High-resolution fluorescence endomicroscopy for rapid evaluation of breast cancer margins

Breast cancer is a major public health problem world-wide and the second leading cause of cancer-related female deaths. Breast conserving surgery (BCS), in the form of wide local excision (WLE), allows complete tumour resection while maintaining acceptable cosmesis. It is the recommended treatment for a large number of patients with early stage disease or, in more advanced cases, following neoadjuvant chemotherapy. About 30% of patients undergoing BCS require one or more re-operative interventions, mainly due to the presence of positive margins. The standard of care for surgical margin assessment is post-operative examination of histopathological tissue sections. However, this process is invasive, introduces sampling errors and does not provide real-time assessment of the tumour status of radial margins. The objective of this thesis is to improve intra-operative assessment of margin status by performing optical biopsy in breast tissue. This thesis presents several technical and clinical developments related to confocal fluorescence endomicroscopy systems for real-time characterisation of different breast morphologies. The imaging systems discussed employ flexible fibre-bundle based imaging probes coupled to high-speed line-scan confocal microscope set-up. A preliminary study on 43 unfixed breast specimens describes the development and testing of line-scan confocal laser endomicroscope (LS-CLE) to image and classify different breast pathologies. LS-CLE is also demonstrated to assess the intra-operative tumour status of whole WLE specimens and surgical excisions with high diagnostic accuracy. A third study demonstrates the development and testing of a bespoke LS-CLE system with methylene blue (MB), an US Food and Drug Administration (FDA) approved fluorescent agent, and integration with robotic scanner to enable large-area in vivo imaging of breast cancer. The work also addresses three technical issues which limit existing fibre-bundle based fluorescence endomicroscopy systems: i) Restriction to use single fluorescence agent due to low-speed, single excitation and single fluorescence spectral band imaging systems; ii) Limited Field of view (FOV) of fibre-bundle endomicroscopes due to small size of the fibre tip and iii) Limited spatial resolution of fibre-bundle endomicroscopes due to the spacing between the individual fibres leading to fibre-pixelation effects. Details of design and development of a high-speed dual-wavelength LS-CLE system suitable for high-resolution multiplexed imaging are presented. Dual-wavelength imaging is achieved by sequentially switching between 488 nm and 660 nm laser sources for alternate frames, avoiding spectral bleed-through, and providing an effective frame rate of 60 Hz. A combination of hand-held or robotic scanning with real-time video mosaicking, is demonstrated to enable large-area imaging while still maintaining microscopic resolution. Finally, a miniaturised piezoelectric transducer-based fibre-shifting endomicroscope is developed to enhance the resolution over conventional fibre-bundle based imaging systems. The fibre-shifting endomicroscope provides a two-fold improvement in resolution and coupled to a high-speed LS-CLE scanning system, provides real-time imaging of biological samples at 30 fps. These investigations furthered the utility and applications of the fibre-bundle based fluorescence systems for rapid imaging and diagnosis of cancer margins.Open Acces

Effect of early maternal newborn skin to skin contact in labour room on third stage of labour and success at breastfeeding

Background: Immediate postpartum period and birth pose many challenges for the mother and the new-born. Initiation of early skin to skin contact in the labour room can be beneficial to both of them.Methods: Randomized control trial conducted over a period of 7 months in a tertiary care centre enrolling 400 laboring women.200 in the control group were given routine care. In the 200women in the study group, the newborn was given immediate skin to skin contact by placing him/her on the mother’s chest.Results: Duration of third stage of labour was less than 10 minutes in 95%women of study group compared to 56% women in the control group(p<0.01). Placenta was expulsed as a whole in 98% cases in the study group compared to 81% in the control group. Successful breastfeeding was observed in 88% women in study group compared to 54%in the control group(p<0.01). Breastfeeding was initiated within 30 minutes of birth in 96%women in the study group compared to 41% in the control group.Conclusions: Uterus could contract faster with the complete expulsion of placenta and shortening of the third stage of labour with early skin to skin contact. The newborn showed early initiation, success at breastfeeding and longer first breastfeeding with early skin to skin contact

Virosome: A vector in vaccine delivery

These days vaccines are considered the best economical and effective technique for preventing and handling bacterial infections, like human papillomavirus (HPV) or meningitis, allergies, autoimmune Relevant Disorders, Microbial Infections, and many more viral diseases. Annually millions of lives are saved from death-causing infections and other relevant disorders with the help of proper immunization with the help of vaccines. But still, many diseases are not yet preventable by vaccines and there is a chance of the improvement of current vaccines with help of many approaches. One of these is a compound known as a virosome, which is produced when pure haemagglutinin and neuraminidase projections of the influenza virus surface are removed from viral envelope and deposited on the exterior of unilamellar liposomes.Studies have shown that in order to attain the desired therapeutic effect, a physical attachment between the target antigen and the virosomal carrier is necessary. A virosome can bind to and “infect” host cells and deliver the antigen directly into the processing pathway which is an Alternatively, the virosome may be phagocytosed by an APC. So Our review gives a novel idea of the vector technology for vaccine delivery, its properties, method of preparation, and importance as well as some applications of different virosomal vaccine discoveries

Methylene-blue aided rapid confocal laser endomicroscopy of breast cancer

Breast conserving surgery allows complete tumor resection while maintaining acceptable cosmesis for patients. Safe and rapid intraoperative margin assessment during the procedure is important to establish the completeness of tumor excision and minimizes the need for reoperation. Confocal laser endomicroscopy has demonstrated promise for real-time intraoperative margin assessment using acriflavine staining, but it is not approved for routine in-human use. We describe a custom high-speed line-scan confocal laser endomicroscopy (LS-CLE) system at 660 nm that enables high-resolution histomorphological imaging of breast tissue stained with methylene-blue, an alternative fluorescent stain for localizing sentinel nodes during breast surgery. Preliminary imaging results on freshly excised human breast tissue specimens are presented, demonstrating the potential of methylene-blue aided rapid LS-CLE to determine the oncological status of surgical margins in-vivo

En-face optical coherence tomography/fluorescence endomicroscopy for minimally invasive imaging using a robotic scanner

We report a compact rigid instrument capable of delivering en-face optical coherence tomography (OCT) images alongside (epi)-fluorescence endomicroscopy (FEM) images by means of a robotic scanning device. Two working imaging channels are included: one for a one-dimensional scanning, forward-viewing OCT probe and another for a fiber bundle used for the FEM system. The robotic scanning system provides the second axis of scanning for the OCT channel while allowing the field of view (FoV) of the FEM channel to be increased by mosaicking. The OCT channel has resolutions of 25  /  60  μm (axial/lateral) and can provide en-face images with an FoV of 1.6  ×  2.7  mm2. The FEM channel has a lateral resolution of better than 8  μm and can generate an FoV of 0.53  ×  3.25  mm2 through mosaicking. The reproducibility of the scanning was determined using phantoms to be better than the lateral resolution of the OCT channel. Combined OCT and FEM imaging were validated with ex-vivo ovine and porcine tissues, with the instrument mounted on an arm to ensure constant contact of the probe with the tissue. The OCT imaging system alone was validated for in-vivo human dermal imaging with the handheld instrument. In both cases, the instrument was capable of resolving fine features such as the sweat glands in human dermal tissue and the alveoli in porcine lung tissue

A novel method for diameter estimation of small opaque objects using Fraunhofer diffraction

A novel optical method is proposed and demonstrated, for real-time dimension estimation of thin opaque cylindrical objects. The methodology relies on free-space Fraunhofer diffraction principle. The central region, of such tailored diffraction pattern obtained under suitable choice of illumination conditions, comprises of a pair of `equal intensity maxima', whose separation remains constant and independent of the diameter of the diffracting object. An analysis of `the intensity distribution in this region' reveals the following. At a point symmetrically located between the said maxima, the light intensity varies characteristically with diameter of the diffracting object, exhibiting a relatively stronger intensity modulation under spherical wave illumination than under a plane wave illumination. The analysis reveals further, that the said intensity variation with diameter is controllable by the illumination conditions. Exploiting these `hitherto unexplored' features, the present communication reports for the first time, a reliable method of estimating diameter of thin opaque cylindrical objects in real-time, with nanometer resolution from single point intensity measurement. Based on the proposed methodology, results of few simulation and experimental investigations carried-out on metallic wires with diameters spanning the range of 5 to 50 mu m, are presented. The results show that proposed method is well-suited for high resolution on-line monitoring of ultrathin wire diameters, extensively used in micro-mechanics and semiconductor industries, where the conventional diffraction-based methods fail to produce accurate results

Time-frequency analysis of human motion during rhythmic exercises

Biomechanical signals due to human movements during exercise are represented in time-frequency domain using Wigner Distribution Function (WDF). Analysis based on WDF reveals instantaneous spectral and power changes during a rhythmic exercise. Investigations were carried out on 11 healthy subjects who performed 5 cycles of sun salutation, with a body-mounted Inertial Measurement Unit (IMU) as a motion sensor. Variance of Instantaneous Frequency (I.F) and Instantaneous Power (I.P) for performance analysis of the subject is estimated using one-way ANOVA model. Results reveal that joint Time-Frequency analysis of biomechanical signals during motion facilitates a better understanding of grace and consistency during rhythmic exercise