Thierry Boulesteix

Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy

We recorded one-photon excited fluorescence (1PEF) and two-photon excited fluorescence (2PEF) spectra of purified keratin from human epidermis, and determined the action cross section of this endogenous chromophore. We used this spectroscopic analysis to analyse multiphoton images of skin biopsies and assign the intrinsic fluorescence signals in the epidermis. We observed a good agreement between in situ and in vitro 2PEF spectra of keratin. This study provides a comprehensive characterization of the 2PEF signal of the keratins from the epidermis, and will be of practical interest for multiphoton imaging of the skin.

Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy

We extend second-harmonic generation (SHG) microscopy to the measurement of sarcomere length in unstained living cardiac myocytes with 20-nm accuracy. We quantify individual sarcomere shortening in the presence of saxitoxin and find that it is in agreement with mechanical measurements of atrial tissue contracture. This functional application of SHG microscopy is generally applicable to quantify the physiological effects of drugs on contractile tissue. Our data also suggest that packed myosin heads in sarcomere thick filaments are responsible for the large second-harmonic endogenous signal in muscle tissue.

Micrometer scale Ex Vivo multiphoton imaging of unstained arterial wall structure

We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels.

Strong chiroptical effects in surface second harmonic generation obtained for molecules exhibiting excitonic coupling chirality

We measured nonlinear optical activity in second harmonic generation (SHG) by reflection on a thin film of chiral molecules. We selected the structure of our molecules in order to obtain very large chiroptical effects. We indeed measured an angle of rotation of the second harmonic polarization as large as 66° for an acridine substituted Troger base. This is in good agreement with our theoretical expectations for such an excitonic coupling chirality.

Spectroscopic analysis of skin intrinsic signals for multiphoton microscopy

We recorded multiphoton images of human skin biopsies using endogenous sources of nonlinear optical signals. We detected simultaneously two-photon excited fluorescence (2PEF) from intrinsic fluorophores and second harmonic generation (SHG) from collagen. We observed SHG from fibrillar collagens in the dermis, whereas no SHG was detectable from the non fibrillar type IV collagen in the basal laminae. We compared these distinct behaviours of collagens I and IV in SHG microscopy to polarization-resolved surface SHG experiments on thin films of collagens I and IV molecules. We observed similar signals for both types of molecular films, except for the chiroptical contributions which are present only for collagen I and enhance the signal typically by a factor of 2. We concluded that SHG microscopy is a sensitive probe of the micrometer-scale structural organization of collagen in biological tissues. In order to elucidate the origin of the endogenous fluorescence signals, we recorded 2PEF spectra at various positions in the skin biopsies, and compared these data to in vitro spectroscopic analysis. In particular, we studied the keratin fluorescence and determined its 2PEF action cross section. We observed a good agreement between 2PEF spectra recorded in the keratinized upper layers of the epidermis and in a solution of purified keratin. Finally, to illustrate the capabilities of this technique, we recorded 2PEF/SHG images of skin biopsies obtained from patients of various ages.

Multiphoton microscopy using intrinsic signals for pharmacological studies in unstained cardiac and vascular tissue

We report two novel applications of multiphoton microscopy for pharmacological studies of unstained cardiovascular tissue. First, we show that second harmonic generation (SHG) microscopy of unstained cardiac myocytes can be used to determine the sarcomere length with sub-resolution accuracy, owing to the remarkable contrast of the SHG signal originating from myosin filaments. A measurement precision of 20 nm is achieved, taking the sample variability into account. We used this technique to measure sarcomere contracture in the presence of saxitoxin, and results were in agreement with mechanical measurements of atrial tissue contracture. Second, we characterized multiphoton microscopy of intact unlabeled arteries. We performed simultaneous detection of two-photon-excited fluorescence (2PEF) from elastin laminae and SHG from collagen fibers upon 860 nm excitation. Combined 2PEF/SHG images provide a highly specific, micron scale description of the architecture of these two major components of the vessel wall. We used this methodology to study the effects of lindane (a pesticide) on the artery wall structure and evidenced structural alteration of the vessel morphology.

Chiral chromophores for second harmonic microscopy

The use of chiral harmonophores in second harmonic generation (SHG) microscopy of lipid bilayers should enable one to obtain a signal even when the distribution of the chromophores is centrosymmetric. In order to determine optimal chiral molecules, we performed polarization-resolved second harmonic reflection experiments. We found that chirality must arise from an excitonic coupling rather than from an asymmetric center. We selectively labeled giant unilamellar lipid vesicles and cell membranes with such a molecule, namely an acridine substituted Trogers base, as demonstrated by two-photon-excited fluorescence microscopy. We performed preliminary SHG microscopy experiments, but the poor efficiency of the current form of our molecule does not allow us to demonstrate chirality effects.