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.
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.
Optical chromatography is a powerful technique, capable of separating micron-sized particles within a fluid flow, based
on their intrinsic properties, including size, shape and refractive index. Briefly, particles in a fluid flow are subject to two
forces, the Stokes drag force due to the fluid and then an introduced optical force as supplied by a laser beam, acting in
opposite but collinear directions. According to the particle's intrinsic hydrodynamic and optical properties, equilibrium
positions may form where the two forces balance, which is highly dependent on the properties of the particle and as a
result provides a means for spatial separation in a sample mixture. Optical chromatography is a passive sorting
technique, where pre-tagging of the particles of interest is not required, allowing for non-discrete distributions to be
evaluated and/or separated. Firstly we review the current stage of optical chromatography. We present a new advance in
optical chromatography potentially enabling the unique beam delivery properties of photonic crystal fiber (PCF) to be
employed and integrated into microfluidic chips. Also, for the first time a finite element method is applied to the optical
field in the theoretical analysis of optical chromatography, which is found to be in excellent agreement with the current
ray optics model, even for particles much smaller than the optical wavelength. This will pave the way for the technique
to be extended into the nanoparticle regime.
The dual beam fiber trap is an important tool in the field of optical micromanipulation. The characteristics of these traps
are governed by the fibers used. Photonic crystal fibers have emerged in recent years and may be engineered to have vastly
different properties to conventional fibers. In particular, endlessly single-mode photonic crystal fiber (ESM-PCF) will
guide any wavelength of light in a single-mode and is commercially available in core diameters up to 35 μm. By utilising
these unique properties, we show that it is possible to create novel dual beam ESM-PCF traps for micron size particles.
Firstly, we characterise an ESM-PCF trap when using a near-infrared laser coupled into a 25 μm core fiber when trapping
a sample in a square capillary. We calculate the trap stiffness for polymer micro-spheres and show that aside from the
expected confining potential, it is possible to create line and repulsive potentials. Interference effects due to the capillary
are observed. Secondly, we create a dual wavelength standing wave trap which can selectively move two sizes of particles
in an optical conveyor belt. Finally, we use a supercontinuum source to create the first white light dual beam fiber trap and
show that the low coherence length of the source results in interference free potentials. Overall PCF has great promise for
future studies.
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