ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT. financed by the program

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1 TÁMOP C-13/1/KONV projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program keretében finanszírozott ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT financed by the program Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities Dátum / Date: OKTÓBER 11. / OCTOBER 11, 2017 Helyszín / Place: MTA SZBK BIOFIZIKAI INTÉZET, TANÁCSTEREM / LECTURE ROOM, INST. OF BIOPHYSICS, BIOLOGICAL RESEARCH CENTRE SZEGED, TEMESVÁRI KRT. 62. Az előadás címe / Title of the presentation: OPTICAL SPECTROSCOPY TOOLS TO INVESTIGATE THE MOLECULAR ORGANIZATION OF PROTEIN COMPLEXES Előadó / Speaker: PETAR LAMBREV Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB

2 Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMOP C-13/1/KONV Optical Spectroscopy Tools to Investigate the Molecular Organization of Protein Complexes Petar Lambrev Institute of Plant Biology October 11, 2017 OUTLINE T h e e s s e n c e o f o p t i c a l s p e c t r o s c o p y P h o t o s y n t h e t i c m e m b r a n e p r o t e i n c o m p l e x e s L i g h t-h a r v e s t i n g c o m p l e x e s R e a c t i o n c e n t e r s B a s i c t h e o r e t i c a l a s p e c t s M o l e c u l a r e x c i t e d s t a t e s a n d o p t i c a l t r a n s i t i o n s E x c i t o ni n t e r a c t i o n s a n d e n e r g y t r a n s f e r P o l a r i z e d l i g h t s p e c t r o s c Co D p, y L D, A C D, F P T i m e -r e s o l v e d s p e c t r o s c o p y U l t r a f a s t t r a n s i e n t a b s o r p t i o n s p e c t r o s c o p y T i m e -r e s o l v e d f l u o r e s c e n c e s p e c t r o s c o p y THE ESSENCE OF SPECTROSCOPY p r o t e i n 1. F i n d a n o b j e c t o f i n t e r e s t l i g h t 2. S e n d a b e a m o f e l e c t r o m a g n e t i c r a d i a t i o n t o t h e o b j e c t 3. O b s e r v e t h e o u t c o m i n g r a d i a t i o ln i g h t 4. L e a r n s o m e t h i n g a b o u t t h e o b jp er co t e i n 1

3 THE ELECTROMAGNETIC SPECTRUM INTERACTIONS OF EM RADIATION WITH MATTER Absorption Emission Reflection U V / V I S s p e c t r o s c o p y p r o b e s e l e c t r o n i c e x c i t e d s t a t e s e l e c t r o n i c s p e c t r o s c o p y I R s p e c t r o s c o p y p r o b e s m o l e c u l a r v i b r a t i o n s v i b r a t i o n a l s p e c t r o s c o p y PHOTOSYNTHETIC ANTENNA COMPLEXES g r e e n s u l f u r b a c t e r i a p u r p l e b a c t e r i a c y a n o b a c t e r i a d i a t o m s c h l o r o s o m e s d i n o f l a g e l l a t e s L H 2 p h y c o b i l i s o m e s p l a n t s F u c o x a n t h i n- c h l o r o p h y l-p l r o t e i n s ( F C P ) P C P L H C 2

4 PHOTOSYNTHETIC PIGMENTS Chlorophyll a Carotenoid (β-carotene) C h l o r o p h y l l s a n d b a c t e r i o c h l o r o p h y l l s m a j o r p h o t o s e n s i t i v e p i g m e n t s h e t e r o c y c l i c m a c r o c y c l e p l a n a r r i n g o f c o n j u g a t-b eod n d s c e n t r a l M g a t o m d i f f e r e n t t y p e s d e p e n d i n g o n s u b s t i t u t e n t s C h l s a b s o r b b l u e ( n m ) a n d r e d ( n m ) l i g h t C a r o t e n o i d s x a n t h o p h y l l s O - c o n t a i n i n g c a r o t e n o i d s l i n e a r c h a i n o f c o n j u g a t e d C = C b o n d s 7 PHOTOSYNTHETIC PIGMENT MOLECULES The Emerson & Arnold experiment: P r i m a r y p h o t o c h e m i s t r y o n l y t a k e s p l a c e i n r e a c t i o n c e n pt ie gr m e n t s M a j o r i t y o f p i g m e n t s d o n o t p e r f o r m p h o t o c h e m i s t r y t h e y a r e p a r t l oi f g h-t h a r v e s t i n g a n t e n n a c o m p l e x e s L H A s d e l i v e r a b s o r b e d l i g h t e n e r g y t o R C v i a e x c i t a t i o n e n e r g y t r a n s f e r A t s a t u r a t i n g i n t e n s i t i e s, 2 o n e O m o l e c u l e i s p r o d u c e d p e r C h l s q u a n t a a r e u s e d p e2 r O a t l e a s t 4 b y e a c h p h o t o s y s t e m s e v e r a l h u n d r e d m o l e c u l e s a s s o c i a t e d w i t h e a c h p h o t o s y s t e m r e a c t i o n c e n t e r 8 THE FUNNEL CONCEPT Energy b l u e-a b s o r b i n g g r e e n-a b s o r b i n g o r a n g e-a b s o r b i n g r e d-a b s o r b i n g R C E n e r g y i s p r e f e r e n t i a l l y t r a n s f e r r e d d o w n h i l l f r o m h i g h e r-e n e r g y s t a t e s t o l o w e-e r n e r g y o n e s H i g h e r-e n e r g y-a b s o r b i n g p i g m e n t s a r e l o c a t e d i n t h e p e r i p h e r a l L H A s L o w e r -e n e r g y-a b s o r b i n g p i g m e n t s a r e l o c a t e d c l o s e r t o t h e R C c o r e E n s u r i n g f a s t d i r e c t i o n a l t r a n s f e r t o w a r d s t h e R C A c c o r d i n g t o t h e tz B omla n n d i s t r i b u t i o n, i n t h e t h e r m a l l y e q u i l i b r a t e d a n t e n n a l o-e wne er r g y s t a t e s h a v e h i g h e r p o p u l a t i o n 9 3

5 ENERGY FUNNELS IN PHOTOSYNTHETIC ORGANISMS P u r p l e b a c t e r i a C y a n o b a c t e r i a LIGHT-HARVESTING COMPLEXES IN PLANTS Light-harvesting complex II T h e L H C s u p e r f a m i l y I n t e g r a l m e m b r a n e p r o t e i n s 3 o r 4 t r a n s m e m b r a n e h e l i c e s c h l o r o p h y l l s a s m a i n p i g m e n t s 2-4 x a n t h o p h y l l sa s a c c e s s o r y p i g m e n t s M o n o m e r i c o r o l i g o m e r i c T h e p r o t e i n d e t e r m i n e s t h e p i g m e n t s o p t i c a l p r o p e r t i e s D y n a m i c r e g u l a t i o n o f t h e l i g h t h a r v e s t i n g f u n c t i o n LIGHT-HARVESTING COMPLEXES IN PLANTS L h c b 1 ( f o r m s t r i m e r s ) L h c b 3 ( m o n o m e r i c ) 4

6 LHCII TRIMERS C h l s i n L H C I I a r e r o u g h l y a r r a n g e d i n r i n g s, o p t i m i z i n g l i g h t a b s o r p t i o n f r o m a l l d i r e c t i o n s a n d e n e r g y t r a n s f e r T h e e n e r g i e s o f t h e p i g m e n t s i t e s v a r y d u e t o t h e p r o t e i n e n v i r o n m e n t a n d e x c i t o n i c i n t e r a c t i o n s T h i s c r e a t e s a w i d e r a b s o r p t i o n b a n d m o r e e f f i c i e n t l-i g h t h a r v e s t i n g L o w e s t e n e r g y p i g m e n t s l o c a t e d i n t h e p e r i p h e r y o f t h e c o m p l e x t r a n s f e r e n e r g y a w a y. PHOTOSYSTEM II CORE top side s u b u n i t s D 1 (P s b A ) D 2 (P s b D ) C y t b (P s b E / F ) A c c e s s o r y s u b u n i t s (P s b F -P s b Z ) C P 4 3 (P s b B ) C P 4 7 (P s b C ) W O C ( P s b O, P s b U, P s b V ) 3 5 C h l o r o p h y l l a 2 P h e o p h y t i n 1 1 β -c a r o t e n e 2 P l a s t o q u i n o n e 2 H e m e F e 1 N o n -h e m e F e 4 M n, 3-4 C a, 3 C l, H3 C, O l i p i d s PHOTOSYSTEM II SUPERCOMPLEXES C 2 S 2 C 2 S 2 M 2 Nield & Barber, BBA, 2012, 1757: Pagliano et al., BBA, 2013, /j.bbabio

7 PSII REACTION CENTER ELECTRON-TRANSPORT CHAIN Müh & Zouni, Front. Biosci., 2011, 16: PHOTOSYNTHETIC PROTEIN COMPLEXES SUMMARY P r o t e i n s a c t as m a r t s c a f f o l d t h a t b i n d a l a r g e n u m b e r o f -a l ibgsho tr b i n g p i g m e n t s d y n a m i c a l l y c o n t r o l t h e p r o p e r t i e s o f t h e b o u n d p i g m e n t s T h e s a m e p i g m e n t m o l e c u l e s c a n h a dv ie f fa e r e n t f u n c t i o n d e p e n d i n g o n t h e p r o t e i n e n v i r o n m e n t L i g h t-h a r v e s t i n g ( i n a n t e n n a p r o t e i n s ) L i g h t-d i s s i p a t i o n ( i n a n t e n n a p r o t e i n s u n d e r h i g h l i g h t ) P h o t o c h e m i c a l r e a c t i o n ( i n r e a c t i o n c e n t e r p r o t e i n s ) M i n i s c u l e c h a n g e s pirn o t e i n c o n f o r m a t i o n c a n c h a n g e t h e p i g m e n t f u n c t i o n s T h e p h o t o p h y s i c a l a n d p h o t o c h e m i c a l r e a c t i ou nl s t raarf e a s t 1 f s = s LIGHT AS A WAVE 6

8 POLARIZATION MOLECULAR ENERGY E t o t a l = E v i b r a t i o n a l + E e l e c t r o n i c E S S 0-0 S 0 -X e l e c t r o n i c g r o u n d s t a t e S 1 -X e l e c t r o n i c e x c i t e d s t a t e S X -0 v i b r a t i o n a l g r o u n d s t a t e S X -1 v i b r a t i o n a l e x c i t e d s t a t e ABSORPTION OF LIGHT A b s o r p t i o n o f U V / V I S l i g h t i s a m o l e c u l a r t r a n s i t i o n b e t w e e n t w o e l e c t r o n i c l e v e l s hυ S S 0-0 Dipole moment + 7

9 TYPES OF TRANSITIONS * C = C n * C = O ( v e r y w e a k ) d d F e, C u, M n, C o C o m p o u n d λ [ n m ] ε [ M 1 c m -N H -C O - ( * ) N H -C O - (n * ) T r p T y r N A D H C h l a β -c a r o t e n e ] TIME OF THE TRANSITION DECAY OF THE EXCITED STATE Perrin-Jablonski diagram F l u o r e s c e n c e E n e r g y t r a n s f e r I n t e r n a l c o n v e r s i o n E l e c t r o n t r a n s f e r V i b r a t i o n a l r e l a x a t i o n I n t e r s y s t e m c r o s s i n g P h o s p h o r e s c e n c e D e l a y e d f l u o r e s c e n c e 8

10 ABSORPTION AND EMISSION SPECTRA K a s h a -V a v i l o vr u l e : T h e f l u o r e s c e n c e e m i s s i o n i s i n d e p e n d e n t f r o m e x c i t a t i o n w a v e l e n g t h K a s h a s r u l e ( r e p h r a s e d ) : F l u o r e s c e n c e i s e m i t t e d f r o m t h e l o w e s t e l e c t r o n iec x c i t e d l e v e l M i r r o r-i m a g e r u l e F r a n c k-c o n d o n f a c t o r s E x c e p t i o n s t o t h e m i r r o r i m a g e r u l e RATE CONSTANTS, LIFETIMES AND YIELDS INTERMOLECULAR INTERACTIONS I o n i c I o n -i o n i n t e r a c t i o n s I o n -d i p o l e i n t e r a c t i o n s V a n d e r W a a l s D i p o l e- d i p o l e i n t e r a c t i o n s D i s p e r s i o n i n t e r a c t i o n s D e b y e d i s p e r s i o n i n t e r a c t i o n s L o n d o n d i s p e r s i o n i n t e r a c t i o n s 9

11 THE EXCITONIC DIMER molecule 1 dimer transition dipole moments z + molecule 2 y 2 E 2V E a+v E a V E a 1 x Absorption dimer monomer υ 0a V υ 0a υ 0a+V Monomer and dimer energies Interaction energy (point-dipole approximation) FÖRSTER ENERGY TRANSFER S S 0 A* B A B* THEORETICAL ASPECTS - SUMMARY O p t i c a l s p e c t r o s c o p y ( -m l iagthtt e r i n t e r a c t i o n s ) i s u n d e r s t o o d b y t h e t h e o r y o f q u a n t u m e l e c t r o d y n a m i c s L i g h t i s p o r t r a y e d aes l ea cn t r o m a g n e t i c w a v e M a t t e r i s p o r t r a y e d a s a s e t o f m oqluea cnutluam r e i g e n s t a t e s T h e e l e c t r o m a g n e t i c f i e l d i s c o u tp rl ae nd s ti o t i o n sb e t w e e n e l e c t r o n i c a n d v i b r a t i o n a l e i g e n s t a t e s A b s o r p t i o n a n d f l u o r e s c e n c ee m i s s i o n s p e c t r a r e v e a l i n f o r m a t i o n a b o u t t h e m o l e c u l a r s t a t e s E x c i t e d s t a t e s d e c avy i a r a d i a t i v e a n d -rn ao dn i a t i v e p a t h w a y s T h e e x c i t e d s t a t e r e a c t i o n p a t h w a y s a r e c h a r a c trea rtie z ec d o nbsy t a n t s T h e f l u o r e s c e n c e l i f e t i m e s a n d y i e lr de s v e a l i n f o r m a t i o n a b o u t t h e e -x c i t e d s t a t e d y n a m i c s. D i p o l e -d i p o l e i n t e r a c t i o nbs e t w e e n m o l e c u l e s c r e a t e n e w, s h a r e d e x c i t o n s t a t e s D i p o l e-d i p o l e i n t e r a c t i o n s a r e t h e beans eirs g y o f t r a n s f e r 10

12 WHAT IS POLARIZED LIGHT SPECTROSCOPY? A b s o r p t i o n P o l a r i z e d a b s o r p t i o n L i n e a r d i c h r o i s m C i r c u l a dr i c h r o i s m M a g n e t i c C D A n i s o t r o p i Cc D C h l o r o s o m e s C h l o r o p l a s t s T h y l a k o i d m e m b r a n e s L H C I I F l u o r e s c e n c e F l u o r e s c e n c e p o l a r i z a t i o n F l u o r e s c e n c e a n i s o t r o p y F l u o r e s c e n c-d e e t e c t e d L D / C D 3 1 LINEAR DICHROISM T h e d i f f e r e n c e i n a b s o r p t i o n o f l i g h t p o l a r i z e d p a r a l l e l a n d p e r p e n d i c u l a r t o a n o r i e n t a t i o n a x i s. L D g i v e s d i r e c t s t r u c t u r a l i n f o r m a t i o n, b e c a u s e i t d e p e n d s o n t h e a n g l e o f t h e t r a n s i t i o n d i p o l e m o m e n t LD / 3A θ - a n g l e b e t w e e n t h e t r a n s i t i o n d i p o l e m o m e n t a n d m a i n s y m m e t r y a x i s Angle, 3 2 SAMPLE ORIENTATION (ALIGNMENT) Magnetic orientation Orientation by gel squeezing 11

13 ORIENTATION OF MEMBRANES Face-aligned orientation should preferentially excite transitions in the membrane plane Edge-aligned orientation shows transitions primarily perpendicular to the membrane. However the linear anisotropy generates LD and distorts the CD CIRCULAR DICHROISM T h e d i f f e r e n c e i n a b s o r p t i o n -oaf n d l e f t r i g h t-h a n d e d c i r c u l a r l y p o l a r i z e d l i g h t. I n t r i n s i c C D c h i r a l m o l e c u l e s E x c i t o n i c C D d i p o l e i n t e r a c t i o n s b e t w e e n p i g m e n t s P s i-t y p e C D l o n g-r a n g e i n t e r a c t i o n s i n o r d e r e d p i g m e n t e n s e m b l e s 3 5 THE ORIGIN OF INTRINSIC CD R o s e n f e l d qeu a t i o n: CD Im μ m μ m e l e c t r i c d i p o l e m o m e n t m a g n e t i c d i p o l e m o m e n t P u r e e l e c t r i c a b s o r p t i o n P u r e m a g n e t i c a b s o r p t i o n ( c u r r e n t l o o p ) O p t i c a l a c t i v i t y C a n t o r C. R. S& c h i m m e l P. R., B i o p h y s i c a l C h e m i s t r y, , F r e e m a n & C o. 12

14 EXCITONIC CD (+) CD (-) CD ν T h e t w o e x c i t o n t r a n s i t i o n s i o f t h e d i m e r a n d h a v e C D o f e q u a l m a g n i t u d e b u t o p p o s i t e s i g n T h e C D o f t h e d i m e r i s t h e s u m o f t h e C D o f t h e t w o t r a n s i t i o n s T h e C D i s n o n z e r o b e c a u s e o f t h e e x c i t o n e n e r g y s p l i t T h e m o n o m e r m o l e c u l e s d o n o t n e e d t o b e c h i r a l T h e C D s t r o n g l y d e p e n d s o n t h e g e o m e t r y o f t h e d i m e r MEASURING LD AND CD P h o t o e l a s t imco d u l a t o r : p o l a r i z a t i o n i s c o n t r o l l e d b y p h a s e s h i f t i n g C D / L D i s m e a s u r e d a s t h e a m p l i t u d e o f t h e m o d u l a t e d s i g n a l CD OF CHLOROPLAST MEMBRANES L o n g -r a n g e p i g m e n t- p i g m e n t i n t e r a c t i o n s i n t h e m e m b r a n e m a c r o s t r u c t u r e p r o d u c e p s i-t y p e C D P s i-t y p e C D i s s e n s i t i v e t o t h e m a c r o s t r u c t u r a l o r g a n i z a t i o n D i s r u p t i o n o f t h e l-r o an ng g e i n t e r a c t i o n s r e v e a l s t h e e x c i t o n i c C D o f p i g m-e n t p r o t e i n s C D s p e c t r a o f s t a c k e d a n d w a s h e d t h y l a k o i d m e m b r a n e s 13

15 CD OF INTACT PLANT LEAVES WT C 2 S 2 M 2 CD kocp24 C 2 S 2 Wavelength (nm) CD OF ISOLATED LHCII T h e C D s p e c t r a d e t eo cl t i g o m e r i z a t i oann d c h a n g e s i n t h e m o l e c u l a r e n v i r o n m e n t TIME-RESOLVED SPECTROSCOPY 14

16 TIME-RESOLVED SPECTROSCOPY I n f o r m a t i o n a b o u t f a s t a n d u l t r a f a s t p r o c e s s e s i n t h e s y s t e m e x c i t e d-s t a t e r e a c t i o n s, e t c. T h e s y s t e m i s p e r t u r b e d b y a v e r y s h o r t l a s e r p u l s e a n d t h e t i m e e v o l u t i o n o f t h e s y s t e m s p r o p e r t i e s a f t e r t h e p u l s e i s f o l l o w e d K i n e t i c p r o f i l e : c a n r e s o l v e m u l t i -l p lie v esd h oirntt e r m e d i a t e r e a c t i o n s t a t e s, t h e i r s p e c t r a l p r o p e r t i e s, t r a n s i e n t c o n c e n t r a t i o n s, r e a c t i o n r a t e c o n s t a n t s, e t c. T e m p o r a l r e s o l u t i o n d o w n t o 1 f s = s T e m p o r a l r e s o l u t i o n a n d s p e c t r a l r e s o l u t i o n a r e r e l a t e d b y t h e u n c e r t a i n t y p r i n c i p l e ( s h o r t p u l s e s h a v e b r o a d s p e c t r a l w i d t h ) PUMP-PROBE TRANSIENT ABSORPTION S2 S1 p u m p p r o b e P u m p p u l s e c r e a t e s e x c i t e d s t a t e s ( G S S 1 ) A s u b s e q u e n t p r o b e p u l s e ( S 1 S n ) m e a s u r e s t h e c h a n g e s i n d u c e d b y t h e p u m p T h e t e m p o r a l e v o l u t i o n i s f o l l o w e d b y s c a n n i n g o v e r t h e t i m e b e t w e e n p u m p a n d p r o b e T e m p o r a l r e s o l u t i o n i s o n l y l i m i t e d b y t h e p u l s e d u r a t i o n GS D i f f e r e n t i a l a b s o r p t i o n : Δ A ( t ) = A + p u m p A p u m p PUMP-PROBE TRANSIENT ABSORPTION 15

17 TRANSIENT ABSORPTION OF PHOTOSYNTHETIC ANTENNA COMPLEXES Active LH1 complexes Energy-dissipating LH1 complexes T y p e s o f t r a n s i e n t a b s o r p t i o n s i g n a l s : N e g a t i v e A d u e t o l o s s ( b l e a c h i n g ) o f g r o u n d s t a t e s N e g a t i v e A d u e t o e m i s s i o n f r o m e x c i t e d s t a t e s P o s i t i v e A d u e t o a b s o r p t i o n b y e x c i t e d s t a t e s 4 6 TIME-RESOLVED FLUORESCENCE A F τ f t F l u o r e s c e n c e l i f e t i m e : A b s o l u t e v a l u e I n d e p e n d e n t o n c o n c e n t r a t i o n I n s e n s i t i v e t o a r t i f a c t s M u l t i p l e l i f e t i m e s : H e t e r o g e n e i t y T r u e d y n a m i c s INFORMATION FROM LIFETIME MEASUREMENTS F l u o r o p h o r e e n v i r o n m e n t M u l t i p l e c o n f o r m a t i o n s, c o n f o r m a t i o n a l c h a n g e s M u l t i p l e e n v i r o n m e n t s I n t e r a c t i o n s w i t h n e i g h b o u r i n g r e s i d u e s S o l v e n t r e l a x a t i o n F l u o r e s c e n c e l i f e t i m e s e n s o 2 r + s, ( MCg 2 a + ) R e s o n a n c e e n e r g y t r a n s f e r Lakowicz J.R. (2006) Springer 16

18 TRF QUENCHING T R F c a n d i s t i n g u i s h b e t w e e n d y n a m i c q u e n c h i n g ( c o l l i s i o n a l q u e n c h i nlg i) f e t i m e d e c r e a s e w i t h q u e n c h e r c o n c e n t r a t i o n s t a t i c q u e n c h i (e n g x c i p l e xf o r m a t i o n ) l i f e t i m e i s u n c h a n g e d, a m p l i t u d e d e c r e a s e s T R F c a n d i s t i n g u i s h d i f f eq ru e nt c h e d p o p u l a t i o n s Lakowicz J.R. (2006) Springer DECAY-ASSOCIATED EMISSION SPECTRA k AB = 5 ns A* -1 B* 0.5 ns ns -1 A B METHODOLOGY FOR TRF SPECTROSCOPY D i r e c t F r e q u e n c y- d o m a i n ( C W ) T i m e -d o m a i n ( p u l s e d ) G a t i n g P h a s e m o d u l a t i o n T C S P C S t r e a k c a m e r a U p c o n v e r s i o n T C S P C i s t h e m o s t v e r s a t i l e a n d c o m m o n l y u s e d t e c h n i q u e C a n r e s o l v e l i f e t i m e s f r o m f e w μsp s t o H i g h d y n a m i c r a n g e a n d s i g-t n o-n a l o i s e r a t i o N o w a f f o r d a b l e a n d a c c e s s i b l e t-so p ne oc ni a l i s t u s e r s 17

19 TIME-CORRELATED SINGLE-PHOTON COUNTING threshold Reference pulses from light source Range CFD start zero cross Gain Mem ory Histogram threshold TAC AMP ADC Address (tim e) Pream plifier stop Detector Singlephoton CFD zero cross Offset = control elements data +1 Adder pulses T i m e -t o-a m p l i t u d e c o n v e r s i o n : 1. T h e l a s e r p u l s e s t a r t s a c l o c k 2. T h e d e t e c t e d f l u o r e s c e n c e p h o t o n s t o p s t h e c l o c k 3. T h e t i m e b e t w e e n t h e S t a r t a n d S t o p s i g n a l s i s r e c o r d e d TIME-CORRELATED SINGLE-PHOTON COUNTING Detector Signal: Period 1 Period 2 Period 3 Period 4 Period 5 Period 6 Period 7 Period 8 Period 9 Period 10 Original Waveform (Distribution of photon probability) Time 4. A f t e r m a n y s i n g l e p h o t o n e v e n t s a h i s t o g r a m o f d e c a y t i m e s i s c o l l e c t e d 5. T h i s h i s t o g r a m i s t h e f l u o r e s c e n c e d e c a y k i n e t i c s Period N Result after many Photons OPTICAL SPECTROSCOPY - SUMMARY P o l a r i z e d l i g h t s p e c t r o s c o p y i s a v a l u a b l e t o o l t o m o n i t o r t h e m o l e c u l a r s t r u c t u r e o f p r o t e i n c o m p l e x e s L D r e v e a l s t h e o r i e n t a t i o n o f c h r o m o p h o r e s i n t h e p r o t e i n m a t r i x C D i s s e n s i t i v e t o s-r hao nrgt e i n t e r a c t i o n s o f c h r o m o p h o r e s i n t h e p r o t e i n a n d l o-r n ga n g e i n t e r a c t i o n s i n p r o t e i n m a c r o a s s e m b l i e s T i m e -r e s o l v e d s p e c t r o s c o p y r e v e a l s e-sx tc ai t e d r e a c t i o n d y n a m i c s T r a n s i e n t a b s o r p t i o n c a n m e a s u r e u l t r a f a s t t r a n s i t i o n s b e t w e e n e x c i t e d s t a t e s, e v e n n o n r a d i a t i v e o n e s T i m e -r e s o l v e d f l u o r e s c e n c e m e a s u r e s d i r e c t l y e m i s s i v e e x c i t e d s t a t e s a n d e x c i t e-s d t a t e l i f e t i m e s w i t h u n p a r a l l e l e d p r e c i s i o n, s e n s i t i v i t y a n d d y n a m i c r a n g e 18

20 THANK YOU FOR YOUR ATTENTION! This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities " TÁMOP C-13/1/KONV project. 19