We have applied a CNN to preprocess Raman spectra from fresh tissue samples from brain tumors. The neural network can handle the variations that occur naturally, which enables explorative data analysis methods such as PCA.
Spectroscopic holography refers to techniques in which the detected hologram contains information about specific species in the medium under study. In general, at least two lasers are required with wavelengths chosen carefully to fit the interaction process utilized. In this process, energy from the shorter wavelength laser beam is transferred to the longer wavelength coherently through the process of stimulated emission. Two interaction mechanisms are considered; Stimulated Laser Induced Fluorescence (LIF) and Stimulated Raman Scattering (SRS), which both are species specific with the ability of coherent interaction. In this paper, the fundamental properties of spectroscopic holography is presented and demonstrated with a few idealized experiments. These validation experiments are performed in a gas chamber in which different gases may be blended and the gas pressure changed between 1-12 bars. In addition, two examples of applications are presented. In the first set of experiments, LIF holography is used to image light absorption and laser heating in a dye simultaneously. The second set of experiments is performed in a ow of methane gas. It is demonstrated that the combination of holographic phase measurements and SRS gain images may be used for calibration. This calibration may further be used to measure absolute concentration in a burning flame.
KEYWORDS: Microfluidics, Optical tweezers, Neurons, Spectroscopy, Lab on a chip, Hypoxia, Control systems, Proteins, Optical manipulation, System integration
We present a new approach of combining Lab-on-a-chip technologies with optical manipulation technique for accurate
investigations in the field of cell biology. A general concept was to develop and combine different methods to perform
advanced electrophysiological investigations of an individual living cell under optimal control of the surrounding
environment. The conventional patch clamp technique was customized by modifying the open system with a gas-tight
multifunctional microfluidics system and optical trapping technique (optical tweezers).
The system offers possibilities to measure the electrical signaling and activity of the neuron under optimum conditions of
hypoxia and anoxia while the oxygenation state is controlled optically by means of a spectroscopic technique. A cellbased
microfluidics system with an integrated patch clamp pipette was developed successfully. Selectively, an individual
neuron is manipulated within the microchannels of the microfluidic system under a sufficient control of the environment.
Experiments were performed to manipulate single yeast cell and red blood cell (RBC) optically through the microfluidics
system toward an integrated patch clamp pipette. An absorption spectrum of a single RCB was recorded which showed
that laser light did not impinge on the spectroscopic spectrum of light. This is promising for further development of a
complete lab-on-a-chip system for patch clamp measurements.
We describe the possibility of using a microresonance Raman spectrometer combined with a microfluidic system and optical tweezers to study Escherichia coli (E. coli) overexpressing wild type (wt) neuroglobin (NGB) and its E7Leu mutant, respectively. NGB is a recently discovered heme protein and its function still is a matter of debate. So far, the protein has been studied in its purified form, and in vivo measurements on the single cell level could give more information. To study the feasibility of the combined techniques, the possibilities of the setup are investigated by taking spectra from single cells and clusters of cells. We find that the microresonance Raman technique enables studies of the wt NGB protein in a living cell under fluctuating aerobic and anaerobic conditions. E. coli cells overexpressing wt NGB are stable, and the reversible oxygenation-deoxygenation can be studied over a long period of time. Further, the experiment indicates the presence of an enzymatic system in the bacteria reducing the ferric form NGB. The study of E. coli cells overexpressing E7Leu NGB, on the other hand, gives insight into limiting factors of the setup, such as cell lysis, photoinduced chemistry, and protein concentrations.
In recent years there has been a growing interest in the use of optical manipulation techniques, such as optical
tweezers, in biological research as the full potential of such applications are being realized. Biological research is
developing towards the study of single entities to reveal new behaviors that cannot be discovered with more
traditional ensemble techniques. To be able to study single cells we have developed a new method where a
combination of micro-fluidics and optical tweezers was used. Micro-fluidic channels were fabricated using soft
lithography. The channels consisted of a Y-shaped junction were two channels merged into one. By flowing
different media in the two channels in laminar flow we were able to create a sharp concentration gradient at the
junction. Single cells were trapped by the tweezers and the micro-fluidic system allowed fast environmental
changes to be made for the cell in a reversible manner. The time required to change the surroundings of the cell
was limited to how sharp mixing region the system could create, thus how far the cells had to be moved using
the optical tweezers. With this new technique cellular response in single cells upon fast environmental changes
could be investigated in real time. The cellular response was detected by monitoring variations in the cell by
following the localization of fluorescently tagged proteins within the cell.
The average environmental response of red blood cells (RBCs) is routinely measured in ensemble studies, but in such investigations valuable information on the single cell level is obscured. In order to elucidate this hidden information is is important to enable the selection of single cells with certain properties while subsequent dynamics triggered by environmental stimulation are recorded in real time. It is also desirable to manipulate and control the cells under phsyiological conditions. As shown here, this can be achieved by combining optical tweezers with a confocal Raman set-up equipped with a microfluidic system. A micro-Raman set-up is combined with an optical trap with separate optical paths, lasers and objectives, which enables the acquisition of resonance Raman profils of single RBCs. The microfluidic system, giving full control over the media surrounding the cell, consists of a pattern of channels and reservoirs produced by electron beam lithography and moulded in PDMS. Fresh Hepes buffer or buffer containing sodium dithionite are transported through the channels using electro-osmotic flow, while the direct Raman response of the single optically trapped RBC is registered in another reservoir in the middle of the channel. Thus, it is possible to monitor the oxygenation cycle in a single cell and to study photo-induced chemistry. This experimental set-up has high potential for monitoring the drug response or conformational changes caused by other environmental stimuli for many types of single functional cells since "in vivo" conditions can be created.
We introduce a novel setup combining a micro-Raman spectrometer with external optical tweezers, suitable for resonance Raman studies of single functional trapped cells. The system differs from earlier setups in that two separate laser beams used for trapping and Raman excitation are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually to optimize the functionality of the setup and to enable the recording of resonance Raman profiles from a single trapped cell. Trapping is achieved by tightly focusing infrared (IR) diode laser radiation (830 nm) through an inverted oil-immersion objective, and resonance Raman scattering is excited by the lines of an argon:krypton ion laser. The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using different excitation lines (488.0, 514.5, and 568.2 nm) in resonance with the heme moiety and by studying spectral evolution during illumination. We found that great care has to be taken in order to avoid photodamage caused by the visible Raman excitation, whereas the IR trapping irradiation does not seem to harm the cells or alter the hemoglobin Raman spectra. Stronger photodamage is induced by Raman excitation using 488.0- and 514.5-nm irradiation, compared with excitation with the 568.2-nm line.
In this paper we will describe a system designed to combine optical tweezers and laser scalpels with confocal as well as epi-fluorescence microscopy. A continuos wave Nd:YVO4 laser is used to produce a dual optical tweezers, where each trap can be individually controlled. A second optical tweezers setup is based on a tunable titanium sapphire laser, which allows us to adjust the wavelength to minimize the damage to the cell under investigation. A pulsed nitrogen laser working at 337 nm forms a laser scalpel. The tweezers and scalpels are both incorporated in an inverted microscope equipped with epi-fluorescence and confocal imaging capabilities. In order to further control the sample we have developed a technique to tailor make the environment closest to the studied objects. Micrometer-sized structures such as channels and reservoirs have been produced in rubber silicon using lithographic methods. In combination with a micro-manipulator, our system can be used to extract single cells from a population of billions for further studies or growth.
It has recently been shown that the combination of Raman spectroscopy and optical tweezers constitute a powerful tool for biological studies. Raman spectra of single cells immobilized in a sterile surrounding can then be recorded without the risk of surface-induced morphological cell changes. Further, the complete cellular environment can be changed while measuring dynamics in real time. We here introduce a novel Raman tweezers set-up ideal for resonance Raman studies of single cells. The system differs from earlier set-ups in that two separate laser beams, used for trapping and Raman excitation, are combined in a double-microscope configuration. This has the advantage that the wavelength and power of the trapping and probe beam can be adjusted individually, for example in order to optimize the functionality of the set-up or to record resonance Raman profiles from the same trapped cell. Further, the tweezers can be removed from the system without affecting the spectrometer configuration. Trapping is achieved by tightly focusing IR diode laser radiation (830 nm) through an inverted oil immersion objective with high numerical aperture (NA = 1.25), while Raman scattering is excited by the lines of an ArKr ion-laser. The backscattered Raman signal is collected by a single-grating spectrometer equipped with a microscope and a 60x water-immersion objective (NA = 0.9). The functionality of the system is demonstrated by measurements of trapped single functional erythrocytes using differen excitation lines (488, 514.5 568.2 nm) in resonance with the heme moiety and by studying the spectral evolution during illumination.
Hemoglobin (Hb) within single erythrocytes (red blood cells), adsorbed on poly-lysine coated glass surfaces, was studied using resonance Raman spectroscopy and global Raman imaging. The erythrocytes were found to be sensitive to both surface adsorption and to the laser light. Topological changes of the cell membrane were observed immediately after cell adsorption in Raman images. We observed a photo-induced increase of the fluorescence background occurring simultaneously with a decrease in the Hb Raman signal. Concurrent changes in Raman spectra revealed a conversion of oxy-Hb to the met-Hb state. However, at a low accumulated photon dose, the preparation method enabled the recording of Raman spectra during the oxygenation cycle of a single red blood cell in buffer, which shows that Hb was in an in-vivo environment. Thus, Raman spectroscopy of functional Hb in isolated red blood cells is feasible.
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