Femtosecond resolved absorption spectroscopy

P R E S E N T A T I O N B Y : R O S H N I B A N O

2 8 T H O C T O B E R , 2 0 1 1

General use of absorption spectroscopy

Determine absorption spectra of photoactive agents

Characterise excited states, reactive intermediates and photoproducts

Sequence of events, rates of occurrence and factors influencing rates

- Ground state absorption – for stable

(1ms) species, CW light

- Transient absorption – for metastable

- species (fs to ks), pulsed laser + probe

source (CW or laser)

Picture from : http://www.photobiology.info/Nonell_Viappiani.html

Time resolved absorption spectroscopy

Steady state spectroscopy – continuous irradiation, continuous creation of excited states and finally steady state

Time resolved spectroscopy – light source’s intensity fluctuates in time, burst of excited states (only a fraction of them excited), monitor time evolution

-- fourier transform limited, therefore time resolution sacrificed for spectral resolution

Femtosecond time resolution

Timescale of electronic transitions in most photochemical/photophysical processes

Large bandwidth for small pulses

Such sources are prepared using “mode locking”

Can be used to generate probe pulses by chirping

1mJ pulse with 100fs duration – 10 GW !

Picture from :(Lecture notes) Basics of femtosecond laser spectroscopy, Mikhailovsky, UCSB

The pump probe approach

Excitation with a pump laser beam (pulse duration shorter than time constant of reaction)

Sample interrogated with probe before and after excitation Can monitor absorbance change at given wavelength or at several

wavelengths Can delay probe pulse wrt pump pulse for studying time evolution Study disappearance of excited states, formation of reactive

intermediates and photoproducts

Picture from : Berera, PhotosynthResearch, 2009

Picture from : http://os.tnw.utwente.nl/images_new/proj40_2.jpg

Some applications of fs-resolved spectroscopy

Novel spectral features may lead to discovery of reaction intermediates

Figure out reaction mechanisms Rate constant of each kinetic step Temperature dependence, for

example, activation parameters

Picture from : Introduction to femtosecond laser spectroscopy and ultrafast XRD , Wolf, Freie Universitat Berlin

Protein found in membrane of archaean that lives in salt marshes

Aerobic – makes ATP through ETS, Anaerobic – makes bacteriorhodopsinbR and harvests light to set up a proton gradient

Light absorbing chromophore – retinal (polyene compound) linked that isomerises from all-trans to 13-cis form and starts a thermal cascade

In methanol solution, all-trans goes mostly to 11-cis with quantum efficiency of 0.15. In bR, goes to 13-cis with quantum efficiency of 0.6 Stored energy is used to pump protons across the membrane!

Highly specific, quick isomerisation – how?

The model before this paper came…

Photoexcitation triggers ultrafast movement towards excited state minimum (“twisted conformation”)

Twisted conformation can go to all-trans or 13-cis

Ultrafast torsional motion must be

along a repulsive potential in the Franck Condon region

● Contrasts with retinal in solution where FC region = minimum of excited state due to symmetry across C13-C14 bond

● Repulsive potential can result only if protein shifts ground and excited state landscapes wrt each other

But bR mutants have quantum efficiencies similar to bR, even with different excited state lifetimes!

Stimulated emission dynamics

If the FC region of excited potential energy surface is repulsive, can monitor time dependence of stimulated emission. Will redshift as photoexcited retinal progresses towards minimum of surface.

(LEFT) Stimulated emission and excited state absorbance decay, ground state bleach recovers, photoproduct grows. (RIGHT) No red shift in stimulated emission with time !

Excited state dynamics

Both stimulated emission and excited state absorbance come from the same state, therefore dynamics should be the same.

Modelled with biexponential –fast and slow decay components - 370 fs (87%) and 2.1 ps(13%) – indicates multiple species.

-- Since stimulated emission spectra are identical at different delay times, the kinetically different populations are spectroscopically similar.

Average rise time – 10 fs

The missing stimulated emission !

Integrated stimulated emission should be comparable to integrated ground state bleach, but isn’t.

Subtract ground state bleaching and stimulated emission to get only excited state absorption

The stimulated emission is masked by uncharacterisedpositive going absorbance, and hence seems to be lower!

The final three state model

Rise time of stimulated emission (10 fs) suggests flat, not repulsive FC region

Two avoided crossing regions (marked by red arrows)

S1 and S2 strongly overlap – affects crossing of second avoided region

Non exponential relaxation due to conformational heterogeneity of S0

which modulates the barrier leading to reactive surface of S1

(encircled)

In conclusion, protein does not play an important role in photo-isomerisation; just catalyses conversion along C13-C14 bond and inhibits that along others. It is the intrinsic electronic energy landscape of retinal which leads to high efficiency photoisomerisation.

Thank you!

Acknowledgements

Images in first slide from :- (Left) http://sherwingroup.itst.ucsb.edu/img/image006.gif

- (Right) Textbook of Physical Chemistry, P W Atkins

Useful readings :

- http://www.photobiology.info/Nonell_Viappiani.html

- (Lecture notes) Basics of femtosecond laser spectroscopy, Mikhailovsky, UCSB

- (Lecture notes) Introduction to femtosecond laser spectroscopy and ultrafast XRD , Wolf, Freie Universitat Berlin

- (Lecture notes) Time resolved absorption spectroscopy, Penzkofer

- Ultrafast transient absorption spectroscopy : principles and application to photosynthetic systems, Berera et. al., Photosynth Res (2009), 101 : 105-118

Cool Youtube video on femtosecond pump-probe spectroscopywww.youtube.com/watch?v=mdNr6eVBJqk