Jonas Enger

Resonance Raman spectroscopy of optically trapped functional erythrocytes

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.

Optical manipulation and microfluidics for studies of single cell dynamics

Most research on optical manipulation aims towards investigation and development of the system itself. In this paper we show how optical manipulation, imaging and microfluidics can be combined for investigations of single cells. Microfluidic systems have been fabricated and are used, in combination with optical tweezers, to enable environmental changes for single cells. The environment within the microfluidic system has been modelled to ensure control of the process. Three biological model systems have been studied with different combinations of optical manipulation, imaging techniques and microfluidics. In Saccharomyces cerevisiae, environmentally induced size modulations and spatial localization of proteins have been studied to elucidate various signalling pathways. In a similar manner the oxygenation cycle of single red blood cells was triggered and mapped using Raman spectroscopy. In the third experiment the forces between the endoplasmic reticulum and chloroplasts were studied in Pisum sativum and Arabidopsis thaliana. By combining different techniques we make advanced biological research possible, revealing information on a cellular level that is impossible to obtain with traditional techniques.

Direct detection of antimony in environmental and biological samples at trace concentrations by laser-induced fluorescence in graphite furnace with an intensified charge coupled device

The technique of laser-induced fluorescence in graphite furnace (LIF-GF) with intensified charge coupled device (ICCD) detection was used for the detection of Sb at pg ml–1 concentrations in various biological and environmental samples. The ICCD detector permits the simultaneous multi-channel detection of large fluorescence wavelength regions, which gives the user the possibility to control and correct for various background signals (which is important when complex environmental and biological samples are to be analysed). The detection limit for Sb in a pure water solution was found to be 5 fg. Antimony was directly detected in pure aqueous solutions down to fg ml–1 concentrations. A variety of aqueous and solid environmental and biological samples were investigated with respect to their Sb content. Good agreement between the measured and certified Sb contents (at pg ml–1–ng ml–1 levels) was obtained for various certified reference materials, viz., marine sediments (MESS-1 and BCSS-1) and riverine water (SLRS-2). Measurements of the Sb content in non-certified natural drinking water, estuarine water reference material (SLEW-1), serum reference material (Seronorm), and whole blood of healthy Swedish people were also performed. After a thorough investigation and elimination of various sources of contamination (regarding sampling and sample storage), typical levels of Sb in human whole blood at or below several tens of pg ml–1 were obtained. These levels are significantly lower than previously established values of the normal Sb content in human blood. The detection limit for Sb in human whole blood was close to that of pure water.

Holographic optical tweezers combined with a microfluidic device for exposing cells to fast environmental changes

Optical manipulation techniques have become an important research tool for single cell experiments in microbiology. Using optical tweezers, single cells can be trapped and held during long experiments without risk of cross contamination or compromising viability. However, it is often desirable to not only control the position of a cell, but also to control its environment. We have developed a method that combines optical tweezers with a microfluidic device. The microfluidic system is fabricated by soft lithography in which a constant flow is established by a syringe pump. In the microfluidic system multiple laminar flows of different media are combined into a single channel, where the fluid streams couple viscously. Adjacent media will mix only by diffusion, and consequently two different environments will be separated by a mixing region a few tens of micrometers wide. Thus, by moving optically trapped cells from one medium to another we are able to change the local environment of the cells in a fraction of a second. The time needed to establish a change in environment depends on several factors such as the strength of the optical traps and the steepness of the concentration gradient in the mixing region. By introducing dynamic holographic optical tweezers several cells can be trapped and analyzed simultaneously, thus shortening data acquisition time. The power of this system is demonstrated on yeast (Saccharomyces cerevisiae) subjected to osmotic stress, where the volume of the yeast cell and the spatial localization of green fluorescent proteins (GFP) are monitored using fluorescence microscopy.

Detection of titanium in electrothermal atomizers by laser-induced fluorescence. Part 1. Determination of optimum excitation and detection wavelengths

Abstract A detailed investigation has been performed of suitable excitation and detection wavelengths of Ti for the technique of laser-induced fluorescence in electrothermal atomizers. Fluorescence spectra (most often in the 250–350 nm region) from the 39 excitation wavelengths conjectured to be the most important (in the 220–270 nm region) have been investigated in more detail by the use of an intensified CCD detector. The fluorescence spectra were found to have a rich occurrence of peaks (about 50 each), of which many cannot be found in existing atomic wavelength data in the literature. All of the peaks found (with one exception) could be identified thanks to a home-made program that calculates atomic wavelengths from existing energy level data. Most of the spectra are dominated by “indirect” transitions (i.e. fluorescence from an upper level that is different from the one accessed by the laser) despite the prediction of existing “direct” transitions. This indicates that the collisional redistribution processes the excited levels, in general, are faster than typical fluorescence rates for Ti in graphite furnaces. Suitable excitation and detection wavelength combinations are given. One such suitable choice is excitation at 264.662 nm since the fluorescence following this excitation consisted of peaks of almost equal magnitude in three different wavelength regions (around 295, 319 and 338 nm).

Laser-induced fluorescence in a graphite furnace as a sensitive technique for assessment of traces in North Arctic atmospheric aerosol samples

An improved version of the highly sensitive laser-based spectroscopic trace-element detection technique, laser-induced fluorescence in a graphite furnace, LIF–GF (often also referred to as laser-excited atomic fluorescence spectrometry in electrothermal atomizer, ETA–LEAFS) has been used to assess the trace-element content of Al and Pb in size-fractionated aerosol samples from the Norwegian Arctic. The ordinary LIF–GF technique has been modified for improved selectivity by the incorporation of a multi-channel intensified CCD detector (ICCD) which makes constant monitoring of various background signals possible (scattered laser light, concomitant fluorescence light, and black body radiation). It is shown that the sensitivity and selectivity of the LIF–GF–ICCD technique is sufficient for efficient detection of the trace contents of Al and Pb in dissolved aerosol samples from the Norwegian Arctic (0–75 pg for each furnace heating). The Al and Pb concentrations in air from Ny Alesund, Svalbard, at the time of sampling (March–April 1992) were found to be 1–50 ng m–3.

Detection of trace amounts of Ni by laser-induced fluorescence in graphite furnace with intensified charge coupled device

Abstract A laser-induced fluorescence in graphite furnace (LIF-GF) set-up has been equipped with an intensified CCD detector (ICCD) that enables simultaneous multichannel detection of large wavelength regions. The main advantages of such a system in comparison with conventional photomultiplier detection are: simultaneous detection of several fluorescence wavelengths for easy characterization of excitation and fluorescence spectra and for an increase of the absolute sensitivity and spectral selectivity; simultaneous monitoring of background signals, such as those due to matrix interferences, blackbody radiation and scattered laser light; decrease of the susceptibility to radio-frequency pick-ups emitted from the pump laser due to the delayed read-out procedure; time-resolved studies of fluorescence spectra for improved elemental selectivity or for studies of atomization processes, and a possibility to perform two-dimensional imaging of height distributions of atomization and, in combination with an imaging spectrometer, diffusion processes in the furnace. The first work on LIF-GF with ICCD detection has been performed on Ni. The most sensitive and versatile excitation and detection wavelengths have been identified. Detection limits in water solutions by the LIF-GF technique have been improved by two orders of magnitude and are found to be 0.015 pg with ICCD and 0.01 pg using a photomultiplier at the most sensitive excitation and detection wavelengths. Nickel in ng ml concentrations has been detected in aqueous standard reference samples with sodium concentrations ranging from μg ml to % (riverine water and estuarine water) with good accuracy and precision. The Ni contents of standard riverine and estuarine water were determined to 1.00 ± 0.11 and 0.75 ± 0.07 ng/ml, respectively. The certified concentrations are 1.03 ± 0.10 and 0.743 ± 0.078 ng ml .

An experimental setup for combining optical tweezers and laser scalpels with advanced imaging techniques

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.

Sorting particles in a microfluidic system using SLM-reconfigurable intensity patterns

We explore the Generalized Phase Contrast (GPC) approach for optical sorting in microfluidic systems. A microsystem is used in which two streams meet, interact and separate in an X-shaped channel. When the flow in the two arms of the X is balanced, the laminar flow that exists at very low Reynolds numbers ensures minimal stream blending and the fluid separates without mixing (i.e. diffusion is negligible). Optical forces due to an intensity pattern can be fashioned to induce a selective deflection of particles between the two streams. This method is known as optical fractionation (OF). In brief, OF uses the same mechanisms as optical tweezers to exert forces upon microscopic particles. OF has been shown to have an exponential size selectivity. This means that the interaction between the streams can be made to discriminate by particle size at a critical flow velocity. With correctly adjusted flow velocity, particles with a certain size will more often shift to the other stream than another particle size. One method for creating the light pattern is by interference of several beams that are variably attenuated using mechanical means. However, this approach offers low optical efficiency and is not easily reconfigured. The GPC method offers a solution that gives the possibility to instantaneously reconfigure the intensity pattern by a method that is inherently computer-controllable. This enables one to rapidly test various intensity patterns to optimize sorting of particles.

A versatile system for optical manipulation experiments

In this paper a versatile experimental system for optical levitation is presented. Microscopic liquid droplets are produced on demand from piezo-electrically driven dispensers. The charge of the droplets is controlled by applying an electric field on the piezo-dispenser head. The dispenser releases droplets into a vertically focused laser beam. The size and position in 3 dimensions of trapped droplets are measured using two orthogonally placed high speed cameras. Alternatively, the vertical position is determined by imaging scattered light onto a position sensitive detector. The charge of a trapped droplets is determined by recording its motion when an electric field is applied, and the charge can be altered by exposing the droplet to a radioactive source or UV light. Further, spectroscopic information of the trapped droplet is obtained by imaging the droplet on the entrance slit of a spectrometer. Finally, the trapping cell can be evacuated, allowing investigations of droplet dynamics in vacuum. The system is utilized to study a variety of physical phenomena, and three pilot experiments are given in this paper. First, a system used to control and measure the charge of the droplet is presented. Second, it is demonstrated how particles can be made to rotate and spin by trapping them using optical vortices. Finally, the Raman spectra of trapped glycerol droplets are obtained and analyzed. The long term goal of this work is to create a system where interactions of droplets with the surrounding medium or with other droplets can be studied with full control of all physical variables.