontenthadsen- sitivityandspecificitiesover 83%.
25xBCCsections, 25xnormaltissue sections 830nm laser, 80mW power