We have developed a microfluidic cell sorter for mammalian cells expressing intrinsic fluorescent proteins that enables selection of cells with proteins that have enhanced photophysical properties, such as reduced fluorescence photobleaching and/or reversible dark state conversion. Previous ensemble imaging studies have used an acousto-optic modulator (AOM) to provide millisecond pulsed laser illumination for in vivo assays that distinguish reversible darkstate conversion from irreversible photobleaching. However, in the sorter, cells are hydrodynamically focused into a stream, which flows through a series of 4 or 8 line-focused, continuous, 532 nm laser beams, such that each cell experiences a similar millisecond modulated excitation. The amplitude and timing of the fluorescence response from each of the beams are measured by a red-sensitive photomultiplier and analyzed in real time to separately determine initial fluorescence brightness and photobleaching characteristics. In addition, each cell’s flow speed is found from its time of passage through the beams, and if the analysis results are within adjustable limits, a 1064 nm optical trap beam is switched on and moved along an intersecting trajectory at a matching speed, so that the cell becomes deflected by the optical gradient forces towards another exit channel of the microfluidic device. The optical sorting of cells is similar to that demonstrated by others, except that the motion of the trap beam is achieved using a piezo mirror under computer control, rather than an AOM; also, rather than a single-beam brightness measure using a hardwired circuit, a more complex multi-beam analysis is performed in software using the Real-Time module of LabView (National Instruments) on a separate computer to achieve deterministic timing and low latency. The software displays updated statistics of the sort, obtained by counting cells that pass through an extra laser beam in the exit channel. A mixture of cells expressing different proteins was resolved to select those with slowest photobleaching. Cells collected from the instrument were viable and could reproduce.
It has recently been demonstrated that diode laser bars can be used to not only optically trap red blood cells in flowing
microfluidic systems but also, stretch, bend, and rotate them. To predict the complex cell behavior at different locations
along a linear trap, 3D optical force characterization is required. The driving force for cells or colloidal particles within
an optical trap is the thermal Brownian force where particle fluctuations can be considered a stochastic process. For
optical force quantification, we combine diode laser bar optical trapping with Gabor digital holography imaging to
perform subpixel resolution measurements of micron-sized particles positions along the laser bar. Here, diffraction
patterns produced by trapped particles illuminated by a He-Ne laser are recorded with a CMOS sensor at 1000 fps where
particle beam position reconstruction is performed using the angular spectrum method and centroid position detection.
3D optical forces are then calculated by three calibration methods: the equipartition theorem, Boltzmann probability
distribution, and power spectral density analysis for each particle in the trap. This simple approach for 3D tracking and
optical control can be implemented on any transmission microscope by adding a laser beam as the illumination source
instead of a white light source.
We report red blood cell (RBC) stretching using a Zeiss Axioplan microscope, modified for phase contrast and optical
trapping using a 808 nm diode laser bar, as a tool to characterize RBC dynamics along a linear optical trap. Phase
contrast offers a convenient method of converting small variations of refractive index into corresponding amplitude
changes, differentially enhancing the contrast near cell edges. We have investigated the behavior of RBCs within both
static and dynamic microfluidic environments with a linear optical stretcher. Studies within static systems allow
characterization of cell interactions with the line optical force field without the complicating forces associated with
hydrodynamics. In flowing, dynamic systems, cells stretch along the optical trap down microfluidic channels and are
eventually released to recover their original shape. We record the dynamic cell response with a CMOS camera at 250 fps
and extract cell contours with sub-pixel accuracy using derivative operators. To quantify cell deformability, we measure
the major and minor axes of individual cells both within and outside of the trap, which also allows measurement of cell
relaxation. In these studies, we observe that cell rotation, stretching, and bending along the linear optical trap, are tightly
coupled to the modulation of optical power and cell speed inside our microfluidic systems.
The measurement of cell elastic parameters using optical forces has great potential as a reagent-free method for cell classification, identification of phenotype, and detection of disease; however, the low throughput associated with the sequential isolation and probing of individual cells has significantly limited its utility and application. We demonstrate a single-beam, high-throughput method where optical forces are applied anisotropically to stretch swollen erythrocytes in microfluidic flow. We also present numerical simulations of model spherical elastic cells subjected to optical forces and show that dual, opposing optical traps are not required and that even a single linear trap can induce cell stretching, greatly simplifying experimental implementation. Last, we demonstrate how the elastic modulus of the cell can be determined from experimental measurements of the equilibrium deformation. This new optical approach has the potential to be readily integrated with other cytometric technologies and, with the capability of measuring cell populations, enabling true mechanical-property-based cell cytometry.
Photon echo spectroscopy has been used to resolve the amplitudes and time scales of reorganization resulting from electronic excitation of the chromophore in three fluorescein-binding antibodies. The spectral density of nuclear motions derived by fitting the data serves as a characterization of protein flexibility. The three antibodies show motions that range in time scale from tens of femtoseconds to nanoseconds. Relative to the others, one antibody, 4-4-20, possesses a rigid binding site, that likely results from a short and inflexible HCDR3 loop and residue TyrL32 acting as a 'molecular splint,' to rigidify the Ag across its most flexible degree of freedom. The remaining two antibodies possess binding sites that are considerably more flexible, possibly due to the increased length of the HCDR3 loops. These variations in binding site flexibility may result in differing mechanisms of antigen recognition, including lock-and-key, induced-fit, and conformational selection.
Using ultrafast x-ray diffraction from a laser-plasma x-ray source, we have observed coherent photon generation and propagation in bulk(111)-GaAs, (111)-Ge, and thin(111)-Ge- on-Si films. At higher optical pump fluences, ultrafast melting of Ge films is observed.
Optical pump, x-ray diffraction probe measurements have been used to study the lattice dynamics of single crystals with picosecond-milliangstrom resolution by employing a table- top, laser-driven x-ray source. The x-ray source, consisting of an approximately 30 fs, 75 mJ/pulse, 20 Hz repetition rate, terawatt laser system and a moving Cu wire target assembly, generates approximately 5 X 10<SUP>10</SUP> photons (4π steradians s)<SUP>-1</SUP> of Cu K<sub>α</sub> radiation. Lattice spacing changes of as small as 1 X 10<SUP>-3</SUP> Å in a few picoseconds have been detected, utilizing Bragg diffraction from GaAs single crystals. Enhancement of the diffraction intensity associated with degradation of the crystals during and after the laser irradiation has been observed, likely due to a transition from dynamic to kinematic diffraction.