利用双空间滤波提高拉曼成像显微光谱的空间分辨率

拉曼显微光谱由于能够提供微米级样品的快速和无损测量而被广泛使用。该技术还提供了类似于FTIR显微光谱的信息，为分析实验室提供了增强的能力。拉曼显微光谱与傅立叶变换红外显微光谱相比的主要优点是更好的空间分辨率。相比之下，FT-IR测量的最小空间分辨率约为10µm，而拉曼可以轻松实现1µm的空间分辨率。本应用中结论NRS-5000/7000提供了更高的空间分辨率，更接近理论衍射极限。此外，使用DSF光学布置可以实现共焦光学系统中的最大性能。如果特定实验不需要高空间分辨率，则可以使用较大的针孔孔径来提供增加的能量，以获得更好的信噪比结果。Application NoteRaman Spectroscopy The use of dual spatial filtration to improve spatialresolution in micro-Raman imaging spectroscopy2/7Application Note The use of dual spatial filtration to improve spatia l resolution in Raman imaging micro-spectroscopy R am a n mi c r o -spec tr oscopy ha s bee n widely used d u e t o its ab ili t y t o prov i de rapid an d n on -des t r u ct i ve m e a su r e m e n ts of mi c ro n -sized s a mpl es . Th e te c h ni q ue a l so pr o vi des in f o rmation s imil ar t o th at o f FTI R mi cro -spec tr osco p y，offe rin g e n h an ced c a p ab i li t i es fo r the an a lytical l a bo ra tor y .A pr im ary a d v a n t ag e of Raman mi c r o-spec t r osco py o v er F TIR mi c r o-spectroscopy is be tt e r spatial r esolu t io n .C o mp ara tively， th e m ini m u m sp a ti a l r esolu ti o n for FT-I R me a s ur eme n ts i s a ppr o x i mat e ly 10 u m ， w h i le R a ma n c a n easily a ch ieve a spa ti a l resolution of 1 um. In th i s a p pl ica ti o n n ote ， the s p a ti a l reso lu tion o f R aman mi c r o -s pe ctr o sc o p y， it s gen e ral defin iti o n an d ev alu ation me t hod s w i ll be d es crib ed . Th e d u a l s pati a l filtra ti o n (D S F)syste m， a standard f eatu r e of the JASCO N RS-5000/7000seri e s Ram a n m i c r o-s p ec t r ome t e rs ， wi ll a lso be e xp lain ed . Keywords R aman ， Raman mi c rosco py， d u a l s pat ial fi ltr a t io n ， s pa t i al r esolu ti o n ， diff r action limit， NRS-4100， N R S-5100， N RS-5200，N R S-7100，N RS-7200 Definition of spatia l resolution A l a se r of a spec i f i c w a v e le n gth i s u sed a s th e e xc i t a tion so u r c e in R am a n mi cr o-spectros copy. As the l aser be a m spot s i ze on t h e sample i s r educed ， th e spatial r esolu ti on at th e XY p l a n e in cr eas e s . Whe n l i ght w i th a co n s tan t in t e n si ty d i s t rib u ti o n i s i n trod uc ed i n to a n o b j e ctiv e len s， the d i ff r acti o n pat t e rn s h o w n i n Fi gure 1 occu r s (see page 2).Th e br i g h t c ent e r area i s ca ll e d the A ir y -d is k ， an d it s si z e (d )c a n b e d e t e rmin ed f rom the w av elen gth () a n d the n u meri ca l a p erture (N .A.) (see equ ati o n in Figu r e 1). The “d ”te r m re f ers t o the d i ff r a c ti o n li mit that deter min e s th e s pa ti al res o l u t i on of a n o ptica l mi c ros co p e . JASCO INC. 28600 Mary's Court ， Ea s t on， MD 21601 U S A Figure 1. Airy-disk resulting from diffraction with a circular aperture Figure 2. Spatial resolution defined by the Rayleigh criterion 28600 Mary's Cour t ， E asto n ， MD 21601US A Th e def ini tio n of spat i al reso lu tion on an XY p l ane i s based on th e d i stance be t ween two p oi n ts c l ose to each o th e r -the R a y l eigh c r it e ri o n . In the s t andar d c o n f i g u rati o n o f th e J ASCO NR S -5000/7000 (532 n m l as er ， with 100X o bj ec t ive l e ns an d N .A . = 0.90)， th e l a s e r sp ot s i ze a t th e d if f ra cti o n limit is c a lculat ed as d=720 n m. Th e sp a ti al r es ol u ti o n ， a cc o r d in g t o the Rayle i g h criter i o n ， i s th e n eq u a l t o 360 nm. As the equ a t i on in Fig u re 1 s u ggests ， if th e laser w a v elen gth decreases，the s p a ti al reso lu tion th e n i n creases .A dditi o nall y ， if an obj e c t iv e l e n s su ch a s an oi l-i mmer se d l e n s w i th a l a r ge r N .A. i s u sed ， o n e ca n e xp ect a co mp a r a tiv e in crease i n spatial re s o lu ti o n. H o w ever， the defin iti o n base d on FW H M (full w i d t h at half ma ximu m) of the i n t e n si t y of th e in ter f ere n ce ri ng has a l so b een r ecogn i zed . T o avoid co n fus i o n ， w hen m ak in g a compa r ison of spa t ia l r esolut i on fo r the Rama n micro-sp ectrometers prepared b y d iffe r en t m an u factu r e r s， th e d e f in i t io n o f 's p a ti a l re s o lu tion ' wh ich h a s b ee n a pp l ied s h oul d b e co nf irmed .F or th e NR S-5000/7000 se ri es， each o f the values calculated f rom th e two di f f eren t d ef ini tio n s i s sh o w n i n Ta b le 1. Table 1. Spatial resolution on an XY plane Equation 532 nm，N.A.=0.9configuration 355 nm， N.A.=1.4configuration Rayleigh criterion 0.61X/N.A. 360nm 154nm FWHM 0.51从/N.A. 301nm 129nm Evaluation of spatial resolution in confocal optics Th eo r eti c a ll y ， o n e c an c a l cul a te th e s p at ia l r es o l uti on fo r c o n f oca l o pti c s as s h o w n i n T a bl e 1. Ho w e v er ， the a ctu a l v a l ue for optical microscopes may become la r ger due to lens a b e rr ations， the intens i ty d is tr i b ution of th e i nci d ent l ase r l i g ht to th e obje cti ve l e n s ， a s wel l a s o t h e r f a ct o r s . T h e act u al s p a tial r eso l u ti o n c an b e e v a lu ate d by me a s u r i n g the dis t a nce of th e R a man i n t e ns it y pr o fil e w h en it chan ge s from 10% t o 90% by th e meas ure me n t of a s ampl e with a sh arp e d ge a s i llu st r a te d in F i g ure 3. This evaluat i on m eth o d was applied t o th e NRS-5000/7000 ser i e s in s tru ments with t h e s t a n da r d con fi guratio n of a 532nm laser an d an o bj ective l en s of N.A .=0.9. The res u lting value w as 370 nm ， w h ich is very c l ose to the th eo r etical val u e o f 360 nm cal c u l a t ed acco r din g to Ta bl e 1. 28600 Mary's Cour t ， E asto n ， MD 21601US A Resolution in depth in confocal optics In a co nfoc a l o pti c al s y st e m， a pin h o le a p er tu re is l oca t ed a t a r e c ipr oca l foca l po in t of th e o pti c a l s ys t e m. L i ght r a y s co min g f rom p oin ts o t her than the f o cus of t h e o b j ect iv e l en s are e l imi n at ed . The d e p th reso l ution (d， w here z = resol u tion) ca n b e d et e r m in e d b y the e x c itat i on wa v e l e n gth“入 "， th e re fr a ct ive in de x “n ”of the s u b s t rate a n d the nu me r ical ape r tu r e “N.A." of the objective lens (equa ti o n 2). In sert ing th e v alu e s of “x " = 532n m，“n” =1 (a t mo sp he r e) a n d N.A.=0.90， a ca l cu l at ed “d " o f 830 n m i s o b t a i n e d . T h e a ct ual de p th resol u ti o n can a g ain be est i m at ed by m e as u rin g th e FWHM of th e R a m a n in ten s i ty p r o f ile fo r a s i l i con cry s tal wh en it i s sca nne d in the Z d i rectio n . In a s t a n dar d confoca l o p t i ca l sys t em ， availa bl e w ith most of th e commercially ava il able Ra m a n m icro -spec tr ometers， a typi c al r eso lut io n o f 1.5 to 2.0 u m a t FWHM is expected. I n th e NRS-5000/7000， a FWH M of 1 um c an be ex p ected， wh i c h i s mu ch c l oser to th e the o re ti ca l v alue as s ho w n in Figu re 4. Th i s n u m ber c a n b e ac h ie v ed b y th e u se of an i mp r o v ed confoca l aper tu re optimized for Rama n spectroscopy. JASCO INC. Figure 4. Raman intensity profile of silicon in Z depth profiling (532 nm laser， objective lens 100X，N.A=0.9) Dual spatial filtration (DSF) T h e stand ar d d u a l spatia l f iltr a t io n (DSF ) opt i ca l sys t e m in th e NRS-5000/7000 spectrom et e r s has a d u a l aper tu re sy s tem fo r c o n f oc a l op t i c s . T h i s DSF sy s t e m c an el i m i n at e a n y s t ray ligh t th at was n ot remo v ed a t the f ir s t a pe r tu r e . Th e DSF me th od ca n impr o v e the Z re s ol u ti o n a n d at th e s a me time， a n i m pr o v e m e nt c a n be e xpe cte d i n th e sp at ia l r es ol ut i on a t the XY pla n e . Figure 5. a) standard confocal optics with pinhole aperture； b) JASCO DSF confocal optics 28600 Mary's Cour t ， E asto n ， MD 21601US A The standard s in g l e aper tu re system ('a’in Figure 5) a n d the J ASCO DSF o p tics ('b ’in F i g ur e 5) we r e applied to the me a s u r e me n t of t i t a n i u m o x i d e (T i O，) p a r t i cles o n a s il ico n su b str a t e. Th e R a ma n im a ging of t h e pe ak in t e n sity prof i le f o r b o t h res u lts a r e sh own in F ig u r e 7. In t h i s c a se ， the s p a ti a l reso lu ti o n in th e X Y pl ane w a s i m pr o v ed b y th e u se of D S F opt i cs (righ t i m ag e in F i g ur e 7). The image in ten s ity p r o fi le was also close r to th e m i c r osco p e image of the sample (F i gu r e 6). Figure 6. Microscope image of a TiO2 particle on a silicon substrate Figure 7. Raman imaging of a TiO2 particle on a silicon substrate (I ntensity profile of the TiO2 peak height) (left； by ordinary confocal system ('a' of Figure 5)， r ight； the JASCO DSF system ('b’of Figure 5 The si n g l e apertu r e con f oca l system an d t h e D S F s y ste m w ere appl i ed t o t h e non -d e stru c tive Z sca n of a polyvinyl-a lcohol (PVA) la y e r o f a po lari zer f i l m to o bse r ve t h e c h ang e in t h e R am a n pe a k i n tensit y o f a n iodide compo u n d (25 u m th i c k n ess ) san dwi che d b etw ee n the P VA la y e r s. (F ig ur e 8) Th eo r eti ca l ly， th e i n tens i ty of t he i od id e c omp o u nd pe a k s ho u ld rapi d l y in c rea s e w hen re ac hin g the i od i d e c o m p o u nd laye r then r a p i dly decrease w hen again reach in g the PVA laye r. T h is is show n by the blue d as h ed l i ne in t h e Raman i n ten si t y pr ofi le di s pl a y e d as Fi g ure 8. T h e re su lt s de m o n st rate th a t the pro f i le u sin g the D SF o p ti cs (sol i d red l in e i n Fi g ur e 8) is c lose r t o th e th eo re ti c al p rof ile th a n th e s ta n d ar d co n focal syste m (so l i d gr e en l i n e i n F i gu r e 8). This c l ea rly i nd icate s th at t h e D S F o p tical s y ste m ca n greatly i mprove the Z r esolu ti on of a co n focal optical system. Conclusion A s o u tli n ed a b ove， th e N R S-5000/7000 of f ers a h ig h er s p a ti a l reso lu tion c l ose r to the th eoretica l dif f rac t i o n limit . Also，the ma xi m u m pe rfo rm a nce i n a co n fo c al opt ical s y s t e m ca n b e achie v ed us ing th e D S F op t i ca l a rra ngem e n t. I f hi gh spa t ia l resolut i on i s n ot requ ir ed fo r spe c ific expe r i me n ts， a l a rger pin h o l e apert u re can be used t o p rov i de in creased energy for b etter s i g n a l-t o-n oise r a tio res u lts. Note The me a s u r e me n t p o s i ti o n i n the Z s ca n be c om e s s h a ll o wer (t hin n e r) th a n th e a ctu al s amp le p os i ti o n (th i c k n e s s ) d ue t o the in fl ue n c e o f the re fr a c tive in de x of th e su b s t ra t e. In th e c a se of a 25 u m th ick ness o f the i o di d e c o m p o u n d l a y er ， th e apparen t th i c k n ess w ill b e approximately 15.3 um w h e n the re f ractive in dex i s 1.64. In the NRS-5000/7000， a [Ref r active In dex Co r r ecti o n ] fun ct i o n i s a v a il abl e in the mapp in g data a n al y sis pr og r a m s o tha t a co rrec t i on fo r t h e d if fe r e n ce b e tween th e tru e an d m eas u re d va lue s c an b e pe r f o rmed References 1) T . Wi l son and C . J. R . Sh e ppard： Theory and Practice of S can nin g Opti c a l Mi c ros c opy， Acad e m i c P ress (1984) 2) G. Kino： Co n foc a l Sc a n ning Optica l Mi c roscop y a n d Re l a t ed Im a g i ng System s， Academ i c Pr ess (1996) 3) C o n fo c al Laser S ca n nin g M ic r osco py P rinci p les ： ZE ISS 4) H. Ku bota： Hado-K ougak u (w ritten in Japa n ese ) (1971) 5) N eil J. Eve ra l l ， Applied Spectros c o p y， Vol. 54， Issue 6， p p. 773-782 (2000) JASCO INC. 28600 Mary's Cour t ， E asto n ， MD 21601US A T el ： (800) 333-5272， Fa x ： (410) 822-7526 A ppli c at io n Li brary： htt p ：//www.j as c o i n c .co m/applica t ion s