Rapid On-Site Histology of Lung and Pleural Biopsies Using Higher Harmonic Generation Microscopy and Artificial Intelligence Analysis.

Lung cancer is one of the most prevalent and lethal cancers. To improve health outcomes while reducing health care burden, it becomes crucial to move toward early detection and cost-effective workflows. Currently, there is no method for the on-site rapid histologic feedback on biopsies taken in diagnostic, endoscopic, or surgical procedures.

Self-Supervised Image Denoising of Third Harmonic Generation Microscopic Images of Human Glioma Tissue by Transformer-Based Blind Spot (TBS) Network.

Third harmonic generation (THG) microscopy shows great potential for instant pathology of brain tumor tissue during surgery. However, due to the maximal permitted exposure of laser intensity and inherent noise of the imaging system, the noise level of THG images is relatively high, which affects subsequent feature extraction analysis. Denoising THG images is challenging for modern deep-learning based methods because of the rich morphologies contained and the difficulty in obtaining the noise-free counterparts.

Uniaxial mechanical stretch properties correlated with three-dimensional microstructure of human dermal skin.

The intact and healthy skin forms a barrier to the outside world and protects the body from mechanical impact. The skin is a complex structure with unique mechano-elastic properties. To better direct the design of biomimetic materials and induce skin regeneration in wounds with optimal outcome, more insight is required in how the mechano-elastic properties emerge from the skin's main constituents, collagen and elastin fibers.

Leukocyte differentiation in bronchoalveolar lavage fluids using higher harmonic generation microscopy and deep learning.

Background: In diseases such as interstitial lung diseases (ILDs), patient diagnosis relies on diagnostic analysis of bronchoalveolar lavage fluid (BALF) and biopsies. Immunological BALF analysis includes differentiation of leukocytes by standard cytological techniques that are labor-intensive and time-consuming. Studies have shown promising leukocyte identification performance on blood fractions, using third harmonic generation (THG) and multiphoton excited autofluorescence (MPEF) microscopy.

Fast intraoperative histology-based diagnosis of gliomas with third harmonic generation microscopy and deep learning.

Management of gliomas requires an invasive treatment strategy, including extensive surgical resection. The objective of the neurosurgeon is to maximize tumor removal while preserving healthy brain tissue. However, the lack of a clear tumor boundary hampers the neurosurgeon's ability to accurately detect and resect infiltrating tumor tissue.

Compact portable multiphoton microscopy reveals histopathological hallmarks of unprocessed lung tumor tissue in real time.

During lung cancer operations a rapid and reliable assessment of tumor tissue can reduce operation time and potentially improve patient outcomes. We show that third harmonic generation (THG), second harmonic generation (SHG) and two-photon excited autofluorescence (2PEF) microscopy reveals relevant, histopathological information within seconds in fresh unprocessed human lung samples. We used a compact, portable microscope and recorded images within 1 to 3 seconds using a power of 5 mW.

Confinement in crystal lattice alters entire photocycle pathway of the Photoactive Yellow Protein.

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time.

Effective enzymatic debridement of burn wounds depends on the denaturation status of collagen.

The treatment of burn wounds by enzymatic debridement using bromelain has shown promising results in our burn center. However, inadequate debridement occurred in a few cases in which the etiology of the burn was attributed to relatively low temperature burns. We hypothesized that bromelain is ineffective in burns in which collagen denaturation, which occurs approximately at 65°C, has not taken place.

Label-free stimulated Raman scattering imaging reveals silicone breast implant material in tissue.

Millions of women worldwide have silicone breast implants. It has been reported that implant failure occurs in approximately a tenth of patients within 10 years, and the consequences of dissemination of silicone debris are poorly understood. Currently, silicone detection in histopathological slides is based on morphological features as no specific immunohistochemical technique is available.

Quantitative Third Harmonic Generation Microscopy for Assessment of Glioma in Human Brain Tissue.

Distinguishing tumors from normal brain cells is important but challenging in glioma surgery due to the lack of clear interfaces between the two. The ability of label-free third harmonic generation (THG) microscopy in combination with automated image analysis to quantitatively detect glioma infiltration in fresh, unprocessed tissue in real time is assessed. The THG images reveal increased cellularity in grades II-IV glioma samples from 23 patients, as confirmed by subsequent hematoxylin and eosin histology.

Second and third harmonic generation microscopy visualizes key structural components in fresh unprocessed healthy human breast tissue.

Real-time assessment of excised tissue may help to improve surgical results in breast tumor surgeries. Here, as a step towards this purpose, the potential of second and third harmonic generation (SHG, THG) microscopy is explored. SHG and THG are nonlinear optical microscopic techniques that do not require labeling of tissue to generate 3D images with intrinsic depth-sectioning at sub-cellular resolution.

Photoactivation Mechanism, Timing of Protein Secondary Structure Dynamics and Carotenoid Translocation in the Orange Carotenoid Protein.

The orange carotenoid protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCP; blue light illumination results in the red active state, known as OCP. The OCP state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain.

Structural and Mechanical Comparison of Human Ear, Alar, and Septal Cartilage.

Background: In the human ear and nose, cartilage plays a key role in establishing its form and function. Interestingly, there is a noticeable paucity on biochemical, structural, and mechanical studies focused on facial cartilage. Such studies are needed to provide elementary knowledge that is fundamental to tissue engineering of cartilage.

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images.

Third harmonic generation (THG) microscopy is a label-free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all-nuclei-highlighted fluorescence images, acquired simultaneously from the same tissue area.

Extracting morphologies from third harmonic generation images of structurally normal human brain tissue.

Motivation: The morphologies contained in 3D third harmonic generation (THG) images of human brain tissue can report on the pathological state of the tissue. However, the complexity of THG brain images makes the usage of modern image processing tools, especially those of image filtering, segmentation and validation, to extract this information challenging.

Results: We developed a salient edge-enhancing model of anisotropic diffusion for image filtering, based on higher order statistics.

Unfolding of the C-Terminal Jα Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy.

Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal Jα helix that is docked on a β-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds.

Cartilage Tissue Engineering: Preventing Tissue Scaffold Contraction Using a 3D-Printed Polymeric Cage.

Scaffold contraction is a common but underestimated problem in the field of tissue engineering. It becomes particularly problematic when creating anatomically complex shapes such as the ear. The aim of this study was to develop a contraction-free biocompatible scaffold construct for ear cartilage tissue engineering.

Short hydrogen bonds and negative charge in photoactive yellow protein promote fast isomerization but not high quantum yield.

Biological signal transduction by photoactive yellow protein (PYP) in halophilic purple sulfur bacteria is initiated by trans-to-cis isomerization of the p-coumaric acid chromophore (pCa) of PYP. pCa is engaged in two short hydrogen bonds with protein residues E46 and Y42, and it is negatively charged at the phenolate oxygen. We investigated the role in the isomerization process of the E46 short hydrogen bond and that of the negative charge on the anionic phenolate moiety of the chromophore.

Ultrafast infrared spectroscopy in photosynthesis.

In recent years visible pump/mid-infrared (IR) probe spectroscopy has established itself as a key technology to unravel structure-function relationships underlying the photo-dynamics of complex molecular systems. In this contribution we review the most important applications of mid-infrared absorption difference spectroscopy with sub-picosecond time-resolution to photosynthetic complexes. Considering several examples, such as energy transfer in photosynthetic antennas and electron transfer in reaction centers and even more intact structures, we show that the acquisition of ultrafast time resolved mid-IR spectra has led to new insights into the photo-dynamics of the considered systems and allows establishing a direct link between dynamics and structure, further strengthened by the possibility of investigating the protein response signal to the energy or electron transfer processes.

Excitation energy trapping and dissipation by Ni-substituted bacteriochlorophyll a in reconstituted LH1 complexes from Rhodospirillum rubrum.

Bacteriochlorophyll a with Ni(2+) replacing the central Mg(2+) ion was used as an ultrafast excitation energy dissipation center in reconstituted bacterial LH1 complexes. B870, a carotenoid-less LH1 complex, and B880, an LH1 complex containing spheroidene, were obtained via reconstitution from the subunits isolated from chromatophores of Rhodospirillum rubrum . Ni-substituted bacteriochlorophyll a added to the reconstitution mixture partially substituted the native pigment in both forms of LH1.

Early bacteriopheophytin reduction in charge separation in reaction centers of Rhodobacter sphaeroides.

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on BA and HA via the appearance of the BA and HA anion bands.

Photoionization and electron radical recombination dynamics in photoactive yellow protein investigated by ultrafast spectroscopy in the visible and near-infrared spectral region.

Photoinduced ionization of the chromophore inside photoactive yellow protein (PYP) was investigated by ultrafast spectroscopy in the visible and near-infrared spectral regions. An absorption band that extended from around 550 to 850 nm was observed and ascribed to solvated electrons, ejected from the p-hydroxycinnamic acid anion chromophore upon the absorption of two 400 nm photons. Global kinetic analysis showed that the solvated electron absorption decayed in two stages: a shorter phase of around 10 ps and a longer phase of more than 3 ns.

Short-coherence off-axis holographic phase microscopy of live cell dynamics.

We demonstrate a single-shot holographic phase microscope that combines short-coherence laser pulses with an off-axis geometry. By introducing a controlled pulse front tilt, ultrashort pulses are made to interfere over a large field-of-view without loss of fringe contrast. With this microscope, quantitative phase images of live cells can be recorded in a full-field geometry without moving parts.