Optofluidic rotation of living cells for single‐cell tomography

Flow cytometry provides a high throughput, multi‐dimensional analysis of cells flowing in suspension. In order to combine this feature with the ability to resolve detailed structures in 3D, we developed an optofluidic device that combines a microfluidic system with a dual beam trap. This allows for the rotation of single cells in a continuous flow, around an axis perpendicular to the imaging plane. The combination of both techniques enables the tomographic reconstruction of the 3D structure of the cell. In addition this method is capable to provide detailed 3D structural data for flow cytometry, as it improves the reconstructed z‐resolution of a standard microscopy system to produce images with isotropic resolution in all three axes.

     

Label‐free and non‐invasive monitoring of porcine trophoblast derived cells: differentiation in serum and serum‐free media

Traditional approaches to characterize stem cell differentiation are time‐consuming, lengthy and invasive. Here, Raman microspectroscopy (RM) and atomic force microscopy (AFM) – both considered as non‐invasive techniques – are applied to detect the biochemical and biophysical properties of trophoblast derived stem‐like cells incubated up to 10 days under conditions designed to induce differentiation. Significant biochemical and biophysical differences between control cells and differentiated cells were observed. Quantitative real time PCR was also applied to analyze gene expression. The relationship between cell differentiation and associated cellular biochemical and biomechanical changes were discussed.

Monitoring trophoblast cells differentiation     

The use of optical trap and microbeam to investigate the mechanical and transport characteristics of tunneling nanotubes in tumor spheroids

The use of optical trap and microbeam for investigating mechanical and transport properties of inter cellular tunneling nanotubes (TnTs) in tumor spheroids has been demonstrated. TnTs in tumor spheroids have been visualized by manipulating TnT connected cells using optical tweezers. Functionality of the TnTs for transferring cytoplasmic vesicles and injected dye molecules by optoporation method has been studied. Further, the TnTs could be longitudinally stretched by manipulating the connected cells and their elastic response was studied.

Manipulation of cells at the surface of tumor spheroid using optical tweezers and injection of fluorescent dye into a trapped cell using optoporation technique.     

Light‐emitting diode therapy in exercise‐trained mice increases muscle performance,cytochrome c oxidase activity,ATP and cell proliferation

Light‐emitting diode therapy (LEDT) applied over the leg, gluteus and lower‐back muscles of mice using a LED cluster (630 nm and 850 nm, 80 mW/cm², 7.2 J/cm²) increased muscle performance (repetitive climbing of a ladder carrying a water‐filled tube attached to the tail), ATP and mitochondrial metabolism; oxidative stress and proliferative myocyte markers in mice subjected to acute and progressive strength training. Six bi‐daily training sessions LEDT‐After and LEDT‐Before‐After regimens more than doubled muscle performance and increased ATP more than tenfold. The effectiveness of LEDT on improving muscle performance and recovery suggest applicability for high performance sports and in training programs.

Positioning of the mice and light‐emitting diode therapy (LEDT) applied on mouse legs, gluteus and lower‐back muscles without contact.     

GJIC Enhances the phototoxicity of photofrin‐mediated photodynamic treatment by the mechanisms related with ROS and Calcium pathways

Despite initially positive responses, recurrences after Photodynamic treatment (PDT) can occur and there is need for improvement in the effectiveness of PDT. Our study uniquely showed that there was a significantly gap junctional intercellular communication (GJIC)‐dependent PDT cytotoxicity. The presence of GJIC composed of Connexin 32 increased the PDT phototoxicity in transfected HeLa cells and in the xenograft tumors, and the enhanced phototoxicity of Photofrin‐mediated PDT by GJIC was related with ROS and calcium pathways. Our study indicates the possibility that up‐regulation or maintenance of gap junction functionality may be used to increase the efficacy of PDT.

The phototoxicity effect of Photofrin was substantially greater in Dox‐treated cells, which expressed the Cx32 and formed the GJ, than Dox‐untreated.     

Improved detectability of microcirculatory dynamics by laser speckle flowmetry

Mechanisms of renal autoregulation generate oscillations in arterial blood flow at several characteristic frequencies. Full‐field laser speckle flowmetry provides a real‐time imaging of superficial blood microcirculation. The possibility to detect changes in oscillatory dynamics is an important issue in biomedical applications. In this paper we show how laser power density affects quality of the recorded signal and improves detectability of temporal changes in microvascular perfusion.

     

Isotropic high resolution optoacoustic imaging with linear detector arrays in bi‐directional scanning

Optoacoustic (photoacoustic) imaging is often performed with one‐dimensional transducer arrays, in analogy to ultrasound imaging. Optoacoustic imaging using linear arrays offers ease of implementation but comes with several performance drawbacks, in particular poor elevation resolution, i.e. the resolution along the axis perpendicular to the focal plane. Herein, we introduce and investigate a bi‐directional scanning approach using linear arrays that can improve the imaging performance to quasi‐isotropic transverse resolution. We study the approach theoretically and perform numerical simulations and phantom measurements to evaluate its performance under defined conditions. Finally, we discuss the features and the limitations of the proposed method.

The poor elevation resolution in a linear scan (left image) is overcome by the proposed bi‐directional scanning approach that yields isotropic transverse resolution (right).     

CTAB enhancement of FRET in DNA structures

The effect of cetyl‐trimethylammonium bromide (CTAB) on enhancing the fluorescence resonance energy transfer (FRET) between two dye‐conjugated DNA strands was studied using fluorescence emission spectroscopy and dynamic light scattering (DLS). For hybridized DNA where one strand is conjugated with a TAMRA donor and the other with a TexasRed acceptor, increasing the concentration of CTAB changes the fluorescence emission properties and improves the FRET transfer efficiency through changes in the polarity of the solvent, neutralization of the DNA backbone and micelle formation. For the DNA FRET system without CTAB, the DNA hybridization leads to contact quenching between TAMRA donor and TexasRed acceptor producing reduced donor emission and only a small increase in acceptor emission. At 50 µM CTAB, however, the sheathing and neutralization of the dye‐conjugated dsDNA structure significantly reduces quenching by DNA bases and dye interactions, producing a large increase in FRET efficiency, which is almost four fold higher than without CTAB.

     

Hyperspectral imaging in medicine: image pre‐processing problems and solutions in Matlab

The paper presents problems and solutions related to hyperspectral image pre‐processing. New methods of preliminary image analysis are proposed. The paper shows problems occurring in Matlab when trying to analyse this type of images. Moreover, new methods are discussed which provide the source code in Matlab that can be used in practice without any licensing restrictions.

The proposed application and sample result of hyperspectral image analysis.     

Cell nuclei have lower refractive index and mass density than cytoplasm

Common perception regards the nucleus as a densely packed object with higher refractive index (RI) and mass density than the surrounding cytoplasm. Here, the volume of isolated nuclei is systematically varied by electrostatic and osmotic conditions as well as drug treatments that modify chromatin conformation. The refractive index and dry mass of isolated nuclei is derived from quantitative phase measurements using digital holographic microscopy (DHM). Surprisingly, the cell nucleus is found to have a lower RI and mass density than the cytoplasm in four different cell lines and throughout the cell cycle. This result has important implications for conceptualizing light tissue interactions as well as biological processes in cells.

     

Comparing Raman and fluorescence lifetime spectroscopy from human atherosclerotic lesions using a bimodal probe

Fluorescence lifetime imaging (FLIm) and Raman spectroscopy are two promising methods to support morphological intravascular imaging techniques with chemical contrast. Both approaches are complementary and may also be used in combination with OCT/IVUS to add chemical specificity to these morphologic intravascular imaging modalities. In this contribution, both modalities were simultaneously acquired from two human coronary specimens using a bimodal probe. A previously trained SVM model was used to interpret the fluorescence lifetime data; integrated band intensities displayed in RGB false color images were used to interpret the Raman data. Both modalities demonstrate unique strengths and weaknesses and these will be discussed in comparison to histologic analyses from the two coronary arteries imaged.

     

Holographic imaging of unlabelled sperm cells for semen analysis: a review

Male reproductive health in both humans and animals is an important research field in biological study. In order to characterize the morphology, the motility and the concentration of the sperm cells, which are the most important parameters to feature them, digital holography demonstrated to be an attractive technique. Indeed, it is a label‐free, non‐invasive and high‐resolution method that enables the characterization of live specimen. The review is intended both for summarizing the state‐of‐art on the semen analysis and recent achievement obtained by means of digital holography and for exploring new possible applications of digital holography in this field.

Quantitative phase maps of living swimming spermatozoa.     

Photodamage in deep tissue two‐photon optical biopsy of human skin

Photodamage, induced by femtosecond laser radiation, was studied in thick samples of human skin tissue (healthy skin and neoplastic lesions). Photobleaching, photoionization, and thermomechanical damage effects were characterized comparatively. The laser power dependence of the damage rates allowed to connect macroscopic effects to underlying molecular processes. Optical effects were correlated to histopathological changes. Tissue alterations were found only from thermomechanical cavitation and limited to superficial layers of the epidermis. From the depth‐dependencies of all damage thresholds a depth‐dependent power‐compensation scheme was defined allowing for damage‐free deep tissue optical biopsy.

Damage‐induced luminescence pattern for different excitation powers and a corresponding threshold analysis.     

Significance of the follicular pathway for dermal substance penetration quantified by laser Doppler flowmetry

The understanding of transdermal substance penetration pathways remains an important field for the development of future topical drugs and cosmetics. Laser Doppler flowmetry is a well‐established method for evaluating cutaneous perfusion. In a study on 6 healthy male volunteers, we topically applied the vasoactive substance benzyl nicotinate on two test areas with open and obturated hair follicles and measured changes in the blood flow by Doppler flowmetry. Contrary to occluded follicles, the application onto the test area with open follicles led to a statistically significant perfusion increase within the first 5 minutes, emphasizing the importance of the follicular pathway for epidermal penetration.

     

Ex vivo investigations on the potential of optical coherence tomography (OCT) as a diagnostic tool for reproductive medicine in a bovine model

Routine infertility investigations in the male and female include imaging techniques such as ultrasonography and endoscopy (fertiloscopy). However, these techniques lack the resolution to localize vital sperm or to reveal detailed morphological analysis of the oviduct which is often the cause of infertility in females. Therefore we set out to evaluate the efficiency of optical coherence tomography (OCT) as a diagnostic imaging tool for micron‐scale visualization of the male and female genital tract. Using the bovine as a model, the optical features of the Telesto^TM, Ganymede^TM (both Thorlabs) and Niris^TM (Imalux) OCT imaging systems were compared.

Comparative visualization of ex vivo bovine testicular tissue by the Telesto^TM microscopic optical coherence tomography system (left) and corresponding H&E staining (right).     

Dye distance mapping using waveguide evanescent field fluorescence microscopy and its application to cell biology

Previous studies have measured the distance between cells and the substratum at sites of adhesion via the emission of a fluorescent dye and waveguide methods. Here, we demonstrate a novel approach to measure the position of fluorescent dyes above a waveguide surface in the 10–200 nm distance range throughout an entire area, yielding a 2D dye distance map or a 3D contour plot. The dye is located in a multilayered Langmuir Blodgett (LB) film or in the plasma membrane of a cell. Waveguide evanescent field fluorescence (WEFF) images obtained using two different waveguide modes are employed allowing, with a simple mathematical approach, the calculation of dye distance maps. Ultra‐thin steps made using LB technology, adhesion distances and the bending of the plasma membrane between focal adhesions of osteoblastic cells are shown as examples. The errors are discussed.

False color representation of a dye distance map with four osteoblasts. The inset represents an overexposed WEFF image of the same field of view.     

Cell perforation mediated by plasmonic bubbles generated by a single near infrared femtosecond laser pulse

We report on transient membrane perforation of living cancer cells using plasmonic gold nanoparticles (AuNPs) enhanced single near infrared (NIR) femtosecond (fs) laser pulse. Under optimized laser energy fluence, single pulse treatment (τ = 45 fs, λ = 800 nm) resulted in 77% cell perforation efficiency and 90% cell viability. Using dark field and ultrafast imaging, we demonstrated that the generation of submicron bubbles around the AuNPs is the necessary condition for the cell membrane perforation. AuNP clustering increased drastically the bubble generation efficiency, thus enabling an effective laser treatment using low energy dose in the NIR optical therapeutical window.

Schematic representation of single femtosecond laser pulse plasmonic bubble generation in the vicinity of a cell.     

easySTORM: a robust,lower‐cost approach to localisation and TIRF microscopy

TIRF and STORM microscopy are super‐resolving fluorescence imaging modalities for which current implementations on standard microscopes can present significant complexity and cost. We present a straightforward and low‐cost approach to implement STORM and TIRF taking advantage of multimode optical fibres and multimode diode lasers to provide the required excitation light. Combined with open source software and relatively simple protocols to prepare samples for STORM, including the use of Vectashield for non‐TIRF imaging, this approach enables TIRF and STORM imaging of cells labelled with appropriate dyes or expressing suitable fluorescent proteins to become widely accessible at low cost.

     

Raman spectroscopy as a diagnostic tool for monitoring acute nephritis

Both acute nephritis and chronic nephritis account for substantial morbidity and mortality worldwide, partly due to the lack of reliable tools for detecting disease early and monitoring its progression non‐invasively. In this work, Raman spectroscopy coupled with multivariate analysis are employed for the first time to study the accelerated progression of nephritis in anti‐GBM mouse model. Preliminary results show up to 98% discriminant accuracy for the severe and midly diseased and the healthy among two strains of mice with different susceptibility to acute glomerulonephritis. This technique has the potential for non‐invasive or minimally‐invasive early diagnosis, prognosis, and monitoring of renal disease progression.

     

Dual‐polarization Raman spectral imaging to extract overlapping molecular fingerprints of living cells

Raman spectral imaging is gaining more and more attention in biological studies because of its label‐free characteristic. However, the discrimination of overlapping chemical contrasts has been a major challenge. In this study, we introduce an optical method to simultaneously obtain two orthogonally polarized Raman images from a single scan of the sample. We demonstrate how this technique can improve the quality and quantity of the hyperspectral Raman dataset and how the technique is expected to further extend the horizons of Raman spectral imaging in biological studies by providing more detailed chemical information.

The dual‐polarization Raman images of a HeLa cell.