Christian P. Pfeffer

Two-Dimensional Nanoscale Structural and Functional Imaging in Individual Collagen Type I Fibrils

The piezoelectric properties of single collagen type I fibrils in fascia were imaged with sub-20 nm spatial resolution using piezoresponse force microscopy. A detailed analysis of the piezoresponse force microscopy signal in controlled tip-fibril geometry revealed shear piezoelectricity parallel to the fibril axis. The direction of the displacement is preserved along the whole fiber length and is independent of the fiber conformation. It is shown that individual fibrils within bundles in skeletal muscle fascia can have opposite polar orientations and are organized into domains, i.e., groups of several fibers having the same polar orientation. We were also able to detect piezoelectric activity of collagen fibrils in the high-frequency range up to 200 kHz, suggesting that the mechanical response time of biomolecules to electrical stimuli can be approximately 5 micros.

The structural origin of second harmonic generation in fascia.

Fascia tissue is rich in collagen type I proteins and can be imaged by second harmonic generation (SHG) microscopy. While identifying the overall alignment of the collagen fibrils is evident from those images, the tridimensional structural origin for the observation of SHG signal is more complex than it apparently seems. Those images reveal that the noncentrosymmetric (piezoelectric) structures are distributed heterogeneously on spatial dimensions inferior to the resolution provided by the nonlinear optical microscope (sub-micron). Using piezoresponse force microscopy (PFM), we show that an individual collagen fibril has a noncentrosymmetric structural organization. Fibrils are found to be arranged in nano-domains where the anisotropic axis is preserved along the fibrillar axis, while across the collagen sheets, the phase of the second order nonlinear susceptibility is changing by 180 degrees between adjacent nano-domains. This complex architecture of noncentrosymmetric nano-domains governs the coherent addition of 2ω light within the focal volume and the observed features in the SHG images taken in fascia.

Multimodal nonlinear optical imaging of collagen arrays

We report multimodal nonlinear optical imaging of fascia, a rich collagen type I sheath around internal organs and muscle. We show that second harmonic generation (SHG), third harmonic generation (THG) and Coherent anti-Stokes Raman scattering (CARS) microscopy techniques provide complementary information about the sub-micron architecture of collagen arrays. Forward direction SHG microscopy reveals the fibrillar arrangement of collagen type I structures as the main matrix component of fascia. SHG images detected in the backward direction as well as images of forward direction CARS microscopy show that the longitudinal collagen fiber bundles are further arranged in sheet-like bands. Forward-THG microscopy reveals the optically homogeneous content of the collagen sheet on a spatial scale of the optical wavelength. This is supported by the fact that the third harmonic signal is observed only at the boundaries between the sheets as well as by the CARS data obtained in both directions. The observations made with THG and CARS microscopy are explained using atomic force microscopy images.

Second harmonic generation imaging of fascia within thick tissue block

Comparing the SHG image formation for thin sections of tail tendon fascia and skeletal muscle fascia, we demonstrate that the forward (F) and backward (B) SHG images are vastly different. In addition, despite the different arrangement of the collagen Type I fibrillar architecture forming these two fascias, their ratios of forward over backward signal (F/B) are nearly equal. SHG images of thick tissue blocks of the fascia-muscle unit show only backward features, as opposed to SHG images of tissue blocks of the fascia-tendon unit. These images are an amalgamation of forward and backward features due to the backscattering of forward components within tendon. These forward features disappear when this tissue block is immersed in glycerol as backscattering is hereby suppressed.

Imaging the noncentrosymmetric structural organization of tendon with Interferometric Second Harmonic Generation microscopy.

We image the relative orientation of organized groups of noncentrosymmetric molecules (like collagen or myosin) at the micron scale in biological tissues by combining interferometry and Second Harmonic Generation (SHG) microscopy.

Editorial: Journal of Negative Results in Biomedicine

We are pleased to introduce you to the Journal of Negative Results in Biomedicine (JNRBM). A journal, very unique in its kind, as it publishes articles, fully PubMed indexed that challenge current models, tenets and dogmas. The articles are based on rigorous, and well documented results that do not support these models or even disprove them. It publishes methods and techniques that are found to be unsuitable for studying a particular phenomenon. JNRBM strongly promotes and encourages the publication of clinical trials that fall short of demonstrating an improvement over current treatments. JNRBMs immediate goal is to provide scientists and physicians with responsible and balanced information in order to improve experimental designs and clinical decisions.

Imaging skeletal muscle using second harmonic generation and coherent anti-Stokes Raman scattering microscopy.

We describe experimental results on label free imaging of striated skeletal muscle using second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy. The complementarity of the SHG and CARS data makes it possible to clearly identify the main sarcomere sub-structures such as actin, myosin, acto-myosin, and the intact T-tubular system as it emanates from the sarcolemma. Owing to sub-micron spatial resolution and the high sensitivity of the CARS microscopy technique we were able to resolve individual myofibrils. In addition, key organelles such as mitochondria, cell nuclei and their structural constituents were observed revealing the entire structure of the muscle functional units. There is a noticeable difference in the CARS response of the muscle structure within actin, myosin and t-tubule areas with respect to laser polarization. We attribute this to a preferential alignment of the probed molecular bonds along certain directions. The combined CARS and SHG microscopy approach yields more extensive and complementary information and has a potential to become an indispensable method for live skeletal muscle characterization.

Extracorporeal life support and left ventricular unloading in a non-intubated patient as bridge to heart transplantation.

Introduction Veno-arterial extracorporeal life support (ECLS) is a well-established bridging therapy in patients with cardiac or pulmonary failure to maintain organ function and is frequently performed in patients who are not intubated. However, severly impaired cardiac function can occur pulmonary edemy in these patients, necessitating left ventricular unloading. Methods and Results In this study we report a 37-year old female patient with familiar dilated cardiomyopathy suffering from acute biventricular heart failure. After implantation of a peripheral ECLS, the decreased ventricular led to refractory pulmonary edema. To unload the left ventricle, an percutaneous balloon atrioseptostomy was performed without intubating the patient. The left ventricle was vented by the venous cannula resting inside the atrioseptostomy. After twelve days on ECLS, the patient underwent orthotopic heart transplantation. The postoperative course was uneventful and the patient discharged from intensive care unit four days after surgery. Conclusions In this report we present a patient in which the hybrid technique of ECLS with secondary left ventricular unloading was successfully used as a bridge to transplant therapy. This procedure may offer an alternative bridge-to-decision options in selected patients, including those that were not intubated or anaesthetized.

Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components

Using an optical system made from fused silica catalogue optical components, third‐order nonlinear microscopy has been enabled on conventional Ti:sapphire laser‐based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of ∼300 nm and in multimodal nonlinear optical imaging experiments using third‐order sum frequency generation and coherent anti‐Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from ∼300 nm to ∼660 nm.

Aortic valve replacement surgery reveals previously undiagnosed alkaptonuric ochronosis

alkaptonuric ochronosis Christian Pfeffer*, Erik Bagaev, Karl Sotlar and Christian Hagl a Herzchirurgische Klinik und Poliklinik der Ludwig-Maximilians-Universitat Munchen, Munich, Germany b Pathologisches Institut der Ludwig-Maximilians-Universitat Munchen, Munich, Germany * Corresponding author. Herzchirurgische Klinik und Poliklinik der Ludwig-Maximilians-Universitat Munchen, Marchioninistrase 15, 81377 Munich, Germany. Tel: +49-89-70952951; fax: +49-89-70958898; e-mail: christian.pfeffer@med.uni-muenchen.de (C. Pfeffer). Received 3 January 2014; received in revised form 13 February 2014; accepted 18 February 2014