Multiphoton imaging (MMPI) has become one of thee key non-invasive light microscopy techniques. This technique allows deep tissue imaging with high resolution and less photo-damage than conventional confocal microscopy. MPI is type of laser-scanning microscopy that employs localized nonlinear excitation, so that fluorescence is excited only with is scanned focal volume. For many years, Ti: sapphire femtosecond lasers have been the leading light sources for MPI applications. However, recent developments in laser sources and new types of fluorophores indicate that longer wavelength excitation could be a good alternative for these applications. Mode-locked VECSEELs have the potential to be low cost, compact light sources for MPI systems, with the additional advantage of broad wavelength coverage through use of different semiconductor material systems. Here, we use a femtosecond fibber laser to investigate the effect average power and repetition rate has on MPI image quality, to allow us to optimize our mode-locked VVECSELs for MPI.
Optical spectroscopy and imaging methods have proved to have potential to discriminate between normal and abnormal tissue types through minimally invasive procedures. Raman spectroscopy and Optical Coherence Tomography (OCT) provides chemical and morphological information of tissues respectively, which are complementary to each other. When used individually they might not be able to obtain high enough sensitivity and specificity that is clinically relevant. In this study we combined Raman spectroscopy information with information obtained from OCT to enhance the sensitivity and specificity in discriminating between Colonic Adenocarcinoma from Normal Colon. OCT being an imaging technique, the information from this technique is conventionally analyzed qualitatively. To combine with Raman spectroscopy information, it was essential to quantify the morphological information obtained from OCT. Texture analysis was used to extract information from OCT images, which in-turn was combined with the information obtained from Raman spectroscopy. The sensitivity and specificity of the classifier was estimated using leave one out cross validation (LOOCV) method where support vector machine (SVM) was used for binary classification of the tissues. The sensitivity obtained using Raman spectroscopy and OCT individually was 89% and 78% respectively and the specificity was 77% and 74% respectively. Combining the information derived using the two techniques increased both sensitivity and specificity to 94% demonstrating that combining complementary optical information enhances diagnostic accuracy. These results demonstrate that a multimodal approach using Raman-OCT would be able to enhance the diagnostic accuracy for identifying normal and cancerous tissue types.
Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person’s state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical.
Raman spectroscopy has proven to be a powerful tool for discriminating between normal and abnormal tissue types.
Fiber based Raman probes have demonstrated its potential for in vivo disease diagnostics. Combining Raman
spectroscopy with Magnetic Resonance Imaging (MRI) opens up new avenues for MR guided minimally invasive optical
biopsy. Although Raman probes are commercially available, they are not compatible with a MRI environment due to the
metallic components which are used to align the micro-optic components such as filters and lenses at the probe head.
Additionally they are not mechanically compatible with a typical surgical environment as factors such as sterility and
length of the probe are not addressed in those designs. We have developed an MRI compatible fiber Raman probe with a
disposable probe head hence maintaining sterility. The probe head was specially designed to avoid any material that
would cause MR imaging artefacts. The probe head that goes into patient’s body had a diameter <1.5 mm so that it is
compatible with biopsy needles and catheters. The probe has been tested in MR environment and has been proven to be
capable of obtaining Raman signal while the probe is under real-time MR guidance.
Femtosecond photoporation is an optical, non-invasive method of injecting membrane impermeable substances
contained within the surrounding medium into cells. The technique typically addresses individual cells in a static
monolayer. While this gives excellent selectivity, it can be time consuming or impractical to treat larger samples. We
build on previous work using a microfluidic platform, which allows for a suspension of cells to be dosed with
femtosecond light as they flow through a microfluidic channel. A reusuable quartz chip is designed with an 's'-bend with facilitates the delivery of a 'non-diffracting' femtosecond Bessel beam along the centre of the channel. By implementing off-chip hydrodynamic focusing, cells are confined to the central region of the channel and pass along the Bessel beam core where they are photoporated. This new parallel approach allows for higher flow rates to be used compared to the previous, orthogonal, design whilst maintaining the necessary dwell time in the Bessel beam core. Optical injection of the cell membrane impermeable stain propidium iodide has been successful with two cell lines. These have yielded viable injection efficiencies of 31.0±9.5% Chinese hamster ovary cells (CHO-K1) and 20.4±4.2% human promyelocytic cells (HL60) with a cell throughput of up to 10 cells/second. This marks an order of magnitude increase compared to the previous microfluidic design.
KEYWORDS: Raman spectroscopy, Luminescence, Tissues, Modulation, Signal to noise ratio, Bone, Tissue optics, Principal component analysis, Spectroscopy, Chemical analysis
In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time ( ∼ 30 s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.
Raman spectroscopy is a non-invasive technique offering great potential in the biomedical field for label-free
discrimination between normal and tumor cells based on their biochemical composition. First, this contribution describes
Raman spectra of lymphocytes after drying, in laser tweezers, and trapped in a microfluidic environment. Second,
spectral differences between lymphocytes and acute myeloid leukemia cells (OCI-AML3) are compared for these three
experimental conditions. Significant similarities of difference spectra are consistent with the biological relevance of the
spectral features. Third, modulated wavelength Raman spectroscopy has been applied to this model system to
demonstrate background suppression. Here, the laser excitation wavelength of 785 nm was modulated with a frequency
of 40 mHz by 0.6 nm. 40 spectra were accumulated with an exposure time of 5 seconds each. These data were subjected
to principal component analysis to calculate modulated Raman signatures. The loading of the principal component shows
characteristics of first derivatives with derivative like band shapes. The derivative of this loading corresponds to a
pseudo-second derivative spectrum and enables to determine band positions.
Cell transfection is the process in which extra cellular nucleic acids such as DNA, RNA, Si-RNA can be deliberately
injected into the cytoplasm of the cell. This technique of cell transfection forms a central tool in the hands of a cell
biologist to explore the mechanism within the cell. In optical transfection a well focused laser spot alters the permeability
of the cell membrane so as to allow the entry of extra-nuclear materials into the cell. Femto-second optical transfection
have proved to be better than other laser based cell transfection, owing to the three dimensionally confined multi-photon
effects on the cell membrane thereby leaving the rest of the cell unaffected. Even though the femto-second optical
transfection has proved to be sterile, non-invasive and highly selective, it has to improve in terms of efficiency, and
throughput to address real life problems. We report here a method to achieve significant enhancement in the efficiency of
femto-second optical transfection. The protocol of the transfection procedure is modified by adding a suitable biochemical
reagent - Nupherin-neuron - into the cell medium during the transfection, which can assist the delivery of
DNA into the nucleus once the DNA gets injected into the cytoplasm of the cell. We achieved a 3 fold enhancement in
the transfection efficiency with this modified protocol. Also we report for the first time the transfection of recently
trypsinised cells with a very high transfection efficiency, which would pave way to the development of high throughput
microfluidic optical transfection devices.
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