3.4 Experimental setup
To perform fluorescence microscopy and fluorescence-excitation spectroscopy the samples were illuminated with a continuous-wave tunable Titanium-Sapphire (Ti:Sa) laser (3900S, Spectra Physics, Mountain View CA, USA) pumped by a frequency doubled continuous-wave Neody-nium-Yttrium-Vanadat (Nd:YVO4) laser (Millennia Vs, Spectra Physics, Mountain View CA, USA) using a home build microscope that can be operated either in widefield or confocal mode.
To obtain a well-defined variation of the wavelength of the Ti:Sa laser the intracavity bire-fringent filter has been rotated with a motorised micrometer screw (MM4005 and 850f, New-port, Irvine CA, USA). For calibration purposes a wavemeter (Wavemeter jr., EXFO Burleigh Products Group, Victor NY, USA) has been used and an accuracy as well as a reproducibil-ity of 1 cm-1 for the laser frequency have been verified. A fluorescence-excitation spectrum of an individual light-harvesting complex was obtained in two steps. First a widefield image of the sample was taken and a spatially well-isolated complex was chosen. Subsequently a fluorescence-excitation spectrum of this complex was obtained by scanning the laser and simul-taneously collecting the fluorescence.
Widefield fluorescence microscopy
A 40×40µm2 wide-field image of the sample was taken by exciting the sample through a simple planoconvex lens with a large focal length (f = 140mm) creating a∼ 100×100µm2 spot on the film (Fig. 3.2A). Thereby, the excitation wavelength was chosen to coincide with an absorption maximum of the complexes (800 nm for LH2 and 870 nm for LH1-RC) whereas the emitted light was collected by an objective lens (single aspheric lens,f =1.45 mm, NA= 0.55, Thorlabs, Newton NJ, USA) mounted inside the cryostat immersed in liquid helium and focused onto a back-illuminated CCD camera (512 SB, Roper Scientific, Trenton NJ, USA) after passing suitable bandpass filters (∆λ ≈ 20nm, Dr. Hugo Anders, AHF Analysentech-nik, T¨ubingen, Germany) which blocked the residual laser light. The 3D-representation of the fluorescence — as depicted in the lower part of Fig. 3.2A — shows several peaks each repre-senting the diffraction limited airy pattern of individual LH complexes. The lateral resolution was determined to 1µm corresponding to the theoretical expected resolution using the Rayleigh criterion (e.g., [142]). From this image a spatially well-isolated complex was selected.
Nd:YVO4
Figure 3.2: Experimental setup for low-temperature single molecule spectroscopy. (A) Widefield arrangement of the microscope. In the lower part a 3D-representation of an example image is shown in which each peak corresponds to the diffraction limited image of an individual LH complex. (B) Confocal mode of the microscope.
A typical fluorescence-excitation spectrum of an individual LH2 complex fromRs. molischianumis shown in the lower part. See text for more details.
Confocal fluorescence-excitation spectroscopy
A fluorescence-excitation spectrum of this complex was obtained by switching to the confocal mode of the set-up (Fig. 3.2B). In this mode the excitation light was passed through an exci-tation pinhole and focused onto the sample by the objective lens inside the cryostat creating a diffraction limited excitation volume of less than 1µm3. This confocal volume was made to coincide with the complex by tilting the direction of the excitation beam with the scan mirror.
A pair of telecentric lenses ensured a precise and well controlled displacement of the focus on the sample while maintaining alignment with the confocal aperture.
The fluorescence was collected by the same objective lens and focused onto a single-photon counting avalanche photodiode (APD) (SPCM-AQR-16, Perkin Elmer Optoelectronics, Fre-mont CA, USA) which also fulfilled the role of the detection pinhole. Instead of obtaining a spectrum by slowly scanning the laser once, many spectra were recorded in rapid succession
3.4: Experimental setup 37 by scanning repetitively the spectral range of interest and storing the different traces separately.
Thereby, not only light-induced fluctuations of the fluorescence intensity on a timescale of sec-onds could be diminished as reported in [143] but also information about the spectral evolution in time could be gained. With a scan speed of the laser of 3 nm per second (≈ 50cm-1/s) and an acquisition time of 10 ms per data point, this yields a nominal resolution of 0.5 cm-1 ensur-ing that the linewidth is limited by the spectral bandwidth of the laser (1 cm-1). The confocal-detection mode features a superior background suppression that allowed to record fluorescence-excitation spectra with high signal-to-noise ratios as shown for instance in the lower part of Fig.
3.2B [118].
Widefield fluorescence-excitation spectroscopy
Due to the sequential character of the experiment confocal fluorescence-excitation spectroscopy is limited to the investigation of one to two complexes per day. Therefore, it would be advan-tageous to use a CCD camera instead of the APD in Fig. 3.2B to monitor the fluorescence in-tensity of many complexes simultaneously while scanning the frequency of the excitation light.
Consequently the spectra of as many complexes as can be imaged on the CCD are obtained si-multaneously as depicted in Fig. 3.3. Here, the CCD-frame number corresponds to wavelength
λ1
λ1λ2λ3λ4λ5λ6λ7
λ2 λ3 λ4 λ5 λ6 λ7 λ8
λ8
wavelength
intensity
CCD Frame
1 2 3 4 5 6 7 8
wavelength
x
x x x x x x x x
x x x x x x x
Figure 3.3:Widefield fluorescence-excitation spectroscopy. An EMCCD camera records the fluorescence intensity of a sample containing several individual complexes depicted by the differently bright dots. During the image acquisition, the wavelength of the laser is scanned. The spectra of the complexes can then easily be reconstructed as each frame corresponds to a certain wavelengthλi and the total fluorescence in this frame is obtained by integration over the image of the individual complexes.
and by integrating the total intensity of the fluorescence image of an individual complex on the CCD as a function of the read-out frame a fluorescence-excitation spectrum can be obtained.
In the past, basically two types of CCDs existed, (i) fast cameras with high read-out noise and (ii) slow cameras with low read-out noise. Fast cameras typically have signal-to-noise ratios (SNR) that is to poor for single LH spectroscopy whereas the slow cameras — as also used for the widefield microscopy — generally have very long read-out times. Nowadays, a new generation of CCD cameras, so call electron multiplying CCDs (EMCCD) exist that eliminate the read-out noise by amplifying the number of counts in a pixel directly on the CCD chip itself. The key feature of such cameras is that they combine high sensitivity and low noise without sacrificing fast read-out.
However, using similar SNR as a target criterion it can be stated that the cycle time (i.e., image acquisition time plus read-out time) of the EMCCD (DV465, Andor, Belfast Ireland) used in this thesis was still about a factor of 50 higher than of the APD in the confocal mode. This is due to the fact that no detection pinhole is present anymore so that the confocal setup is no longer fulfilled and stray light from out-of focus regions is imaged onto the CCD. Also the quantum efficiency of the CCD (15% at 900 nm) is a factor of 3 lower than for the APD (40%
at 900 nm). Furthermore, the read-out time of the APD is basically zero whereas the EMCCD still needs 100-150 ms per frame. Nevertheless, the new parallel detection scheme of widefield fluorescence-excitation spectroscopy which was used for the experiments described in chapter 6 enhanced the efficiency of the experimental work by a factor of 50 compared to confocal operation with an APD.
Polarisation dependent spectroscopy
To examine the polarisation dependence of the spectra, a 12λ plate was put in the confocal excitation path. This plate was rotated in steps of 0.9 degrees between two successive scans by which the angle of polarisation of the excitation light changes with twice this value. Limited by the signal noise, this allowed to determine polarisation angles to within 5◦accuracy.
Chapter 4
The B800 band of LH2 from Rhodospirillum molischianum
In this chapter, a study is presented on the B800 absorptions of individual LH2 complexes from Rhodospirillum molischianum, which are sensitive to the variations in the electronic coupling between individual BChlamolecules. The electronic coupling is determined by the interaction strengthV between the pigments and the energetic disorder, characterised by the width ∆of a Gaussian distribution of site energies. For the B800 band the ratioV /∆ 1is consistent with the picture that the chromophores feature a weak to intermediate coupling and that the excitations are mainly localised on individual chromophores.
The observations are also affected by apparent, temporal variations in the local environment of individual pigments. These can be caused by conformational fluctuations within the pro-tein scaffold. The embedded chromophores react on these fluctuations with changes of their electronic energies which makes them suited to monitor the dynamics of a protein with optical spectroscopy.
The objective is to establish a relationship between spectroscopic observations, the molecular structure, and the statistical variations of the interaction strength and the site energies.
39
4.1 Structure of the B800 ring
As mentioned in the introduction, the LH2 complex fromRhodospirillum molischianum is an octameric hollow cylinder of 90 ˚A diameter featuring a C8-symmetry [3]. It is built up from eight monomer units each comprising two membrane spanning apoproteins (α inside and β outside) binding non covalently three bacteriochlorophyll a and presumably one carotenoid molecules, see Fig. 4.1A. In the complex, two distinct BChla assemblies can be distinguished, one comprising eight BChla molecules, named B800 ring owing to its main absorption peak around 800 nm, the other comprising 16 BChl a molecules named B850 ring owing to its ab-sorption around 850 nm. The arrangement of the chromophores is shown in Fig. 4.1C together with the orientations of their Qyabsorption dipole moments. From the C8symmetry it is evident that the mutual orientations between uncoupled absorption transition-dipole moments equals 0 modulo 45 degrees. Important characteristics of the B800 system are the equivalence of the 8 binding sites, the relatively narrow absorption line widths, and the nearest-neighbour interaction strengthV of about 20 cm-1[54].
(A) (B) (C)
B800 BChl a
B850 BChl a β-apoprotein
α-apoprotein
B800 ring 10 Å V ≈ 20 cm-1
45° 22 Å 135°
Figure 4.1: Structure of the B800 ring of LH2 fromRhodospirillum molischianum. (A) Subunit containing one B800, two B850 bacteriochlorophyllaand one carotenoid (not shown) molecules non-covalently bound by two apoproteins. (B) Side view of the LH2 complex. (C) Top view of the B800 ring containing eight B800 BChla molecules arranged in a C8 symmetry. The arrays indicate the orientations of their Qyabsorption dipole moment.