Laser−Micropipet Combination for Single-Cell Analysis

Abstract

Due to its potential for exquisite mass detection limits and resolving power, capillary electrophoresis is used for biochemical measurements on single cells; however, accurate measurements of many physiological parameters require sampling strategies that are considerably faster than those presently available. We have developed a laser-based technique to lyse single, adherent, mammalian cells on millisecond time scales. The cellular contents are then introduced into a capillary where electrophoretic separation and detection are performed. Improved temporal resolution of biological measurements results from the extremely rapid lysis made possible by this method. Additionally, the cell is not perturbed by mechanical or electrical stresses prior to sampling. Such disturbances can alter cellular physiology, resulting in inaccurate measurements. The fast cell lysis, the absence of cellular stresses prior to lysis, and the application to adherent mammalian cells are significant refinements to CE-based measurements on single cells. With this lasermicropipet combination, it will be possible to measure the intracellular concentration of molecules that change on subsecond to second time scales, for example, substrates of many cellular enzymes.

Myristoyl-Based Transport of Peptides into Living Cells

“…This CE-based method provides a quantitative measure of the fluorescent species obtained from single cells as previously described (37,43,45,46). After trypsin and TCEP exposure, the electrophoretic traces obtained from these cells (n = 12) demonstrated predominately a single peak that comigrated with the nonphosphorylated standard for the ABL peptide domain used in these experiments (Fig.…”

“…Cells were maintained at 37°C and constantly perfused with warmed ECB. A cell was selected for analysis, lysed using a Nd:YAG-laser, and loaded into the capillary by electrophoresis (30 kV, negative polarity) as previously described (37). At 10 seconds after cell sampling, electrophoresis was halted, the capillary was moved to the run buffer (100 mM Tris, 100 mM tricine, pH = 8.0), and electrophoresis was continued.…”

Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

“…A sample set of this data is shown in Figure 6b. Conservation of mass in this system provides the following relationship between the external fluid velocity V ∞ (r, t) and the bubble dynamics as: (1) Conservation of momentum was then applied to obtain the following expression for the wall shear stress τ w (r, t) generated by the cavitation bubble expansion [20]: (2) where ρ and ν are is the density and kinematic viscosity of the fluid medium [20], Equation (2) is valid at any radial position r and time 0 ≤ t ≤ t′ where t′ is the time of arrival of the bubble wall at position r.…”

“…As a result, there is an increasing interest to use pulsed laser microbeams for precise cellular manipulation, including laserinduced cell lysis [1], cell microdissection and catapulting [2][3][4][5], cell collection, expansion, and purification [6][7][8], cellular microsurgery [9][10][11], and cell membrane permeabilization for the delivery of membrane-impermeant molecules into cells [12 15], The processes of laser-induced optoinjection and optoporation offer the ability to load cells with a variety of biomolecules on short time scales (milliseconds to seconds) through optically produced cell membrane permeabilization [12,14,15], Despite the innovative utilization of laser microbeams in cell biology and biotechnology, only recently have studies provided insight regarding the mechanisms that mediate the interactions of highly focused pulsed laser beams with cells [16][17][18][19][20][21][22], A better understanding of these processes will prove critical to the continued development of laser microbeams for both research and practical applications. In previous studies, we provided a detailed characterization of the physics involved in the interaction of highly-focused nanosecond laser microbeams with cells [19,20], However, it is important to relate these physical effects to the biological response of the cells.…”

Chemical cytometry on a picoliter-scale integrated microfluidic chip

“…This volume compares favorably with the nanoliter volumes reported for derivatization reactions in capillary-based chemical cytometry methods (1)(2)(3)(4)(5), although smaller volumes have been reported (26). The capacity to confine a lysing, reacting plug of cell and reagents is an advantage that motivated prior work by us (8) and others (18) to use valves formed by multilayer soft lithography to confine approximately nanoliter volumes.…”