Download Download PDF

FACILE IMAGE-BASED CELL SORTING USING OPTOFLUCS (OPTO-FLUIDIC CELL SORTING)
1
Joseph Kovac1 and Joel Voldman1
Electrical Engineering and Computer Science,
Massachusetts Institute of Technology, USA
Abstract
We present a new approach for sorting cells based upon visual information,
termed opto-fluidic cell sorting (OPTO-FluCS). This technique uses a combination of
hydrodynamic and optical scattering forces to array, image, and then sort many
individual cells. Our device combines the strengths of microscopy–imaging in space
and time–with the ability to array cell populations and isolate cells of interest in an
intuitive, user-friendly fashion. We successfully demonstrate image-based sorting
based on fluorescence localization, a sort unavailable with flow-assisted cell sorting.
Keywords: Cell sorting, microwell array, optical manipulation, optofluidics
1. Introduction
We have developed technology to sort cells based upon visual information. Our
opto-fluidic architecture obtains sorting information through microscopy, predicating
sorts on temporal and spatial behavior of cells. While flow-assisted cell sorting (FACS)
allows higher throughput, it bases sorts on whole-cell fluorescence at a single timepoint.
Our approach scales effortlessly, requiring no electrical interconnects or support
electronics to address cells [1]. Sorting of a single cell takes seconds, allowing for
collection
of
larger
numbers
of
cells
than
is
practical
with
micropipette/micromanipulator systems [2]. While laser capture microdissection
(LCM) has been used to sort non-viable cells from microwell arrays [3], its live-cell
sorts require the use of proprietary films. Our opto-fluidic array architecture scales
effortlessly to array sizes in excess of 20,000 sites, contains no interconnects, allows for
diverse surface functionalization, requires seconds to remove a cell of interest, and
provides for simple retrieval of released viable cells.
2. Architecture Design
There are two important elements to our system. First is a microfluidic chamber
(Figure 1) with a PDMS well array that we use for simple passive cell loading via
sedimentation and subsequent flushing (Figure 2A-B). After visualizing cells using any
desired microscopy technique (phase, fluorescence, etc.), we note the positions of cells
of interest. Second, we use a straightforward optical system to couple a laser onto the
imaging axis. We remove a target cell by focusing the laser through an inexpensive
objective onto the cell; the optical scattering force pushes the cell into the flow (Figure
2C). The optical intensity and energy-density levels that we apply to cells are orders of
magnitude below those found harmful in conventional, high-numerical aperture optical
tweezers, and thus our approach is unlikely to damage cells [4].
The 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS2006)
November 5-9, 2006, Tokyo, Japan
4-9903269-0-3-C3043 © 2006 Society for Chemistry and Micro-Nano Systems
1483
CCD
Tubing
F TL M2
Glass
PDMS
Glass
O
Laser
M1
BE BS
Figure 1: Device structure and layout. M1,M2: Mirror; BS: Beamsplitter;
BE: Beam Expander TL: Tube Lens; F: Filter; O: Objective, 10x, 0.25 NA
(A)
(B)
(C)
Figure 2: Device operation. A) We inject cells into the device, where some
cells settle into wells. B) We clear cells outside wells via flow. We then use
any microscopy technique to locate cells of interest. C) Using the optical
scattering force, we briefly push cells of interest into the flow field for
downstream collection. Here we schematically show a sort based on
fluorescent probe location (membrane vs. cytoplasm), a sort that cannot be
performed using FACS.
3. Results and Discussion
As a first demonstration, we loaded orange- and green-labeled HL60 cells
randomly into the device, imaged them, and then sorted out the orange-labeled cells
(Figure 3).
Phase +
Phase +
Fluorescence Fluorescence Fluorescence Fluorescence
(A) Before
(B) After
Figure 3: Cell sorting. A) Random mixture of orange/green-labeled HL60 cells
(orange cell locations circled). B) Images after removing orange cells.
The 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS2006)
November 5-9, 2006, Tokyo, Japan
1484
(A) Before
(B) After
Phase
+
Fluor.
Fluor.
Figure 4: Image-based cell sorting. A) We added a small fraction of nuclearand membrane-labeled cells to nuclear-stained cells. Here we use microscopy
to find the rare membrane-labeled cells, which are circled above. B) We
selectively removed the membrane-labeled cells, demonstrating image-based
sorting based upon localization. The use of different color stains is merely for
verification; the assay would work if the dyes were the same color.
We demonstrated true image-based sorting by loading the device with nuclearstained cells and a small fraction of nuclear- and membrane-stained cells (Figure 4).
Using microscopy, we identified the cells with membrane stain and sorted them from
the cells only labeled with nuclear stain. This demonstrates image-based sorting based
upon localization, an assay that cannot be performed using flow cytometry.
4. Conclusions
Our results demonstrate the feasibility of sorting cells via microscopy in a userfriendly fashion. The simplicity of the optical system allows for straightforward
incorporation of the system into widely used microscopes. Using this novel
architecture, we aim to sort cells predicated on complex phenotypes in realms where
traditional flow-assisted cell sorting (FACS) falls short.
Acknowledgements: We thank David Appleyard, Dr. Stanley Hong, Prof. Matthew
Lang, and Prof. Dennis Freeman for their advice. This work was supported in part by
the NIH, the Singapore-MIT Alliance, and a National Defense Science and Engineering
Graduate Fellowship.
References
1.
Taff, B.M. and J. Voldman, A scalable addressable positive-dielectrophoretic
cell-sorting array. Anal. Chem., 2005. 77(24): p. 7976-7983.
2.
Yamamura, S., et al., Single-cell microarray for analyzing cellular response.
Anal. Chem., 2005. 77(24): p. 8050-8056.
3.
Revzin, A., et al., Development of a microfabricated cytometry platform for
characterization and sorting of individual leukocytes. Lab Chip, 2005. 5(1): p. 30-37.
4.
Wang, M.M., et al., Microfluidic sorting of mammalian cells by optical force
switching. Nat. Biotechnol., 2005. 23(1): p. 83-87.
The 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS2006)
November 5-9, 2006, Tokyo, Japan
1485