IN VIVO PHOTOACOUSTIC SPECTROSCOPY OF THE SKIN

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Submitted on 1 Jan 1983

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IN VIVO PHOTOACOUSTIC SPECTROSCOPY OF THE SKIN

P. Poulet, J. Chambron

To cite this version:

P. Poulet, J. Chambron. IN VIVO PHOTOACOUSTIC SPECTROSCOPY OF THE SKIN. Journal

de Physique Colloques, 1983, 44 (C6), pp.C6-413-C6-418. �10.1051/jphyscol:1983668�. �jpa-00223227�

I N V I V O P H O T O A C O U S T I C SPECTROSCOPY O F THE S K I N P. P o u l e t and J . Chambron

I n s t i t u t de Physique BioZogique, FacuZte' de Me'decine, 4 , rue KirschZeger,

6 7 0 8 5 Strasbourg Cedex, France

Resume - Un spectrometre photoacoustique u t i l i s a n t un d e t e c t e u r d i f f e r e n t i e l permet de mesurer i n v i v o l e s spectres d ' a b s o r p t i o n o p t i q u e de l a peau humai- ne. Sa conception e t ses c a r a c t e r i s t i q u e s sont d e c r i t e s ; le s premiers r e s u l - t a t s experimentaux sont presentes.

A b s t r a c t - b!e describe the conception and t h e c h a r a c t e r i s t i c s of an ooen- ended photoacoustic d e t e c t o r developed f o r doing i n v i v o measurements o f s k i n o p t i c a l absorption. P r e l i m i n a r y r e s u l t s a r e presented.

As soon as he introduced the modern photoacoustic spectroscopy, Rosencwaig attempted t o demonstrate the usefulness of t h i s new method i n medical sciences such as derma- t o l o g y . He soon mentionned t h e f e a s i b i l i t y ⁰⁶ performing i n v i v o measurements on hu- man skin, by the use of an open-ended c e l l ' .

I n s p i t e of t h i s , almost a l l the cutaneous a p p l i c a t i o n s o f photoacoustic d e t e c t i o n have been done on excised epidermal samples. These s t u d i e s have shown t h a t t h e pho- t o a c o u s t i c spectrum r e v e a l s t h e a b s o r p t i o n band o f p r o t e i n s a t about 280 nanometres and depends on t h e h y d r a t i o n o f t h e sample through i t s thermal p r o p e r t i e s . The pho- t o a c o u s t i c s i g n a l s produced by drugs o r sunscreens g i v e t h e i r o ~ t i c a l absorption i n s i t u , t h a t i s on and i n the epidermis and t h e i r d i f f u s i o n c o e f f i c i e n t i n stratum corneum' .

At t h e time being, o n l y Campbell e t a12 and Pines and Cunningham3 r e p o r t e d i n v i v o measurements. The major d i f f i c u l t y i n doing i n v i v o photoacoustic measurements 1 ie s i n t h e f a c t t h a t the microphone i n s e n s i t i v e t o the body's movements. We undertook t o study t h e r e a l n o s s i b i l i t i e s o f such i n v i v o cutaneous photoacoustic spectroscopy.

A f t e r v a r i o u s approaches, t h i s l e d us t o c o n s t r u c t a photoacoustic d e t e c t o r u s i n g a d i f f e r e n t i a l microphone between two i d e n t i c a l c e l l s . I t s c h a r a c t e r i s t i c s a l l o w us t o measure d i f f e r e n t i n v i v o s i g n a l s w i t h good r e p r o d u c i b i l i t y and s i g n a l t o n o i s e r a t i o .

I - EXPERIFIENTAL SET-UP

The o r i g i n a l i t y of t h e spectrometer r e s i d e s i n t h e conception o f t h e photoacoustic c e l l which must be considered so as t o o p t i m i z e t h e s i g n a l t o n o i s e r a t i o . One o f t h e important features o f t h e conception o f t h i s c e l l i s t o maximizf i t s s e n s i t i v i - t y by t h e o p t i m i z a t i o n o f i t s dimensions. According t o Aamodt e t a1 and t o Quimby and Yen5, the optimal dimensions o f t h e c e l l a t 20 Hz modulation frequency and i n standard temperature and pressure c o n d i t i o n s a r e 1.6 mm l o n g and 7 mm l a r g e . This 20 Hz t h r e s h o l d was chosen because t h e v i b r a t i o n s o f t h e s k i n a r e t o o s u b s t a n t i a l a t any lower frequencies. This choice a l s o means t h a t i t i s p o s s i b l e t o use t h e o r e t i c a l models of t h e photoacoustic e f f e c t based on the Rosencwaig-Gersho theory6. The m i - crophone should be i n c o n t a c t w i t h t h e gaseous volume s p e c i f i e d above by means o f a small a c o u s t i c pipe.

Having defined t h e dimensions which o n t i m i z e t h e s e n s i t i v i t y o f t h e c e l l , we must now consider t h e second aspect o f i t s conception : t o minimize t h e noise and syn- chronous background s i g n a l produced by t h e microphone. The synchronous background i s u s u a l l y t h e most d i f f i c u l t t o reduce. I t can be generated by a b s o r p t i o n o f l i g h t w i t h i n t h e c e l l by a body o t h e r than t h e s t u d i e d sample, such as the s i d e w a l l s o r t h e microphone i t s e l f . Another synchronous background can be produced by t h e l i g h t modulator.

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1983668

C6-414 JOURNAL DE PHYSIQUE

Using an open c e l l r a i s e s problems o f damping e x t e r n a l sounds and v i b r a t i o n s . When t h e c e l l i s a p p l i e d t o t h e body under s c r u t i n y , the gaseous volume d e l i m i t e d by t h e c e l l and t h e surface being analysed must be p e r f e c t l y sealed w i t h r e g a r d t o t h e o u t - s i d e atmosphere i n o r d e r t o o b t a i n a p h o t o a c o u s t i c s i g n a l . As l o n g as t h i s hermetic seal can be achieved, i n s u l a t i o n a g a i n s t o u t s i d e sounds w i l l be sat.isfactory. On the o t h e r hand, i t i s imoossible t o i n s u l a t e the c e l l from t h e v i b r a t i o n s o f t h e body under s c r u t i n y , the s u r f a c e o f which i n e v i t a b l y forms one o f t h e i n t e r n a l w a l l s of t h e c e l l . It i s t h e r e f o r e e s s e n t i a l t o use a m o d u l a t i o n frequency a t which the v i - b r a t i o n s o f t h e s k i n a r e small, a l t h o u g h t i t must n o t be f o r g o t t e n t h a t as t h e mo- d u l a t i o n frequency r i s e s , t h e s i g n a l becomes weaker. I t i s a l s o advantageous t o seek o u t zones o f s k i n where v i b r a t i o n i s weakest. I n s p i t e o f a l l these precautions, t h e l e v e l o f noise detected on t h e s u r f a c e o f t h e s k i n made t h e f i r s t photoacoustic c e l l s v i r t u a l l y impossible t o use.

MICROPHONE

F i g . 1 - The photoacoustic d e t e c t o r f o r s k i n spectroscopy

I n o r d e r t o reduce the e f f e c t o f the s k i n v i b r a t i o n s on t h e measured s i g n a l , a d i f - f e r e n t i a l method was a p p l i e d , u s i n p a c l o s e - t a l k i n g microphone : t h e KnowlesBW1789.

This microphone i n c l u d e s two sounds p o r t s , one on each s i d e o f t h e diaphragm, and t h e s i g n a l i t d e l i v e r s depends on t h e p r e s s u r e d i f f e r e n c e between t h e t w o s i d e s of t h i s diaphragm. The microphone i s f i t t e d t o t h e cutaneous d e t e c t o r , i n between two i d e n t i c a l c e l l s , one o f which i s c l o s e d by t h e l i g h t guide, t h e o t h e r b y a volume- a d j u s t i n g screw. T h i s screw should enable t h e responses o f t h e two c e l l s t o be' equa- l i z e d , a t t h e frequency i n use, so as t o m i n i m i z e noise. The sketch o f t h e photoa- c o u s t i c d e t e c t o r i s shown i n F i g . 1 . I t s dimensions have been o p t i m i z e d according t o t h e c r i t e r i a s e t o u t above. It i s a t t a c h e d t o t h e s u r f a c e o f t h e s k i n b y a double -

sided adhesive tape i n which two h o l e s have been bored o p p o s i t e t h e c e l l s , and t h e

whole apparatus can be fastened t o t h e forearm by means o f an armband.

a p p l i c a t i o n s which a r e considered. The s e n s i t i v i t y o f t h e d e t e c t o r was measured on a t h i n black body, obtained by blackening a p l e x i g l a s s h o l d e r over a flame. A t 20 h e r t z , t h e s e n s i t i v i t y o f t h e c e l l i s 430 pascals per watt, and expresses t h e r a t i o between the r.m.s. values o f v a r i a t i o n s i n a c o u s t i c pressure and i n c i d e n t l i g h t po- wer. This s e n s i t i v i t y v a r i e s as a f u n c t i o n o f t h e modulation frequency. Thetheore- t i c a l decrease i n l / f i s v e r i f i e d a t frequencies h i g h e r than 25 h e r t z , as c o u l d be foreseen from t h e s i z e o f t h e c e l l . A t lower frequencies, t h e thermal losses occu- r i n g i n t h e w a l l s o f t h e c e l l weaken t h e s i g n a l . The spectrum o f t h e noise d e n s i t y d e l i v e r e d by t h e microphone i s estimated from t h e r.m.s. value o f t h e output v o l t a g e o f t h e l o c k i n a m p l i f i e r , w i t h a time constant o f 100 m i l l i s e c o n d s , which c o r r e s - ponds t o a bandwidth o f 1.25 h e r t z .

While the noise was being measured, a l l t h e instruments were operating, t h e c e l l sea- l e d a g a i n s t t h e s t u d i e d sample and t h e modulated l i g h t beam shut o f f by a b l i n d . Va- r i o u s tyoes o f n o i s e a r e detected on a v i b r a t i o n - f r e e sample : a wide-band n o i s e w i t h frequencies above 150 h e r t z , 50, 100 and 260 h e r t z p a r a s i t e s , a l / f noise w i t h a knee a t approximately 30 h e r t z , and a narrow-band n o i s e between 30 and 60 h e r t z . This spectrum i s the d i r e c t r e s u l t o f t h e d i f f e r e n t sources o f n o i s e r e f e r r e d t o a- bove, except f o r t h e synchronous background, which would appear t o be produced by t h e l i g h t s c a t t e r e d o r r e f l e c t e d by t h e sample i t s e l f , and which depends on t h e na- t u r e o f t h e l a t t e r .

W h i l s t the c e l l was attached t o the forearm o f a volunteer, t h e n o i s e l e v e l i s higher, whatever t h e frequency used, although a much more s u b s t a n t i a l l e v e l can be observed a t low frequencies (below 20 h e r t z ) , and t h i s i s due t o i n v o l u n t a r y movements of the forearm.

I I I I I

- ^{200- ,2000}

- 1600

- ¹²⁰⁰

W

10 20 5 0 100 200 500 1000

MODULATION FREQUENCY (Hz)

Fig. 2 - S e n s i t i v i t y t o noise d e n s i t y r a t i o as a f u n c t i o n o f t h e modulation frequen- cy w i t h t h e d e t e c t o r attached t o a forearm (e) and i s o l a t e d (+-)

F i g . 2 shows t h e s e n s i t i v i t y t o noise d e n s i t y r a t i o s corresponding t o the two fore- going s i t u a t i o n s . When t h e c e l l i s placed on an i s o l a t e d base, t h i s r a t i o reaches i t s maximum values a t 30 h e r t z (approximately 2000) and 90 h e r t z (approximately 1500).

When the c e l l i s attached t o a forearm, t h i s r a t i o i s , i n a t y p i c a l case, t e n time smaller, and has two maxima, one a t 25 h e r t z (about 200) and the other a t 80 h e r t z (about 120). The s e n s i t i v i t y t o n o i s e d e n s i t y r a t i o i s poor a t low frequencies (40 a t 10 h e r t z ) and a t h i g h frequencies ( l e s s than 50 over 300 h e r t z ) .

F i q . 3 gives t h e diagram of t h e complete spectrometer r e a l i z e d . The l i g h t source i s

a 00 W xenon lamp w i t h a b u i l t - i n p a r a b o l i c r e f l e c t o r which i s suuplied e i t h e r w i t h

C6-416 JOURNAL DE PHYSIQUE

a constant o r w i t h a sinusoSda1 c u r r e n t d e l i v e r e d by t h e power supply u n i t c o n t r o l - l e d by a f u n c t i o n generator. When t h e lamp i s s u p p l i e d w i t h a constant c u r r e n t t h e 1 ig h t i s modulated by t h e use o f a mechanical chopper placed before t h e monochroma- t o r o r the i n t e r f e r e n c e f i l t e r . The energy o f t h e light-beam produced i s 50 watts, o f which s o r e than 30 w a t t s c o n s i s t o f i n f r a - r e d r a d i a t i o n s , which are f i l t e r e d o u t by a watertank 5 centimetres t h i c k w i t h q u a r t z windows. The beam i s then focused by a UV s i l i c a lens, onto e i t h e r t h e entrance s l i t o f a h o l o g r a p h i c - g r a t i n g monochroma- t o r , w i t h a l i g h t - g u i d e a t t h e e x i t s l i t , o r d i r e c t l y on t h e l i g h t guide placed be- h i n d an i n t e r f e r e n c e f i l t e r . The used l i g h t - g u i d e i s made o f UV s i l i c a , i s 1 metre long, and t h e diameter o f t h e bundle of f i b e r s i s 4 m i l l i m e t r e s . The end o f t h e l i g h t guide forms one o f t h e w a l l s o f t h e cutaneous photoacoustic d e t e c t o r .

Lens Wheel

F i g . 3 - Diagram o f the ~ h o t o a c o u s t i c spectrometer

The e l e c t r i c a l s i g n a l produced by t h e microohone i n t h e c e l l i s analyzed by a two- phase l o c k - i n a m p l i f i e r . The amplitude and phase of t h e photoacoustic s i g n a l a r e then p l o t t e d on an X Y recorder. The X - scan i s c o n t r o l l e d e i t h e r b y t h e time-base o f t h e monochromator, o r by t h e sweep o u t p u t o f t h e f u n c t i o n generator d u r i n g a f r e - quency a n a l y s i s . A AC/DC and l o g a r i t h m i c c o n v e r t e r can be used f o r r e c o r d i n g t h e n o i s e spectra a t t h e o u t p u t o f the l o c k i n a m p l i f i e r , as w e l l as t h e frequency-va- r i a t i o n graphs o f t h e photoacoustic s i g n a l , expressed i n l o g a r i t h m i c coordinates.

As t h e amplitude o f t h e photoacoustic s i g n a l i s p r o p o r t i o n a l t o t h e energy of t h e sample, each spectrum must be c o r r e c t e d by a p o i n t by p o i n t d i v i s i o n w i t h a previous- l y recorded spectrum o f a b l a c k body.

RESULTS AND DISCUSSION

P r e l i m i n a r y a p p l i c a t i o n s o f t h e cutaneous d e t e c t o r were performed on small - s i z e d S O -

l i d s attached t o t h e c e l l w i t h double-sided adhesive tape. Spectra were recorded f o r

d i f f e r e n t s o l i d s : couloured cards, glass o p t i c a l f i l t e r s , whole oreen leaves on t h e

p l a n t . They a l l show t h a t the open d e t e c t o r can e a s i l y be used f o r making spectros-

c o p i c s t u d i e s o f the surface o f i n e r t and v i b r a t i o n - f r e e s o l i d s . Simply using dou-

b l e - s i d e d adhesive tape provides s a t i s f a c t o r y i n s u l a t i o n o f t h e c e l l from e x t e r n a l

sounds. The d e l o c a l i z a t i o n o f photoacoustic spectroscopy r e s u l t i n g from t h e use of

a l i g h t guide and an open c e l l enable t h e method t o be a p p l i e d t o v a r i o u s o r i g i n a l

s i t u a t i o n s , as i t no l o n g e r r e q u i r e s the use o f small samples enclosed w i t h i n a c e l l .

The c h a r a c t e r i s t i c s o f the cutaneous d e t e c t o r and t h e thermal p r o p e r t i e s of s k i n

enable one t o envisage the p o s s i b i l i t y o f t a k i n g cutaneous measurements. The t h e r -

mal e f f u s i v i t y o f s k i n i s close t o t h a t o f water7, and the maximum photoacoustic s i -

gnal measured a t t h e surface of t h e s k i n ought t o be about t h r e e times weaker - r a t i o

o f t h e thermal e f f u s i v i t i e s - t h a n t h e s i g n a l produced by the reference mentionned i n

a t 25 h e r t z and 25 a t 80 h e r t z .

The mean energy a v a i l a b l e i n UVB (280-320 nm) and UVC (200-28D nm) r a d i a t i o n , c o r r e - l a t e w i t h i n t e n s i t i e s o f more than 100 w a t t s Der square metre and the e f f e c t o f the- se i n t e n s i t i e s on the exposed areas must be c a r r e f u l l y c o n t r o l l e d . I t i s w e l l known t h a t such r a d i a t i o n g i v e s r i s e t o numerous p h o t o b i o l o g i c a l r e a c t i o n s , such as t h e i n h i b i t i o n o f n u c l e i c a c i d and p r o t e i n synthesis, t h e i n d u c t i o n o f s k i n cancer, and the formation o f erythemas8.

WAVELENGTH (nm)

F i g . 4 - I n v i v o photoacoustic spectrum o f human epidermis : non corrected spectrum (-) and spectrum c o r r e c t e d from v a r i a t i o n s o f t h e l i g h t i n t e n s i t y v e r s u s wavelength (+-A). The i n s e r t shows t h e s i g n a l a t 470 nm recorded d u r i n g 5 minutes. 80 Hz ; 16 nm ; 10s.

The p r e l i m i n a r y experimental r e s u l t s we present were obtained on t h e forearm o f vo- l u n t e e r s who remained s t a t i o n n a r y throughout t h e measurements. F i g . 4 shows the pho- t o a c o u s t i c spectrum o f t h e s k i n measured i n v i v o . The non-corrected soectrum i s shown, as w e l l as t h e s i g n a l a t 470 nm recorded d u r i n g 5 minutes, i n o r d e r t o g i v e prominence t o t h e good s i g n a l t o n o i s e r a t i o we o b t a i n . The spectrum c o r r e c t e d from t h e wavelength v a r i a t i o n s o f l i g h t i n t e n s i t y reveals, f o r the f i r s t time, t h e ab- s o r p t i o n band o f p r o t e i n s a t about 280 nanometres as measured i n v i v o and can be w e l l comnared w i t h transmission spectrum o f human stratum corneum o r epidermis ob- t a i n e d i n v i t r o by transmission spectroscopy u s i n g i n t e g r a t i n p soheresg o r photoa- c o u s t i c spectroscopy1. According t o t h e thermal d i f f u s i v i t y o f t h e outermost l a y e r s of t h e s k i n 0,5.10-3 square centimetre p e r second and t o theused modulation frequen- cy 80 h e r t z , t h e depth o f t h e s k i n l a y e r which gives r i s e t o the measured s i g n a l i s about 15 micrometres, corresponding t o stratum corneum only.

Fig. 5 shows t h e photoacoustic spectrum o f merbromin (dibromohydroxyrnercurifluores-

c e i n disodium s a l t ) on t h e skin. The spectrum obtained on t h e untreated s k i n i s

shown f o r comparison, as w e l l as t h e photoacoustic spectrum o f t h e used merbromin

aqueous s o l u t i o n ( 2 %), measured w i t h o u r conventional photoacoustic snectrometer

and quantized according t o a p r e v i o u s l y described m e t h o d ~ l o g y ' ~ .

JOURNAL DE PHYSIQUE

1dAVELENGTH ( nm)

F i g . 5 - A b s o r p t i o n s p e c t r a of merbromin: a b s o l u t e a b s o r p t i o n spectrum of t h e 2 % aqueous s o l u t i o n measured i n a c o n v e n t i o n a l photoacoustic c e l l (-)20Hz;

16 nm ; 3s ; and p h o t o a c o u s t i c s p e c t r a o f t h e u n t r e a t e d s k i n (M) and t h e t o p i c a l l y a p p l i e d merbromin (-A-L+) measured on a forearm. 80 HZ ; 16 nm ; 3s.

CONCLUSION

These f i r s t experimental r e s u l t s show t h a t p h o t o a c o u s t i c s i g n a l s can be measured i n vivo, and w i t h s a t i s f a c t o r y s i g n a l - t o - n o i s e r a t i o s . Reduction o f t h e noises caused by t h e s u b j e c t ' s s k i n v i b r a t i o n s has been achieved by u s i n g a d i f f e r e n t i a l micropho- ne, and t h i s i n t u r n has made p o s s i b l e t o o b t a i n t h e f i r s t i n v i v o photoacousticmea- surements o f t h e a b s o r p t i o n o f t h e s k i n i t s e l f , and o f an o p t i c a l absorption spec- trum o f a m e d i c i n a l substance a p p l i e d t o t h e s u r f a c e o f t h e s k i n o f s u b j e c t s having v o l u n t e e r e d t o remain immobile t h r o u g h o u t t h e p e r i o d necessary f o r t h e measurements t o be taken. The d i a g n o s t i c use o f p h o t o a c o u s t i c d e t e c t i o n techniques nevertheless remains l i m i t e d by t h e d e t e c t o r ' s s e n s i t i v i t y t o s k i n v i b r a t i o n s . Another mode o f d e t e c t i o n ought t o be a b l e t o e l i m i n a t e t h i s drawback : photothermal d e t e c t i o n u s i n g i n f r a r e d radiometry1', which was a l r e a d y b e i n g used i n t h e nineteen f i f t i e s f o r mea- s u r i n g t h e thermal p r o p e r t i e s o f t h e skin12.

However, these e a r l y r e s u l t s can be seen t o be encouraging, and from them can be con- s i d e r e d v a r i o u s a p p l i c a t i o n s o f p h o t o a c o u s t i c spectroscopy such as t h e b i l i r u b i n l e - v e l i n newborn's jaundice, p h o t o s e n s i t i v i t y o f t h e s k i n t o UV r a d i a t i o n o r photoche- motherapy o f s k i n diseases l i k e p s o r i a s i s and cancer.

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