Vibrational microscopy and imaging of skin : from single cells to intact tissue Vibrational microscopy and imaging offer several advantages for a variety of dermatological applications , ranging from studies of isolated single cells -LRB- corneocytes -RRB- to characterization of endogenous components in intact tissue . Two applications are described to illustrate the power of these techniques for skin research . First , the feasibility of tracking structural alterations in the components of individual corneocytes is demonstrated . Two solvents , DMSO and chloroform/methanol , commonly used in dermatological research , are shown to induce large reversible alterations -LRB- α-helix to β-sheet -RRB- in the secondary structure of keratin in isolated corneocytes . Second , factor analysis of image planes acquired with confocal Raman microscopy to a depth of 70 μm in intact pigskin , demonstrates the delineation of specific skin regions . Two particular components that are difficult to identify by other means were observed in the epidermis . One small region was formed from a conformationally ordered lipid phase containing cholesterol . In addition , the presence of nucleated cells in the tissue -LRB- most likely keratinocytes -RRB- was revealed by the spectral signatures of the phosphodiester and cytosine moieties of cellular DNA . Introduction Technological developments in infrared and Raman microscopic imaging have permitted several new types of biomedical applications . As demonstrated elsewhere in this volume , the diagnosis of pathological states in a wide range of tissues is now feasible , and is being pursued vigorously in laboratories around the world . The vast volume of spectral data generated from imaging experiments permits sophisticated multivariate statistical analysis of data and enables the experimentalist to discern tissue regions where spectral signatures from pathological states differ from those of healthy tissue . An important attribute of vibrational spectroscopy is the availability of spectra -- structure correlations from many tissue components . This extensive background information can provide a useful supplement for biomedical diagnostics . By way of example , Boskey and her collaborators have characterized a new form of brittle bone disease -LRB- osteoporosis -RRB- based on structural changes in the collagen component of mineralized tissue -LSB- 1 -RSB- . This pathological state differs from the more widely known forms of osteoporosis , which are characterized by altered hydroxyapatite levels/structure . The origin of the spectral changes in the collagen component of pathological tissue biopsies was traced to increased levels of collagen cross links and alterations in their spatial distribution . Evidently , the availability of such information facilitates the development of strategies for tracing the origin -LRB- i.e. , genetic predisposition , external factors , etc. -RRB- of the pathology . Skin is another tissue where the availability of spectra -- structure correlations as well as the ability to detect spectral signals from exogenous constituents opens up new possibilities for vibrational microscopic imaging experiments relevant to pharmacology , physiology , and biochemistry -LSB- 2 -- 4 -RSB- . In this article , we provide two examples depicting the interplay of vibrational microscopic imaging with spectra/structure correlations . The first example uses both IR and Raman microscopy to evaluate structural changes in the keratin of single cells -LRB- corneocytes -RRB- induced by treatment with dimethyl sulfoxide -LRB- DMSO -RRB- or chloroform/methanol -LRB- C/M -RRB- . These solvents have been widely used in dermatology for studies of permeation enhancement and for extracting lipid from the stratum corneum -LRB- SC -RRB- , respectively . The characterization of DMSO-induced structural changes in keratin will aid in the determination of DMSO permeation pathways and in the evaluation of SC barrier recovery . Assessing C/M 's effect on keratin in isolated corneocytes including the reversibility of potential structural changes , will facilitate the delineation of solvent treatment effects from that of lipid removal . In the second example , we demonstrate that confocal Raman microscopy along with multivariate -LRB- factor -RRB- analysis provides the detection of particular skin regions -LRB- stratum corneum , epidermis , dermis -RRB- as well as endogenous sub-areas -LRB- keratinocytes , lipid inclusions -RRB- in the epidermis . The availability of spectra -- structure correlations permits molecular structure information to be deduced from either the spectra or from the factor loadings . Materials and methods Preparation of corneocytes The Rutgers University Internal Review Board has approved all protocols . Corneocytes were collected by tape stripping -LRB- Sellotape , 3M Scotchguard -RRB- human forearm skin . The forearm was flushed with water for several minutes prior to tape stripping . The first two strips were discarded and corneocytes from the third tape strip were isolated as follows . Corneocytes were flushed from the tape with HPLC-grade hexane . The hexane/corneocyte suspension was sonicated for approximately 5 min to break up desmosomes and lipid cohesion between individual corneocytes . Corneocytes were then isolated using nitrocellulose membrane filters with a pore size of 0.22 μm -LRB- GE Osmonics Labstore , Minnetonka , MN -RRB- and resuspended with fresh hexane . This procedure was repeated three times . A small aliquot of this suspension was separately deposited on either CaF2 windows for transmission IR or gold-coated silicon substrates for Raman measurements . Samples were dried overnight under house vacuum prior to spectroscopic measurements . The remaining corneocytes were dried under N2 gas prior to treatment with DMSO or C/M . Corneocytes were placed on substrates that were directly dipped in pure DMSO for about 1 min . Separate aliquots of harvested corneocytes were transferred to glass vials using 5 mL C/M -LRB- 2:1 v/v -RRB- . Non-covalently bound lipids were extracted for 68 h while stirring at room temperature in sealed vials . Corneocytes , collected by centrifugation , were further washed 3 times using fresh C/M before the final suspension was placed on the appropriate substrate . Samples were dried overnight under vacuum prior to spectroscopic measurements . No vibrational bands from residual solvent were observed in corneocyte spectra . Subsequently , corneocytes from both DMSO and C/M treatment were rehydrated on the solid substrates by exposure to 100 % relative humidity overnight before a final set of spectra were acquired . Intact porcine skin preparation Skin biopsies from Yucatan white , hairless pigs were purchased from Sinclair Research Center , Inc -LRB- Columbia , MO -RRB- . The stratum corneum of the biopsies was washed with water and samples were cut to a size of approximately 1 × 1 cm2 , 0.5-cm-thick sections . Sections were placed , SC side up , into a milled brass cell , covered , and sealed with a microscope coverslip for confocal Raman measurements . IR microspectroscopy of corneocytes IR spectra were acquired with the PerkinElmer Spotlight system described elsewhere in detail -LSB- 4 -RSB- . The instrument has imaging capabilities , but in this study the point mode was used . Since the typical diameter of corneocytes is about 40 -- 60 μm , an aperture size of 40 × 40 μm2 was chosen to optimize the spectral quality . A total of 256 scans was acquired for each spectrum using 4 cm − 1 spectral resolution . One level of zero-filling yielded data encoded every 2 cm − 1 . Approximately 50 corneocytes were examined from each sample preparation . Raman microspectroscopy Raman spectra were acquired with a Kaiser Optical Systems Raman Microprobe . The instrument has been described in detail elsewhere -LSB- 5 , 6 -RSB- . A 785-nm diode laser generates approximately 7 -- 12 mW of single mode laser power at the sample with a spot size of about 2 μm using a × 100 objective . The backscattered radiation illuminates a near-IR CCD -LRB- ANDOR Technology , Model DU 401-BR-DD -RRB- . Spectral coverage is from 100 to 3,450 cm − 1 at spectral resolution of 4 cm − 1 . Following linearization , data are encoded every 0.3 cm − 1 . Spectra were acquired from 10 -- 15 corneocytes from each sample preparation using an 80-s exposure time , 4 accumulations , and cosmic ray correction . Optical images of corneocytes were acquired with a Leica microscope -LRB- Model DMLP -RRB- coupled to the Kaiser system with a × 100 objective . Confocal Raman spectra of intact pigskin were acquired using a 60-s exposure time , 2 accumulations , and cosmic ray correction . A × 100 oil immersion objective was used for the intact pigskin samples . To evaluate axial characteristics of the optical set-up used in these experiments , the following observations were made with the oil immersion lens using protocols previously published -LSB- 5 -RSB- . The test sample consisted of five layers of a polymer laminate which incorporated two chemically identical layers -LRB- out of the five -RRB- , each with a thickness of 7.5 μm separated by an intermediate layer of thickness 15 μm . This provides a center-to-center distance of 22.5 μm between the identical layers . Raman intensity profiles show a baseline-resolved width for each of the chemically identical layers of approximately 12 -- 13 μm , a half-width of the intensity profile for each peak of about 8 μm , and a peak-to-peak distance of about 25 μm , in good agreement with the known test sample physical characteristics . The actual axial spatial resolution in the skin samples can not be directly ascertained . Data analysis Grams/32 AI software version 6.0 -LRB- Thermo Galactic , Salem , NH -RRB- was used for processing individual Raman and IR spectra . Raman spectra were Fourier smoothed -LRB- 80 -- 90 % -RRB- and 2-point baselines were applied to spectral regions of interest . This degree of Fourier smoothing could be applied without inducing spectral lineshape distortions . Confocal Raman spectra were analyzed -LRB- e.g. , factor analysis -RRB- and Raman image planes were generated using ISys software version 3.1 -LRB- Spectral Dimensions , Inc. . Olney , MD -RRB- . Factor analysis was performed using the ISys score segregation routine . The analysis seeks to detect simple patterns in the relationships between observed variables in order to reduce the dimensionality of the data . Score segregation begins by normalizing PCA scores which may then be sharpened by raising them to a power specified by the acceleration parameter . The default value of 10 was used . Factor loadings are then calculated according to -LRB- S ′ S -RRB- − 1 * S ′ * X , unless S ′ S is not invertible , in which case iteration stops , and a factor is deleted before continuing . Results and discussion Solvent-induced conformational changes in the keratin of single cells Although its mechanism of action is not well understood , DMSO has often been utilized as an efficient permeation enhancer for a wide variety of drugs , including antiviral agents , steroids , and antibiotics -LSB- 7 -RSB- . It has been suggested to alter protein conformation in the stratum corneum -LRB- SC -RRB- , the outermost layer of skin and the main barrier to permeability . Anigbogu et al. -LSB- 8 -RSB- monitored the protein -LRB- keratin -RRB- α-helix to β-sheet transition in hydrated SC . In addition , Caspers et al. -LSB- 3 -RSB- monitored protein structure following DMSO penetration into human SC in vivo by confocal Raman spectroscopy . A broadening of the Amide I contour was noted , although major new spectral features were not resolved . It is noted that DMSO concentrations differed in the two experiments . IR -LRB- 1,200 -- 3,600 cm − 1 -RRB- and Raman -LRB- 800 -- 1,720 cm − 1 -RRB- spectra , shown in Fig. 1a and b , respectively , demonstrate the quality of data obtained from untreated human corneocytes . Several strong bands characteristic of the protein and lipid constituents are observed in each spectrum . The Amide I and Amide II bands observed at approximately 1,650 and 1,550 cm − 1 in the IR spectra , respectively , are of particular interest . The Amide I band is also one of the strongest bands in the Raman spectrum . In both types of spectra , the measured Amide I frequency reflects the predominantly helical secondary structure found in epidermal keratin . Absorbance in the Amide I region due to ceramides and other components , some containing amide groups and others C = C , is expected to be low compared to keratin absorbance in corneocytes isolated from the topmost layers of the stratum corneum -LSB- 6 -RSB- . Raman spectral quality is very good considering the thickness of an individual corneocyte -LRB- 0.5 -- 0.8 μm -RRB- and is comparable to spectra obtained from much thicker stratum corneum samples and intact skin -LSB- 2 , 5 , 8 -RSB- . It is likely that the observed signal has been enhanced due to a surface-enhanced Raman scattering -LRB- SERS -RRB- effect between the corneocytes and gold-coated substrate . Raman band assignments pertinent to stratum corneum samples have been listed previously -LSB- 5 , 9 , 10 -RSB- . Fig. 1Vibrational microscopy of human corneocytes . a IR point mode spectrum -LRB- 1,150 -- 3,600 cm − 1 region -RRB- acquired using a 40 μm2 aperture of a single corneocyte isolated from the third sequential tape strip applied to human forearm skin . The inset shows the optical image of the corneocyte -LRB- bar = 10 μm -RRB- . b Raman spectrum -LRB- 800 -- 1,720 cm − 1 region -RRB- of a similarly isolated corneocyte displaying bands characteristic of the lipid and protein -LRB- keratin -RRB- components IR and Raman spectra of isolated corneocytes prior to and following immersion in DMSO , and after an overnight rehydration period -LRB- top to bottom trace -RRB- are displayed in Fig. 2a and b , respectively . A similar series of IR and Raman spectra acquired from corneocytes treated with C/M are shown in Fig. 2c and d. Both solvents induce several alterations in the spectra . The most significant change in the IR spectra is the appearance of an intense low frequency Amide I component observed for the DMSO-treated sample at 1,626 cm − 1 in conjunction with a weak high frequency shoulder at approximately 1,695 cm − 1 . This pattern is diagnostic for formation of antiparallel β-sheet structure -LSB- 11 -RSB- . A redistribution of the maximum intensity in the IR Amide II band from 1,550 to 1,517 cm − 1 for the same two spectra is also consistent with β-sheet formation -LSB- 11 -RSB- . Fig. 2Solvent-induced conformational changes in the keratin of single corneocytes . a and b IR and Raman spectra , respectively , of an isolated corneocyte prior to and following immersion in DMSO , and after an overnight rehydration period -LRB- top to bottom , respectively -RRB- . c and d IR and Raman spectra , respectively , of a corneocyte prior to and following C/M treatment , and after an overnight rehydration period -LRB- top to bottom , respectively -RRB- . In the IR spectra -LRB- a and c -RRB- , α-helical and β-sheet components of the Amide I -LRB- ca. 1,650 cm − 1 -RRB- and II -LRB- ca. 1,500 -- 1,550 cm − 1 -RRB- bands are noted . In the Raman spectra -LRB- b and d -RRB- , α-helical and β-sheet components of the Amide I are marked along with disordered constituents at 1,685 cm − 1 . One component of the Amide III band is marked at 1,240 cm − 1 Helix : sheet transitions are well documented for different types of keratin , and have been detected by IR spectroscopy . In particular , Oertel -LSB- 12 -RSB- used IR to observe similar DMSO-induced changes in human SC . The current work extends observation of this conformational transition to isolated human corneocytes . It is also noted that , based on the IR Amide I and II band contours , a fair amount of helical structure remains subsequent to DMSO treatment . The Raman spectrum -LRB- Fig. 2b , middle trace -RRB- corroborates the β-sheet assignment with the appearance of a high frequency Amide I component at about 1,669 cm − 1 and a strong Amide III component at approximately 1,240 cm − 1 . Both features are within frequency ranges assigned to antiparallel β-sheets -LSB- 13 , 14 -RSB- . The observed changes in the Amide I region of the Raman spectrum are consistent with a previous report detailing the effects of a series of aqueous DMSO solutions on human SC -LSB- 8 -RSB- . Comparisons between individual cells and the SC in the Amide III region are difficult due to overlapped bands from lipid components in the SC sample . In the current work , a second slightly weaker band is also observed for the DMSO-treated cells at approximately 1,270 cm − 1 . Bands in this region are usually assigned to α-helical structure -LSB- 13 , 14 -RSB- . Generally , caution is required in associating characteristic frequency ranges with conformation for the Amide III mode given that the NH in-plane bend internal coordinate contributes to a number of modes in the 1,200 -- 1,400 cm − 1 region -LSB- 15 -RSB- and secondary-structure-dependent coupling occurs between the Amide III and the Cα -- H bending vibration -LSB- 16 , 17 -RSB- . This complexity , in addition to the inherent heterogeneity of corneocytes and variable lipid content of the samples examined herein , precludes an in-depth analysis of the Amide III contour in the current context . The majority of the DMSO-induced secondary structure change is reversed upon rehydration of the corneocytes as demonstrated by comparison of the top and bottom spectra shown in Fig. 2a and b . Some residual broadening remains on the low frequency side of the Amide I band in the IR spectrum . The average full width at half height for the Amide I band in the IR spectra increased from 44 cm − 1 for untreated corneocytes to 52 cm − 1 following DMSO treatment and rehydration , indicating that some residual sheet structure may be present . An accurate measure of the Amide I bandwidth in the Raman spectra is precluded by the presence of a weak side chain vibration at approximately 1,608 cm − 1 . The general reversibility of the conformational change upon hydration was also observed by Oertel -LSB- 12 -RSB- for isolated SC . C/M solutions are commonly used to extract lipids from isolated SC and intact skin samples , but solvent effects on the structure of the remaining constituents have not been detailed . The utility of vibrational spectroscopy for evaluation of keratin secondary structure after lipid removal is somewhat hampered by the native ceramide contribution to the Amide I and II band prior to solvent exposure . In SC samples it is therefore difficult to accurately access the contribution ceramides make to Amide I and II band intensities and position . In the current work , this concern is mostly overcome by acquiring spectra of isolated corneocytes in which the ceramide contribution to the Amide modes is substantially reduced . Several changes are apparent in the IR and Raman spectra of corneocytes upon exposure to C/M -LRB- Fig. 2c and d , compare top to middle traces -RRB- . Some of the solvent-induced alterations are similar to those just discussed for the DMSO-treated cells , e.g. , the low -LRB- high -RRB- frequency Amide I component in the IR -LRB- Raman -RRB- spectrum at 1,626 cm − 1 -LRB- 1,670 cm − 1 -RRB- and the appearance of an Amide III band at 1,240 cm − 1 in the Raman spectrum . These features provide strong evidence that C/M also induces antiparallel β-sheet formation . Differences are also evident when spectra from the two treatments are compared . Broadening on the high frequency side of the Amide I band in the Raman spectra is much more intense after C/M treatment vs. DMSO -LRB- compare the middle trace in Fig. 2d to b -RRB- . Amide I Raman intensity in this region -LRB- ca. 1,685 cm − 1 -RRB- has been assigned to non-hydrogen-bonded disordered structures -LSB- 13 -RSB- . The broadening in the Raman Amide I band is not completely reversible upon rehydration of the sample that had been treated with C/M , even though the intensity of the β-sheet component at about 1,670 cm − 1 has diminished -LRB- Fig. 2d , bottom trace -RRB- . In addition , upon rehydration , intensity remains in the Amide III band at approximately 1,240 cm − 1 , although this band also broadens . Finally , measurements of the Amide I bandwidth in IR spectra upon rehydration of several C/M-treated corneocytes are essentially the same -LRB- ± 2 cm − 1 -RRB- as widths measured for untreated cells . The close proximity of Amide I bands arising from helical and random coil structures makes it difficult to differentiate between these two forms . Considering both the Raman and IR spectral data after rehydration , it seems safe to conclude that the majority of keratin exists in a helical form with some disordered structure remaining . The extent of the C/M-induced protein structural changes has been observed to vary among corneocytes . IR spectra of some cells with significant lipid bands -LRB- i.e. , those characterized by relatively intense methylene stretching modes near 2,850 and 2,918 cm − 1 -RRB- still exhibit notable protein conformation changes -LRB- data not shown -RRB- , while some cells from which the lipid has been essentially completely extracted do not show significant changes in protein secondary structure . It thus appears that the extent of lipid extraction is not directly related to C/M-induced protein conformational changes . Similar results have been noted in IR studies of DMSO-induced protein conformation changes in SC samples -LSB- 12 -RSB- . Additional experiments under conditions of controlled hydration are needed to further explore possible origins of these conformational changes . One explanation for the α-helix to β-sheet structure change in keratin is that DMSO displaces bound water necessary for maintaining secondary structure -LSB- 12 -RSB- . DMSO is known to form an association complex with water stronger than that formed between water molecules alone -LSB- 8 -RSB- . It may be that C/M destabilizes keratin 's native secondary structure in a manner similar to DMSO , since the majority of the native structure is restored after both solvent treatments when the corneocytes are exposed to high relative humidity . The importance of the current experiments lies in the fact , that in studies of supposedly native protein structures in the SC , C/M is often used to remove lipid components . As shown in the current work , this procedure is not innocuous , but can induce irreversible changes in protein structure in corneocytes . Confocal Raman microscopy : delineation of skin regions from factor analysis As demonstrated above , Raman microscopy complements the information available from IR . Although Raman scattering is inherently weaker than IR absorbance , the technique offers two major advantages for tissue studies . First , the spatial resolution of the method is inherently better than IR . For an excitation wavelength of 785 nm , Rayleigh 's rule for spatial resolution -LRB- ΔZ = 1.22 λ/2 -LRB- NA -RRB- -RRB- predicts a spatial resolution of approximately 0.5 μm for a wavelength -LRB- λ -RRB- of 785 nm and a numerical aperture -LRB- NA -RRB- of the × 100 objective of 0.95 . In practice , as has been discussed in Materials and methods and elsewhere , the apparent depth and related measured optical properties are degraded by refractive index variations in the tissue -LSB- 5 , 18 , 19 -RSB- . Second , and equally important , spectra may be acquired in a confocal manner . Thus , physical sectioning of samples is not required to obtain molecular structure information from within the tissue . This evidently opens up the possibility for in vivo examination of skin , especially at the low laser powers -LRB- ca. 10 mW -RRB- used in these experiments . Puppels ' group has demonstrated the utility of confocal Raman determination of water levels in vivo in skin -LSB- 20 -RSB- . The benefit of using both a multivariate approach and existing spectra -- structure correlations to characterize the microanatomy of skin is demonstrated in the next few figures . An optical micrograph of a 5-μm-thick pigskin section , shown in Fig. 3a , allows delineation of different skin regions . The outermost layered stratum corneum -LRB- SC -RRB- region appears to be ca. 15-μm thick with the underlying epidermis -LRB- ca. 50-μm thick -RRB- and dermis discernible . The physical sampling used in the confocal Raman measurement precludes the acquisition of a meaningful micrograph from the same sample ; however to allow for comparison , confocal Raman spectra were acquired from a similar pigskin biopsy using approximately the same dimensions . Spectra were acquired from an untreated piece of pigskin , in steps of 2.5 μm in the z direction and 5 μm in the x -LRB- or y -RRB- direction . The area sampled was 75 × 40 μm so that 240 spectra -LRB- 30 × 8 -RRB- were acquired during the experiment . These were analyzed with factor analysis as described in Materials and methods and as applied to liposome-treated pigskin in a previous report -LSB- 21 -RSB- . In the current work , five significant factors were determined as shown in Fig. 3b , while score images for each factor are depicted in Fig. 3c . Three of the score images and corresponding factor loadings in Fig. 3 may be directly correlated with the known regions of skin as follows : Factor 1 corresponds to the SC , Factor 4 to the viable epidermis , and Factor 5 to the dermis . Thus , delineation of the skin regions based on the factor score images is wholly consistent with the optical image . The two remaining factor score images -LRB- Factors 2 and 3 -RRB- highlight particular features within the viable epidermis . Examination of the factor loadings or averaged spectra corresponding to those pixels where the respective scores are high provides an indication of the molecular origin of the components . In general , factor plots tend to offer somewhat higher S/N ratios , since they represent a substantial condensation of data . However , it is important to remember that factors include all sources of variance in the data . For example , baseline distortions may appear as `` features '' in the factor loadings , which however are not related to vibrational modes in the sample . Thus , it is useful to routinely compare factors with appropriately selected spectra . In Fig. 4 an average spectrum of the stratum corneum is overlaid with the average localized to the small spatial region highlighted in Fig. 3c , labeled Factor 2 . The average spectrum labeled `` ordered lipid '' has strong bands near 1,058 , 1,130 , 1,296 , 2,880 , and 2,850 cm − 1 -LRB- Fig. 4b and c -RRB- . These bands are readily assigned -LSB- 9 , 22 -RSB- to lipid C -- C stretching vibrations -LRB- 1,058 , 1,130 cm − 1 -RRB- of a conformationally ordered -LRB- all-trans -RRB- chain , lipid CH2 twist -LRB- 1,296 cm − 1 -RRB- and lipid CH2 stretch -LRB- 2,850 and 2,880 cm − 1 -RRB- . In addition , the bands observed at 605 and 700 cm − 1 -LRB- Fig. 4a -RRB- are indicative of cholesterol -LSB- 23 -RSB- . Thus , the small pocket in Fig. 3c -LRB- Factor 2 -RRB- likely contains an ordered lipid phase . The fact that the same bands are present in the average stratum corneum spectrum -LRB- Fig. 4 , top -RRB- , albeit at lower intensity , strengthens the assignment . Highly ordered lipid phases -LRB- ceramides , fatty acids , and cholesterol -RRB- comprise the inter-corneocyte matrix of the stratum corneum . In addition , infrared spectroscopic evidence exists -LRB- D.J. Moore , unpublished results -RRB- for ordered lipid regions with relatively low protein content in appendages -LRB- i.e. , pores and hair follicles -RRB- well below the stratum corneum . It is possible that the confocal Raman image planes shown in Fig. 3c cut through such an appendage . Fig. 3a An optical micrograph of a microtomed 5-μm-thick pigskin section acquired using the PerkinElmer Spotlight -LRB- bar = 10 μm -RRB- . The results of factor analysis in the 800 -- 1,150 cm − 1 region conducted on a confocal Raman map acquired from intact pigskin . b The five distinct factor loadings generated by the ISys score segregation algorithm -LRB- see Materials and methods -RRB- are offset and labeled Factor 1 -- 5 . c The spatial distribution of factor scores for each of the loadings as marked . Dark blue indicates the lowest score with green , yellow , orange , and red indicative of progressively higher scores . Factor loadings and score images have been assigned to different micro regions in skin as described in the textFig . 4Averaged Raman spectra from within the stratum corneum -LRB- top -RRB- and lipid -LRB- bottom -RRB- regions as noted in Fig. 3b and c as Factor 1 and 2 , respectively . a The 590 -- 790 cm − 1 region with bands assigned to cholesterol marked at 605 and 700 cm − 1 . b The 800 -- 1,380 cm − 1 region displays several bands -LRB- 1,058 , 1,130 , and 1,296 cm − 1 -RRB- characteristic for ordered -LRB- all-trans -RRB- lipid acyl chains . c The CH stretching region -LRB- 2,800 -- 3,030 cm − 1 -RRB- with methylene symmetric -LRB- 2,850 cm − 1 -RRB- and asymmetric -LRB- 2,880 cm − 1 -RRB- stretching modes noted . The frequencies and relative intensity of the bands are consistent with well-ordered lipid acyl chains Finally , Factor 3 -LRB- Fig. 3b -RRB- , whose score image reveals several small-localized pockets in the epidermis , contains one broad spectral feature centered at approximately 1,090 cm − 1 that is not present in the other factor loadings . Based on both the position of this particular band and the spatial distribution of the factor score , the band is tentatively assigned to the phosphodiester mode of DNA -LSB- 24 , 25 -RSB- . Since the identification of DNA is of interest , as it would reveal the location of nuclei within keratinocytes or other cells in the tissue , an additional strategy was employed to acquire better spectra from the epidermis . The experiment , whose results are depicted in Fig. 5 , was carried out as follows . The top ≈ 20 μm of a pigskin section , including most of the SC , were removed by tape stripping prior to confocal Raman imaging . This allows for the acquisition of higher quality spectra in deeper regions of the viable epidermis . The Raman experiment was also carried out with more data points/unit area -LRB- 2-μm step size -RRB- providing a closer examination of the tissue compared to the sample presented in Fig. 3 . The presence of cell nuclei was revealed by factor analysis conducted over two wavenumber regions , each separate analysis restricted to compute only two factor loadings . An additional DNA band assigned to cytosine is detected in the factor loadings shown in Fig. 5b , which also strengthens the previously identified phosphodiester DNA band assignment at about 1,090 cm − 1 -LSB- 24 , 25 -RSB- . As expected , in the tape stripped sample , the phosphodiester band is again observed -LRB- Fig. 5c -RRB- . The score image shown in Fig. 5a is derived from the factor with the strong cytosine band and is essentially the same as the score image generated from the phosphodiester factor -LRB- not shown -RRB- . Thus , the image shown in Fig. 5a , depicting keratinocyte or other epidermal cell -LRB- e.g. , melanocyte -RRB- nuclei , demonstrate the ability of confocal Raman to detect DNA in intact skin tissue . Fig. 5The results of factor analysis conducted in two separate spectral regions for a confocal Raman map of pigskin after stratum corneum removal by tape stripping . Both analyses were performed allowing the generation of only two factors . In each case , one factor contained a spectral feature specific to DNA and the other mapped in a fairly uniform way throughout the epidermal region . a Factor score image for the factor-labeled nuclei shown in -LRB- b -RRB- with red corresponding to the highest score and dark blue to the lowest . b Factor loadings for the 600 -- 820 cm − 1 region with a distinctive vibrational band at approximately 785 cm − 1 assigned to cytosine . c Factor loadings for the 800 -- 1,150 − 1 region with a second characteristic DNA vibrational band due to the phosphodiester backbone stretching mode , at about 1,090 cm − 1 noted Conclusions The current experiments highlight some unique advantages of vibrational microscopic imaging . An understanding of the spectroscopy of the various tissue components permits insight into the molecular origins of the images generated from factor analysis . Thus , identification of the lipid pocket in Fig. 3c -LRB- Factor 2 -RRB- and of the cell nuclei -LRB- as detected from the DNA spectral signatures -RRB- in Fig. 3c -LRB- Factor 3 -RRB- and Fig. 5a were greatly facilitated by the availability of Raman spectra of lipids and of DNA , respectively . In addition , the availability of correlations between lipid spectra and chain conformation , permitted assignment of the lipid inclusions as having arisen from ordered lipid phases . The biological relevance of this observation remains to be determined . From a pharmacological perspective , the detection of cell nuclei -LRB- most likely keratinocytes -RRB- within intact skin permits co-localization experiments to begin to be designed . For example , for classes of drugs targeted to these cells , confocal Raman measurements will permit determination of whether the putative therapeutic agent reaches its intended target . In addition , although all microscopic methods can obviously readily detect single cells , the ability of IR - or Raman-based methods to detect conformational changes within a particular component of a single isolated cell is unique . The example we have chosen in Fig. 2 is of practical importance in skin research , since solvents such as DMSO or chloroform/methanol are used for permeation enhancement and lipid extraction , respectively . In each case these solvents were found to be far from innocuous . Each induced large conformational changes in the cellular keratin . This fact had been known previously from spectra of intact SC , but two aspects of the current measurements are novel . The observation of IR data from single cells provides a sharper spectral assignment of the bands . We note for example that ceramides , a major component of the SC , have strong bands arising from the amide bond located in these lipids . These modes contribute significantly to the spectra of the intact SC and overlap protein Amide I and II vibrations ; however , they are much reduced in relative intensity in spectra of cells . Thus , studies of the reversibility of the solvent-induced conformational changes benefit from the reduced intensity of ceramide interference . Finally , it is feasible to begin to examine biochemical heterogeneity at a single cellular level . Issues such as the relationship of protein structure -LRB- either native or solvent-modified -RRB- to hydration levels or to the levels of natural moisturizing factors , in single cells may be profitably probed . In addition , changes in the spectra of cells from pathological states may be examined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

confocal raman
corneocytes
epidermis
protein secondary structure


Characterization

of

cellular

chemical

dynamics

using

combined

microfluidic

and

Raman

techniques

The

integration

of

a

range

of

technologies

including

microfluidics

surface-enhanced

Raman

scattering

and

confocal

microspectroscopy

has

been

successfully

used

to

characterize

in

situ

single

living

CHO

-LRB-

Chinese

hamster

ovary

-RRB-

cells

with

a

high

degree

of

spatial

-LRB-

in

three

dimensions

-RRB-

and

temporal

-LRB-

s

per

spectrum

-RRB-

resolution

Following

the

introduction

of

a

continuous

flow

of

ionomycin

the

real

time

spectral

response

from

the

cell

was

monitored

during

the

agonist-evoked

flux

process

The

methodology

described

has

the

potential

to

be

used

for

the

study

of

the

cellular

dynamics

of

a

range

of

signalling

processes

Introduction

The

advance

of

miniaturized

microfluidic

systems

for

chemical

and/or

biochemical

applications

based

on

so-called

Lab-on-a-Chip

technology

has

demonstrated

that

such

micro

systems

represent

the

ability

to

shrink

conventional

bench

systems

to

the

size

of

a

few

square

centimetres

with

major

advantages

of

speed

performance

integration

portability

reduced

sample/solvent

quantity

automation

hazard

control

and

lower

cost

-LSB-

-RSB-

These

merits

are

important

for

a

variety

of

applications

in

analytical

chemistry

biochemistry

clinical

diagnosis

medical

chemistry

and

industrial

chemistry

-LSB-

-RSB-

Consequently

numerous

micro

total

analysis

systems

-LRB-

μ-TAS

-RRB-

and

micro

reactor

systems

have

been

developed

and

many

more

are

currently

under

investigation

-LSB-

-RSB-

For

the

study

of

cellular

and

subcellular

systems

a

wide

range

of

analytical

methods

have

been

used

with

fluorescence

techniques

being

the

most

common

In

addition

fluorescence-based

imaging

is

a

highly

attractive

methodology

for

the

study

of

organelle

dynamics

identifying

subcellular

compartments

and

monitoring

biological

kinetics

-LSB-

-RSB-

Over

the

last

two

decades

Raman

spectroscopy

has

become

an

increasingly

important

technology

with

ability

to

study

the

biophysics

and

biochemical

processes

involving

cells

-LSB-

-RSB-

Since

Raman

spectroscopy

is

based

on

vibrational

transitions

where

frequency

shifts

are

associated

with

specific

molecular

vibrations

within

the

sample

of

interest

it

enables

the

identification

of

polarizable

bio/chemical

species

the

elucidation

of

molecular

structure

and

the

investigation

of

interface

reactions

all

in

a

non-destructive

manner

In

addition

unlike

fluorescence-based

techniques

Raman

spectroscopy

does

not

require

labelling

dyes

and

since

water

is

almost

Raman

transparent

the

technique

is

ideally

suited

for

analysing

cell-based

biological

systems

Coupling

a

Raman

spectrometer

with

a

confocal

microscope

enables

the

acquisition

of

full

spectral

information

with

a

high

spatial

-LRB-

μm

-RRB-

resolution

in

three

dimensions

Raman

spectroscopy

however

suffers

from

an

inherent

poor

level

of

sensitivity

compared

for

example

to

fluorescence

which

can

be

orders

of

magnitude

more

sensitive

As

a

consequence

despite

advances

in

detector

technologies

the

technique

can

be

less

than

ideal

for

the

direct

detection

of

intracellular

components

present

at

low

concentrations

which

may

take

from

a

few

tens

of

seconds

to

a

few

minutes

for

spectral

acquisition

-LSB-

-RSB-

With

the

unexpected

discovery

of

surface-enhanced

Raman

scattering

-LRB-

SERS

-RRB-

by

Fleischman

et

al.

-LSB-

-RSB-

Raman

intensity

can

now

be

dramatically

increased

-LRB-

a

factor

of

up

to

-RRB-

with

the

inclusion

of

metallic

nano

structures

either

on

a

substrate

surface

or

in

a

colloidal

solution

-LSB-

-RSB-

For

cellular

and

subcellular

analysis

with

SERS

colloid

nanoparticles

-LRB-

e.g.

silver

or

gold

-RRB-

are

normally

loaded

into

cells

by

different

means

such

as

general

incubation

-LRB-

fluid-phase

uptake

-RRB-

or

ultrasonication-assisted

uptake

-LSB-

-RSB-

Due

to

its

chemical

inactivity

gold

nanoparticles

are

generally

regarded

to

be

more

suitable

for

incorporation

within

living

cells

-LSB-

-RSB-

To

analyse

living

cells

using

Raman

spectroscopy

the

cells

are

usually

fixed

at

a

specific

location

The

most

commonly

used

method

for

cell

fixation

is

to

seed

or

grow

cells

on

a

substrate

e.g.

a

microscope

cover

slide

Other

approaches

such

as

optical

tweezers

have

also

been

reported

-LSB-

-RSB-

However

the

conventional

batch

operation

in

a

static

system

limits

the

in

situ

Raman

analysis

especially

when

a

series

of

reagent

treatments

are

required

With

those

limitations

in

mind

the

development

of

microfluidic

and

associated

Lab-on-a-Chip

technologies

provides

unique

opportunities

for

delivering

and

immobilizing

cells

on

a

microchannel

surface

prior

to

introducing

different

reagents

with

a

continuous

flow

in

a

given

sequence

-LSB-

-RSB-

whilst

under

spectroscopic

investigation

In

this

study

we

report

the

successful

integration

of

a

range

of

techniques

including

microfluidics

surface-enhanced

Raman

scattering

-LRB-

SERS

-RRB-

and

confocal

microspectroscopy

which

enables

in

situ

characterization

of

single

living

CHO

cells

with

high

spatial

and

temporal

resolution

By

using

the

microfluidic

methodology

cells

and

reagents

were

introduced

into

the

chip

in

a

continuous

flow

as

a

series

of

plugs

in

a

given

sequence

where

agonist

-LRB-

ionomycin

-RRB-

evoked

intracellular

fluxes

and

the

cell

's

real

time

spectral

response

was

recorded

Materials

and

methods

Confocal

Raman

microspectroscopy

All

Raman

spectra

were

acquired

with

a

LabRam

inverted

microscope

spectrometer

manufactured

by

Jobin

Yvon

Ltd.

Figure

shows

the

schematic

of

the

experimental

setup

The

spectrometer

was

equipped

with

dual

laser

sources

at

wavelengths

of

nm

-LRB-

diode

laser

mW

-RRB-

and

nm

-LRB-

He

Ne

laser

mW

-RRB-

confocal

optics

a

holographic

transmission

grating

and

a

charge

coupled

device

-LRB-

CCD

-RRB-

detector

with

pixels

The

instrument

included

a

precision

motorized

X

Y

sample

stage

for

automated

mapping

at

spatial

resolution

down

to

less

than

μm

and

extensive

software

support

-LRB-

LabSpec

-RRB-

for

data

processing

In

this

study

an

objective

lens

of

magnification

working

distance

and

numerical

aperture

-LRB-

-RRB-

of

was

used

-LRB-

L

Plan

SLWD

Nikon

Japan

-RRB-

This

objective

lens

was

mounted

on

a

piezo

actuator

-LRB-

Physik

Instrumente

Germany

-RRB-

for

automatic

focussing

of

the

microscope

objective

at

different

depths

in

the

Z

direction

enabling

mapping

A

grating

with

grooves

mm

a

confocal

aperture

of

μm

and

an

entrance

slit

of

μm

were

selected

for

the

experiments

The

Raman

spectrometer

wavelength

range

was

calibrated

using

the

centre

frequency

of

the

silicon

band

from

a

silicon

sample

-LRB-

cm

-RRB-

Using

these

conditions

a

typical

acquisition

time

of

s

was

used

to

collect

SERS

spectra

from

cells

within

the

microchannel

Fig.

of

experimental

setup

Microfluidic

device

fabrication

The

microfluidic

device

was

constructed

using

a

manifold

clamping

method

according

to

published

procedures

with

some

adaptations

-LSB-

-RSB-

The

assembly

of

the

device

is

illustrated

in

Fig.

Briefly

the

microchip

consisted

of

a

PARAFILM

sheet

-LRB-

thickness

μm

American

National

Can

Company

US

-RRB-

with

a

channel

network

and

two

glass

plates

which

sandwiched

the

polymer

film

The

Y-shaped

channel

network

-LRB-

Fig.

-RRB-

cut

through

the

film

was

wide

The

top

glass

plate

-LRB-

mm

-RRB-

had

three

holes

-LRB-

diameter

mm

-RRB-

drilled

through

at

appropriated

positions

in

order

to

link

the

ends

of

the

channels

with

inlet/outlet

tubing

The

bottom

glass

plate

was

a

thin

quartz

coverslip

-LRB-

mm

Agar

Scientific

Ltd

UK

-RRB-

which

had

a

thickness

of

μm

in

order

to

minimise

the

glass

background

during

Raman

measurements

This

sandwich

chip

was

then

clamped

using

two

aluminium

frames

with

screws

The

windows

on

the

frames

were

designed

for

tubing

connections

-LRB-

through

top

frame

-RRB-

and

for

optical

passage

-LRB-

through

bottom

frame

-RRB-

Fig.

-LRB-

a

-RRB-

Assembly

of

microfluidic

device

and

-LRB-

b

-RRB-

Y-shaped

channel

network

-LRB-

channel

depth

μm

width

μm

-RRB-

with

cells

loaded

-LRB-

c

-RRB-

for

examination

Two

KDS

syringe

pumps

-LRB-

KD

Scientific

Inc.

USA

-RRB-

were

used

to

deliver

cells

in

suspension

and

test

solutions

into

the

microchip

channel

-LRB-

Fig.

-RRB-

Ethylene

tetrafluoroethylene

-LRB-

ETFE

-RRB-

polymer

tubing

with

an

inner

diameter

of

μm

on

off

valves

and

appropriate

fittings

and

connectors

all

obtained

from

Upchurch

-LRB-

Upchurch

Scientific

Inc.

USA

-RRB-

were

used

for

plumbing

to

link

the

chip

and

the

syringes

Cell

culture

and

assay

reagents

-LRB-

Chinese

hamster

ovary

Cricetulus

griseus

-RRB-

cells

were

supplied

by

ATCC/LGC

Promochem

-LRB-

ATCC

No

LGC

Promochem

UK

-RRB-

The

cells

were

cultured

routinely

in

DMEM/F

medium

without

L-glutamine

-LRB-

Invitrogen

Ltd

UK

-RRB-

which

was

supplemented

with

fetal

bovine

serum

-LRB-

Invitrogen

Ltd

-RRB-

to

a

final

concentration

of

and

L-glutamine

-LRB-

Invitrogen

Ltd

-RRB-

to

a

final

concentration

of

mM

An

incubator

was

used

at

°C

supplying

The

concentration

of

cells

used

in

loading

the

chips

during

this

experiment

was

in

the

range

of

cells

mL

A

wash

solution

consisting

of

a

modified

Tyrodes

buffer

was

used

to

wash

the

cells

and

to

prepare

the

test

solution

The

Tyrodes

buffer

was

composed

of

mM

NaCl

mM

KCl

mM

HEPES

mM

D-glucose

and

mM

-LRB-

BDH

AnalaR

mM

dissolved

in

Tyrodes

buffer

-RRB-

and

probenecid

-LRB-

Sigma

M

dissolved

in

M

NaOH

aqueous

solution

-RRB-

were

then

added

to

the

Tyrodes

buffer

giving

final

concentrations

of

mM

and

mM

for

and

probenecid

respectively

Ionomycin

test

solutions

were

made

by

adding

ionomycin

stock

-LRB-

mM

in

DMSO

-RRB-

into

wash

solutions

for

a

concentration

of

μM

Ionomycin

was

obtained

from

Calbiochem

-LRB-

Calbiochem

of

EMD

Biosciences

Inc.

USA

-RRB-

and

DMSO

from

Sigma

Aldrich

-LRB-

-RRB-

The

flow

rate

for

the

introduction

of

ionomycin

test

solution

into

the

microchip

was

μL

min

Gold

colloid

with

a

particle

size

of

nm

-LRB-

-RRB-

suspended

in

water

was

supplied

by

BBinternational

Ltd

UK

The

gold

nanoparticles

were

introduced

into

cells

by

a

passive

uptake

method

where

the

cells

were

incubated

with

gold

colloid

solution

at

desired

concentration

-LRB-

colloid

solution

v/v

-RRB-

and

room

temperature

for

mins

Prior

to

loading

into

the

microfluidic

chip

the

cells

were

washed

with

wash

solution

to

remove

the

culture

media

and

gold

nanoparticles

outside

cells

The

cells

were

re-suspended

in

the

wash

solution

for

measurements

Results

and

discussion

SERS

effects

After

loading

cells

into

the

microfluidic

channel

Raman

spectra

were

taken

from

a

selected

single

cell

-LRB-

Fig.

-RRB-

By

using

the

near-infrared

laser

excitation

-LRB-

nm

-RRB-

the

optical

and

thermal

effects

of

laser

illumination

on

the

living

cells

were

minimised

-LSB-

-RSB-

In

addition

using

a

near-infrared

laser

can

significantly

reduce

the

fluorescence

interference

background

and

light

scattering

from

the

quartz

base

plate

It

has

been

reported

that

silver

or

gold

nanoparticles

with

an

individual

size

in

the

range

of

nm

can

yield

significant

enhancement

of

Raman

scattering

for

cellular

analysis

when

nanoparticles

are

introduced

into

cells

-LSB-

-RSB-

This

has

been

confirmed

in

this

study

by

using

gold

nanoparticles

with

a

size

of

nm

Figure

shows

a

typical

spectrum

-LRB-

upper

-RRB-

taken

from

a

cell

incubated

with

gold

colloid

solution

The

spectrum

was

recorded

in

the

range

from

to

cm

with

an

acquisition

time

of

s

We

observed

that

with

the

introduction

of

gold

nanoparticles

the

Raman

spectra

were

clearly

detectable

under

those

conditions

For

comparison

a

spectrum

obtained

from

a

cell

incubated

in

medium

without

gold

colloid

solution

is

also

shown

in

Fig.

-LRB-

lower

-RRB-

It

can

be

seen

that

even

though

the

spectra

acquisition

time

was

increased

to

s

it

was

still

unable

to

obtain

a

good

quality

spectrum

without

gold

nanoparticles

present

This

observation

confirmed

the

significant

enhancement

of

Raman

scattering

with

the

presence

of

gold

nanoparticles

which

also

enabled

a

fast

spectra

acquisition

although

the

exact

mechanism

of

the

SERS

enhancement

of

Raman

signals

is

not

fully

understood

-LSB-

-RSB-

The

introduction

of

gold

nanoparticles

therefore

significantly

shortened

signal

acquisition

times

mainly

owing

to

the

increase

in

sensitivity

which

in

turn

allowed

an

array

of

spectra

to

be

obtained

in

a

relatively

short

time

period

Fig.

spectra

from

cells

following

incubation

in

medium

with

gold

colloid

-LRB-

upper

spectrum

-RRB-

and

without

gold

colloid

-LRB-

lower

spectrum

-RRB-

Characterization

of

single

living

cells

by

mapping

Using

the

SERS

technique

described

above

it

was

possible

to

obtain

spectra

from

a

single

living

cell

which

represented

a

fingerprint

from

which

various

chemical

constituents

in

the

cell

can

be

assigned

Combined

with

an

automated

microscope

stage

which

offers

spatial

resolution

-LRB-

i.e.

μm

-RRB-

mapping

of

an

entire

cell

area

at

a

specific

plane

was

carried

out

It

has

been

suggested

from

previous

studies

that

most

of

the

spectral

bands

associated

with

living

cells

occur

in

the

range

cm

-LSB-

-RSB-

hence

this

range

was

used

in

this

study

for

mapping

Figure

shows

an

image

of

Raman

spectra

obtained

from

the

middle

layer

across

the

single

cell

by

mapping

an

area

of

with

a

spatial

step

of

μm

in

both

X

and

Y

directions

The

mapping

provided

information

on

the

distribution

of

selected

bands

as

seen

in

Fig.

in

the

range

from

to

cm

which

represent

most

of

the

significant

bands

associated

with

DNA

and

proteins

within

a

cell

's

nucleus

and

cytoplasm

-LSB-

-RSB-

Fig.

Spectral

mapping

of

a

single

CHO

cell

on

an

X

Y

plane

and

b

corresponding

spectra

from

three

positions

in

the

area

of

nucleus

cytoplasm

and

membrane

respectively

In

general

the

Raman

spectra

of

single

CHO

cells

showed

contributions

from

all

its

cellular

components

including

nucleic

acids

proteins

lipids

and

carbohydrates

Table

summarises

the

band

assignment

for

the

Raman

spectra

taken

from

CHO

cells

based

on

the

published

data

-LSB-

-RSB-

Comparison

of

the

spectra

taken

from

different

positions

across

the

cell

on

an

X

Y

plane

-LRB-

Fig.

-RRB-

indicated

that

strong

peaks

from

the

nucleus

spectrum

corresponding

to

DNA

sugar

phosphate

backbone

-LRB-

and

cm

-RRB-

and

bases

G

-LRB-

and

cm

-RRB-

A

-LRB-

and

cm

-RRB-

T

-LRB-

and

cm

-RRB-

and

C

-LRB-

cm

-RRB-

were

noticeably

reduced

in

the

cytoplasm

and

membrane

spectra

-LRB-

Table

-RRB-

This

change

was

expected

as

the

nucleus

contains

high

densities

of

DNA

whilst

the

cytoplasm

also

had

significant

quantities

of

RNA

contributing

to

the

corresponding

peaks

As

expected

the

spectrum

taken

from

membrane

area

showed

significant

peaks

corresponding

to

lipids

-LRB-

and

cm

-RRB-

Table

assignment

for

Raman

spectra

of

CHO

cellsBands

-LRB-

cm

-RRB-

BkB

-LSB-

-RSB-

Tyr

-LSB-

-RSB-

BkB

deoxyribose

-LSB-

-RSB-

-LRB-

C

C

-RRB-

α-helix

-LSB-

-RSB-

Phenylalanine

-LSB-

-RSB-

ν

-LRB-

C

O

-RRB-

-LSB-

-RSB-

ν

-LRB-

C

C

-RRB-

chian

-LSB-

-RSB-

ν

-LRB-

C

N

-RRB-

BkB

-LSB-

-RSB-

ν

-LRB-

C

C

-RRB-

chain

-LSB-

-RSB-

Ribose

phosphate

-LSB-

-RSB-

Ribose

phosphate

-LSB-

-RSB-

T

C

G

-LSB-

-RSB-

Phenylalanine

-LSB-

-RSB-

C

-LSB-

-RSB-

Amide

III

-LSB-

-RSB-

δ

-LRB-

C

-RRB-

-LSB-

-RSB-

δ

-LRB-

-RRB-

-LSB-

-RSB-

G

-LSB-

-RSB-

A

-LSB-

-RSB-

T

A

G

-LSB-

-RSB-

δ

-LRB-

CH

-RRB-

-LSB-

-RSB-

δ

-LRB-

CH

-RRB-

-LSB-

-RSB-

A

G

-LSB-

-RSB-

Hemoglobin

-LSB-

-RSB-

A

G

purine

-LSB-

-RSB-

A

C

-LSB-

-RSB-

Amide

I

-LSB-

-RSB-

Abbreviations

BkB

DNA

sugar

phosphate

backbone

Tyr

tyrosine

A

adenine

T

thymine

G

guanine

C

cytosine

ν

stretching

vibrations

δ

deformation

vibrations

Using

the

confocal

optics

of

the

microscope

system

the

chemical

concentration

distribution

in

the

Z

axis

at

three

levels

in

the

nucleus

was

examined

-LRB-

Fig.

-RRB-

and

indicated

that

the

main

peaks

positions

were

generally

identical

but

the

peak

heights

were

markedly

different

indicating

a

concentration

difference

From

Fig.

significant

a

peak

was

observed

at

cm

on

the

spectrum

taken

from

Z

μm

-LRB-

near

to

the

bottom

of

the

cell

-RRB-

which

was

assigned

to

DNA

bases

G

-LSB-

-RSB-

whilst

the

peak

at

cm

from

Z

μm

indicated

a

strong

deformation

from

a

C

H

stretch

in

proteins

-LSB-

-RSB-

Thus

by

collecting

spectra

from

different

spatial

positions

it

provided

an

approach

to

build

a

mapping

of

the

distribution

of

chemicals

within

a

single

cell

Fig.

spectra

taken

at

three

levels

along

the

Z

direction

in

the

nucleus

area

It

should

be

noted

that

the

spectra

taken

from

different

positions

are

distinct

in

terms

of

both

Raman

band

amplitudes

and

band

positions

This

spectral

variation

can

be

attributed

to

the

native

chemical

inhomogeneity

within

a

cell

However

the

possibility

of

non-uniform

distribution

of

gold

nanoparticles

within

the

cell

can

not

be

excluded

-LSB-

-RSB-

In

addition

this

non-uniform

distribution

can

also

take

place

during

the

time-resolved

monitoring

because

dynamic

processes

inside

the

cell

can

lead

to

local

fluctuations

of

the

particle

densities

and

hence

changes

of

the

spectra

To

further

develop

this

spectroscopic

technique

for

both

qualitative

and

quantitative

analysis

technologies

need

to

be

explored

in

order

to

deliver

and

position

nanoparticles

within

cells

in

a

controllable

format

for

a

uniform

distribution

Nevertheless

it

is

still

possible

using

this

promising

methodology

to

examine

a

specific

point

within

a

cell

in

a

dynamic

way

with

time

especially

when

applying

stimulation

e.g.

an

agonist

under

microfluidic

control

conditions

In

situ

monitoring

of

cellular

chemical

dynamics

Finally

we

monitored

the

chemical

dynamics

of

the

cell

when

exposed

to

the

agonist

ionomycin

-LSB-

-RSB-

After

loading

cells

into

the

chip

channel

a

period

of

settlement

was

allocated

before

introducing

the

ionomycin

solution

at

a

constant

flow

rate

of

μL

min

Whilst

focussed

on

a

region

close

to

the

cell

nucleus

a

series

of

spectra

were

taken

at

an

interval

of

s

in

the

range

from

to

cm

-LRB-

Fig.

-RRB-

It

can

be

seen

from

the

spectra

-LRB-

Fig.

-RRB-

that

most

of

the

peaks

corresponding

to

nucleic

acids

proteins

and

lipids

remain

visible

with

time

but

the

peak

heights

vary

noticeably

indicating

the

concentration

change

of

these

compounds

One

of

the

most

significant

changes

is

the

appearance

of

a

peak

at

cm

which

can

be

assigned

to

amide

I

-LSB-

-RSB-

The

time

profile

of

this

peak

-LRB-

Fig.

-RRB-

reveals

the

concentration

change

of

amide

I

within

the

cell

showing

a

similar

trend

to

that

of

the

flux

evoked

by

the

agonist

ionomycin

-LSB-

-RSB-

which

is

commonly

used

in

biomedical

research

to

stimulate

the

intracellular

production

of

proteins

such

as

interferon

-LSB-

-RSB-

Since

amide

I

contributes

spectrally

to

the

Raman

spectrum

of

interferon

it

follows

that

the

in

situ

monitoring

of

such

molecules

could

in

future

be

used

to

produce

characterization

of

protein

expression

dynamics

at

subcellular

levels

Clearly

more

studies

are

required

in

order

to

understand

the

mechanism

and

cause

of

the

concentration

change

of

amide

I

within

the

cell

when

exposed

to

agonist

ionomycin

Fig.

situ

monitoring

of

cellular

dynamics

by

recording

a

series

of

Raman

spectra

-LRB-

a

-RRB-

from

a

specific

area

at

an

interval

of

s

The

first

spectrum

at

time

was

taken

when

no

agonist

was

introduced

-LRB-

b

-RRB-

-LRB-

c

-RRB-

shows

the

time

profile

of

the

peak

at

cm

corresponding

to

the

change

of

amide

I

Conclusions

The

SERS

technique

has

been

used

for

characterisation

of

single

CHO

-LRB-

Chinese

hamster

ovary

-RRB-

cells

with

a

microfluidic

device

where

gold

nanoparticles

were

introduced

into

cells

for

Raman

enhancement

The

use

of

an

inverted

microscope

optical

systems

in

combination

with

a

charge

coupled

device

-LRB-

CCD

-RRB-

detector

allows

the

measurement

of

Raman

spectra

for

simultaneous

analysis

of

bio/chemical

species

within

cells

In

addition

to

a

precision

automated

X

Y

sample

stage

the

confocal

optics

provides

discrimination

between

points

of

different

depths

within

the

cell

enabling

chemical

mapping

in

three

dimensions

By

using

microfluidic

methodology

the

cell

manipulation

and

reagent

delivery

were

performed

in

a

controllable

manner

By

introducing

cells

and

test

reagent

into

the

chip

in

a

continuous

flow

as

a

series

of

plugs

in

a

given

sequence

it

enabled

the

in

situ

chemical

characterization

of

single

CHO

cells

with

a

high

degree

of

spatial

and

temporal

resolution

This

allows

the

real

time

monitoring

of

the

dynamics

of

the

agonist-evoked

flux

response

The

approach

described

has

the

potential

to

be

used

for

the

study

of

the

spatial

dynamics

of

a

range

of

intercellular

processes

O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

surface-enhanced raman scattering (sers)