Jennifer L. Lubbeck

Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability

This paper presents a novel microfluidic cytometer for mammalian cells that rapidly measures the irreversible photobleaching of red fluorescent proteins expressed within each cell and achieves high purity (>99%) selection of individual cells based on these measurements. The selection is achieved by using sub-millisecond timed control of a piezo-tilt mirror to steer a focused 1064-nm laser spot for optical gradient force switching following analysis of the fluorescence signals from passage of the cell through a series of 532-nm laser beams. In transit through each beam, the fluorescent proteins within the cell undergo conversion to dark states, but the microfluidic chip enables the cell to pass sufficiently slowly that recovery from reversible dark states occurs between beams, thereby enabling irreversible photobleaching to be quantified separately from the reversible dark-state conversion. The microfluidic platform achieves sorting of samples down to sub-millilitre volumes with minimal loss, wherein collected cells remain alive and can subsequently proliferate. The instrument provides a unique first tool for rapid selection of individual mammalian cells on the merits of photostability and is likely to form the basis of subsequent lab-on-a-chip platforms that combine photobleaching with other spectroscopic measurements for on-going research to develop advanced red fluorescent proteins by screening of genetic libraries.

Microfluidic flow cytometer for quantifying photobleaching of fluorescent proteins in cells.

Traditional flow cytometers are capable of rapid cellular assays on the basis of fluorescence intensity and light scatter. Microfluidic flow cytometers have largely followed the same path of technological development as their traditional counterparts; however, the significantly smaller transport distance and resulting lower cell speeds in microchannels provides for the opportunity to detect novel spectroscopic signatures based on multiple, nontemporally coincident excitation beams. Here, we characterize the design and operation of a cytometer with a three-beam, probe/bleach/probe geometry, employing HeLa suspension cells expressing fluorescent proteins. The data collection rate exceeds 20 cells/s under a range of beam intensities (5 kW to 179 kW/cm(2)). The measured percent photobleaching (ratio of fluorescence intensities excited by the first and third beams: S(beam3)/S(beam1)) partially resolves a mixture of four red fluorescent proteins in mixed samples. Photokinetic simulations are presented and demonstrate that the percent photobleaching reflects a combination of the reversible and irreversible photobleaching kinetics. By introducing a photobleaching optical signature, which complements traditional fluorescence intensity-based detection, this method adds another dimension to multichannel fluorescence cytometry and provides a means for flow-cytometry-based screening of directed libraries of fluorescent protein photobleaching.

High-speed multiparameter photophysical analyses of fluorophore libraries.

There is a critical need for high-speed multiparameter photophysical measurements of large libraries of fluorescent probe variants for imaging and biosensor development. We present a microfluidic flow cytometer that rapidly assays 10(4)-10(5) member cell-based fluorophore libraries, simultaneously measuring fluorescence lifetime and photobleaching. Together, these photophysical characteristics determine imaging performance. We demonstrate the ability to resolve the diverse photophysical characteristics of different library types and the ability to identify rare populations.

Microfluidic Flow Cytometer for Multiparameter Screening of Fluorophore Photophysics

We present a microfluidic cytometer that sorts mammalian or yeast cells by laser force deflection following real-time multibeam, multiparameter fluorescence measurements, including photobleaching, lifetime and expression level, of the intrinsic fluorophores within each cell.

Real-time analysis of multi-laser-beam fluorescence for timed control of laser tweezers in a microfluidic cell-sorting device

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.