Compact portable higher harmonic generation microscopy for the real time assessment of unprocessed thyroid tissue.

During thyroid surgery fast and reliable intra-operative pathological feedback has the potential to avoid a two-stage procedure and significantly reduce health care costs in patients undergoing a diagnostic hemithyroidectomy (HT). We explored higher harmonic generation (HHG) microscopy, which combines second harmonic generation (SHG), third harmonic generation (THG), and multiphoton excited autofluorescence (MPEF) for this purpose. With a compact, portable HHG microscope, images of freshly excised healthy tissue, benign nodules (follicular adenoma) and malignant tissue (papillary carcinoma, follicular carcinoma and spindle cell carcinoma) were recorded. The images were generated on unprocessed tissue within minutes and show relevant morphological thyroid structures in good accordance with the histology images. The thyroid follicle architecture, cells, cell nuclei (THG), collagen organization (SHG) and the distribution of thyroglobulin and/or thyroid hormones T3 or T4 (MPEF) could be visualized. We conclude that SHG/THG/MPEF imaging is a promising tool for clinical intraoperative assessment of thyroid tissue.

Visualizing dynamic three-dimensional changes of human reticular dermal collagen under mechanical strain.

In clinical practice, plastic surgeons are often faced with large skin defects that are difficult to close primarily. Management of large skin wounds e.g. burns or traumatic lacerations requires knowledge of skin biomechanic properties. Research into skin microstructural adaptation to mechanical deformation has only been performed using static regimes due to technical limitations. Here, we combine uniaxial stretch tests with fast second harmonic generation imaging and we apply this for the first time to investigate dynamic collagen rearrangement in reticular human dermis.Ex vivohuman skin from the abdomen and upper thigh was simultaneously uniaxially stretched while either periodically visualizing 3D reorganization, or visualizing 2D changes in real time. We determined collagen alignment via orientation indices and found pronounced variability across samples. Comparing mean orientation indices at the different stages of the stress strain curves (toe, heel, linear) showed a significant increase in collagen alignment during the linear part of the mechanical response. We conclude that fast SHG imaging during uni-axial extension is a promising research tool for future studies on skin biomechanic properties.

Third harmonic generation imaging for fast, label-free pathology of human brain tumors.

In brain tumor surgery, recognition of tumor boundaries is key. However, intraoperative assessment of tumor boundaries by the neurosurgeon is difficult. Therefore, there is an urgent need for tools that provide the neurosurgeon with pathological information during the operation. We show that third harmonic generation (THG) microscopy provides label-free, real-time images of histopathological quality; increased cellularity, nuclear pleomorphism, and rarefaction of neuropil in fresh, unstained human brain tissue could be clearly recognized. We further demonstrate THG images taken with a GRIN objective, as a step toward in situ THG microendoscopy of tumor boundaries. THG imaging is thus a promising tool for optical biopsies.

Single-shot two-dimensional full-range optical coherence tomography achieved by dispersion control.

We present a full-range Fourier-domain optical coherence tomography (OCT) system that is capable of acquiring two-dimensional images of living tissue in a single shot. By using line illumination of the sample in combination with a two-dimensional imaging spectrometer, 1040 depth scans are performed simultaneously on a sub-millisecond timescale. Furthermore, we demonstrate an easy and flexible real-time single-shot technique for full-range (complex-conjugate cancelled) OCT imaging that is compatible with both two-dimensional as well as ultrahigh-resolution OCT. By implementing a dispersion imbalance between reference and sample arms of the interferometer, we eliminate the complex-conjugate signal through numerical dispersion compensation, effectively increasing the useful depth range by a factor of two. The system allows us to record 6.7 x 3.2 mm images at 5 microm depth resolution in 0.2 ms. Data postprocessing requires only 4 s. We demonstrate the capability of our system by imaging the anterior chamber of a mouse eye in vitro, as well as human skin in vivo.

Charge separation and energy transfer in the photosystem II core complex studied by femtosecond midinfrared spectroscopy.

The core of photosystem II (PSII) of green plants contains the reaction center (RC) proteins D1D2-cytb559 and two core antennas CP43 and CP47. We have used time-resolved visible pump/midinfrared probe spectroscopy in the region between 1600 and 1800 cm(-1) to study the energy transfer and charge separation events within PSII cores. The absorption difference spectra in the region of the keto and ester chlorophyll modes show spectral evolution with time constants of 3 ps, 27 ps, 200 ps, and 2 ns. Comparison of infrared (IR) difference spectra obtained for the isolated antennas CP43 and CP47 and the D1D2-RC with those measured for the PSII core allowed us to identify the features specific for each of the PSII core components. From the presence of the CP43 and CP47 specific features in the spectra up to time delays of 20-30 ps, we conclude that the main part of the energy transfer from the antennas to the RC occurs on this timescale. Direct excitation of the pigments in the RC evolution associated difference spectra to radical pair formation of PD1+PheoD1- on the same timescale as multi-excitation annihilation and excited state equilibration within the antennas CP43 and CP47, which occur within approximately 1-3 ps. The formation of the earlier radical pair ChlD1+PheoD1-, as identified in isolated D1D2 complexes with time-resolved mid-IR spectroscopy is not observed in the current data, probably because of its relatively low concentration. Relaxation of the state PD1+PheoD1-, caused by a drop in free energy, occurs in 200 ps in closed cores. We conclude that the kinetic model proposed earlier for the energy and electron transfer dynamics within the D1D2-RC, plus two slowly energy-transferring antennas C43 and CP47 explain the complex excited state and charge separation dynamics in the PSII core very well. We further show that the time-resolved IR-difference spectrum of PD1+PheoD1- as observed in PSII cores is virtually identical to that observed in the isolated D1D2-RC complex of PSII, demonstrating that the local structure of the primary reactants has remained intact in the isolated D1D2 complex.

Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein.

Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYP's signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is approximately 0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.

Spectroscopic properties of the CP43 core antenna protein of photosystem II.

CP43 is a chlorophyll-protein complex that funnels excitation energy from the main light-harvesting system of photosystem II to the photochemical reaction center. We purified CP43 from spinach photosystem II membranes in the presence of the nonionic detergent n-dodecyl-beta,D-maltoside and recorded its spectroscopic properties at various temperatures between 4 and 293 K by a number of polarized absorption and fluorescence techniques, fluorescence line narrowing, and Stark spectroscopy. The results indicate two "red" states in the Q(y) absorption region of the chlorophylls. The first peaks at 682.5 nm at 4 K, has an extremely narrow bandwidth with a full width at half-maximum of approximately 2.7 nm (58 cm(-1)) at 4 K, and has the oscillator strength of a single chlorophyll. The second peaks at approximately 679 nm, has a much broader bandshape, is caused by several excitonically interacting chlorophylls, and is responsible for all 4 K absorption at wavelengths longer than 685 nm. The Stark spectrum of CP43 resembles the first derivative of the absorption spectrum and has an exceptionally small overall size, which we attribute to opposing orientations of the monomer dipole moments of the excitonically coupled pigments.

Charge separation in the reaction center of photosystem II studied as a function of temperature.

In photosystem II of green plants the key photosynthetic reaction consists of the transfer of an electron from the primary donor called P680 to a nearby pheophytin molecule. We analyzed the temperature dependence of this reaction by subpicosecond transient absorption spectroscopy over the temperature range 20-240 K using isolated photosystem II reaction centers from spinach. After excitation in the red edge of the Qy absorption band, the decay of the excited state can conveniently be described by two kinetic components that both accelerate with temperature. This temperature behavior differs remarkably from that observed in purple bacterial reaction centers. We attribute the first component, which accelerates from 2.6 ps at 20 K to 0.4 ps at 240 K, to charge separation after direct excitation of P680, and explain its temperature dependence by an intermediate that lies in energy above the singlet-excited P680 and that possibly has charge-transfer character. The second component accelerates from 120 ps at 20 K to 18 ps at 240 K and is attributed to charge separation after direct excitation of the "trap" state near-degenerate with P680 and subsequent slow energy transfer from this trap state to P680. We suggest that the slow energy transfer from the trap state to P680 plays an important role in the kinetics of radical pair formation at room temperature.

Purification and spectroscopic characterization of photosystem II reaction center complexes isolated with or without Triton X-100.

The pigment composition of the isolated photosystem II reaction center complex in its most stable and pure form currently is a matter of considerable debate. In this contribution, we present a new method based on a combination of gel filtration chromatography and diode array detection to analyze the composition of photosystem II reaction center preparations. We show that the method is very sensitive for the detection of contaminants such as the core antenna protein CP47, pigment-free and denatured reaction center proteins, and unbound chlorophyll and pheophytin molecules. We also present a method by which the photosystem II reaction center complex is highly purified without using Triton X-100, and we show that in this preparation the contamination with CP47 is less than 0.1%. The results strongly indicate that the photosystem II reaction center complex in its most stable and pure form binds six chlorophyll a, two pheophytin a, and two beta-carotene molecules and that the main effect of Triton X-100 is the extraction of beta-carotene from the complex. Analysis of 4 K absorption and emission spectra indicates that the spectroscopic properties of this preparation are similar to those obtained by a short Triton X-100 treatment. In contrast, preparations obtained by long Triton X-100 treatment show decreased absorption of the shoulder at 684 nm in the 4 K absorption spectrum and an increased number of pigments that trap excitation energy at very low temperatures. We conclude that the 684 nm shoulder in the 4 K absorption spectrum should at least in part be attributed to the primary electron donor of photosystem II.

Protein folding thermodynamics applied to the photocycle of the photoactive yellow protein.

Two complementary aspects of the thermodynamics of the photoactive yellow protein (PYP), a new type of photoreceptor that has been isolated from Ectothiorhodospira halophila, have been investigated. First, the thermal denaturation of PYP at pH 3.4 has been examined by global analysis of the temperature-induced changes in the UV-VIS absorbance spectrum of this chromophoric protein. Subsequently, a thermodynamic model for protein (un)folding processes, incorporating heat capacity changes, has been applied to these data. The second aspect of PYP that has been studied is the temperature dependence of its photocycle kinetics, which have been reported to display an unexplained deviation from normal Arrhenius behavior. We have extended these measurements in two solvents with different hydrophobicities and have analyzed the number of rate constants needed to describe these data. Here we show that the resulting temperature dependence of the rate constants can be quantitatively explained by the application of a thermodynamic model which assumes that heat capacity changes are associated with the two transitions in the photocycle of PYP. This result is the first example of an enzyme catalytic cycle being described by a thermodynamic model including heat capacity changes. It is proposed that a strong link exists between the processes occurring during the photocycle of PYP and protein (un)folding processes. This permits a thermodynamic analysis of the light-induced, physiologically relevant, conformational changes occurring in this photoreceptor protein.

Triplet and fluorescing states of the CP47 antenna complex of photosystem II studied as a function of temperature.

Fluorescence emission and triplet-minus-singlet (T-S) absorption difference spectra of the CP47 core antenna complex of photosystem II were measured as a function of temperature and compared to those of chlorophyll a in Triton X-100. Two spectral species were found in the chlorophyll T-S spectra of CP47, which may arise from a difference in ligation of the pigments or from an additional hydrogen bond, similar to what has been found for Chl molecules in a variety of solvents. The T-S spectra show that the lowest lying state in CP47 is at approximately 685 nm and gives rise to fluorescence at 690 nm at 4 K. The fluorescence quantum yield is 0.11 +/- 0.03 at 4 K, the chlorophyll triplet yield is 0.16 +/- 0.03. Carotenoid triplets are formed efficiently at 4 K through triplet transfer from chlorophyll with a yield of 0.15 +/- 0.02. The major decay channel of the lowest excited state in CP47 is internal conversion, with a quantum yield of about 0.58. Increase of the temperature results in a broadening and blue shift of the spectra due to the equilibration of the excitation over the antenna pigments. Upon increasing the temperature, a decrease of the fluorescence and triplet yields is observed to, at 270 K, a value of about 55% of the low temperature value. This decrease is significantly larger than of chlorophyll a in Triton X-100. Although the coupling to low-frequency phonon or vibration modes of the pigments is probably intermediate in CP47, the temperature dependence of the triplet and fluorescence quantum yield can be modeled using the energy gap law in the strong coupling limit of Englman and Jortner (1970. J. Mol. Phys. 18:145-164) for non-radiative decays. This yields for CP47 an average frequency of the promoting/accepting modes of 350 cm-1 with an activation energy of 650 cm-1 for internal conversion and activationless intersystem crossing to the triplet state through a promoting mode with a frequency of 180 cm-1. For chlorophyll a in Triton X-100 the average frequency of the promoting modes for non-radiative decay is very similar, but the activation energy (300 cm-1) is significantly smaller.

Temperature-dependent triplet and fluorescence quantum yields of the photosystem II reaction center described in a thermodynamic model.

A key step in the photosynthetic reactions in photosystem II of green plants is the transfer of an electron from the singlet-excited chlorophyll molecule called P680 to a nearby pheophytin molecule. The free energy difference of this primary charge separation reaction is determined in isolated photosystem II reaction center complexes as a function of temperature by measuring the absolute quantum yield of P680 triplet formation and the time-integrated fluorescence emission yield. The total triplet yield is found to be 0.83 +/- 0.05 at 4 K, and it decreases upon raising the temperature to 0.30 at 200 K. It is suggested that the observed triplet states predominantly arise from P680 but to a minor extent also from antenna chlorophyll present in the photosystem II reaction center. No carotenoid triplet states could be detected, demonstrating that the contamination of the preparation with CP47 complexes is less than 1/100 reaction centers. The fluorescence yield is 0.07 +/- 0.02 at 10 K, and it decreases upon raising the temperature to reach a value of 0.05-0.06 at 60-70 K, increases upon raising the temperature to 0.07 at approximately 165 K and decreases again upon further raising the temperature. The complex dependence of fluorescence quantum yield on temperature is explained by assuming the presence of one or more pigments in the photosystem II reaction center that are energetically degenerate with the primary electron donor P680 and below 60-70 K trap part of the excitation energy, and by temperature-dependent excited state decay above 165 K. A four-compartment model is presented that describes the observed triplet and fluorescence quantum yields at all temperatures and includes pigments that are degenerate with P680, temperature-dependent excited state decay and activated upward energy transfer rates. The eigenvalues of the model are in accordance with the lifetimes observed in fluorescence and absorption difference measurements by several workers. The model suggests that the free energy difference between singlet-excited P680 and the radical pair state P680+l- is temperature independent, and that a distribution of free energy differences represented by at least three values of about 20, 40, and 80 meV, is needed to get an appropriate fit of the data.