INTRINSIC
AUTOFLUORESCENCE
FREE CELL SORTING
J. Emmelkampl,
OF SINGLE LIVING CELLS FOR LABELIN A MICROFLUIDIC
SYSTEM
R. DaCosta2, H. Andersson1’3, A. van den Berg’
rMESA’ Institute, BIOS group, University of Twente, Eizschede, The Netherlands
“Dept. ofMedical Biophysics, Universit?; qf Toronto, Toronto, Canada
3Dept. of Signals, Sensors and Systems, Royal Institute of Technology, Stockholm, Sweden
Abstract
Results of autofluorescence
(AF) detection of a variety of living human cells in
microfluidic structures are shown to demonstrate its potential for use in a micro cellsorter. Cells were excited with 488 nm and the emission was collected between 505 and
530 nm using a confocal microscope.
A simple microfluidic
three-port
glass
microstructure
was used.
Discrimination
of granulocytes
from red blood cells was
performed with measuring the levels of intrinsic AF. Also the location of AF areas of
melanoma cells is showed.
Keywords: autofluorescence, cytometry, cell sorting, blood cells
1. Introduction
Laser induced autofluorescence
(AF) is a commonly used additional technique for
distinguishing
normal from diseased human tissues.
As normal cells undergo
pathological transformation, distinct changes occur in both the phenotype and genotype of
the cells. Cellular AF reflects changes in the cellular physiology that are associated with
disease progression; and so AF may serve as a diagnostic tool to detect these changes [ 11.
Figure 1. A picture of a fibroblast,
where
the
light
colored
signal
corresponds to the intrinsic AF.
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Figure 1 shows a fibroblast
where the
intracellular AF appears as the light colored
signal.
The AF is restricted to small round
organelles
(probably
mitochondria
and
lysosomes)
clustered
tightly
around
the
nucleus, while the nucleus remains dark [2].
Red blood cells have very low AF signal due
to the lack of organelles.
White blood cells
are, however, highly autofluorescent,
which
allows these two different cell types to be
differentiated.
Microfabricated
cell sorting devices offer a
number
of advantages
over conventional
fluorescence-activated
cell sorter (FACS),
since they are less costly, smaller and more
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efficient detection
[3].
Several examples of micro FACS 0,tFACS) have been
demonstrated
where the cells are manipulated
by hydrodynamic,
electrokinetic,
and
electroosmotic forces [3-51.
Although there is increasing interest in using microfluidic devices for cellomics [6-81,
until today, there have been no reports on studying cellular AF of living cells in such
microstructures.
The ultimate goal of this study is to develop a microfluidic cell sorter
that sorts cells (for example normal from cancer cells) based on difference of their
intrinsic AF. No labeling is required which is an advantage due to a reduction in sample
preparation steps.
In this study we use conventional electro-osmotic flow (EOF) to obtain cell sorting by
switching the flow containing the cells.
2. Experimental
Figure 2(a) shows a schematic of the chip and experimental set-up that we use. Figure
2(b) shows a photo of the microfluidic chip. The hydrofluoric (ELF)-etched channels in
the glass (Borofloat’) chip have a depth of 50 urn and a width of 110 urn. The Pyrex@
(7740, Corning) cover plate of the chip has been back-etched in HF to a thickness of 167
Figure
2
microfluidic
(a). A schematic
cell-sorting set-up.
of
the
Figure 2 (b). A photograph
microfluidic glass chip.
of
the
Using EOF electrodes connected to the inlet and the two outlets of the chip, we can
control the cell sorting by switching the EOF from the inlet to one of the two outlets. As
the switching times for the EOF electrodes are very short, the overall switching time is
still determined by the optical detection and uncertainty
in transfer time from the
detection location to the outlet channels.
The excitation wavelength of 488 run from an argon laser was used to collect the AF
signals of the cells on a confocal fluorescence microscope (Zeiss LSM 510) was used
while the emission was collected between 505 and 530 nm. AF images were overlapped
on to light transmission images of the same cells by an image viewer (Zeiss LSM 5 IO).
Living human melanoma (HTB-67) cells, granulocytes and red blood cells were used.
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Results and Discussion
Figure 3 (a) shows a free flowing HTB-67 cell in a microfluidic
channel.
The
corresponding AF photo of the same cell is shown in Figure 3 (b). In Figure 4, a higher
magnification
of two different HTB-67 cells is shown.
Individual cells are easily
distinguished and the non-fluorescent
nuclei are clearly identifiable; which corresponds
well with the characteristic AF pattern shown in Figure 1. Figure 5 shows a combined
picture of an AF signal overlaid on a transmission light image containing three human
granulocytes in between of red blood cells. This picture shows that the granulocytes have
a very high AF signal, and the red blood cells have almost none.
3.
Figure 3 (a). Photo showing a free
flowing cell in a microfluidic channel.
Figure 3 (b). The corresponding
of the cell shown in (a).
AF signal
Figure 4. A higher magnification
of
two cells.
The AF signal is mainly
originating from the cytosol while the
nuclei are non-fluorescent.
Figure 5. A combined picture of the AF
signal (3 light spots) with transmitted light
from three human granulocytes
amongst
red blood cells.
The blurriness of the pictures is a result of the movement of the cells in the flow channel.
A line intensity scan of the AF signal at 505-530 nm with the transmitted light of 488
nm across two granulocytes and several red blood cells is shown in Figure 6. The image
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viewer has taken the scan over a distance of 206 um in the middle of the microfluidic
channel in the length direction of the charmel. The intensity range was set to 0 - 256.
In the scan two granulocytes
have
a high relative AF signal
intensity
of 256 at a distance of about 40
250
and 90 pm. The scan covers
200
-AF
slgnal
several red blood cells at about
30, 80, 110, 160, 180 and 205
150
-----transmltted
laser
urn. These can be seen as the
intensity of the transmitted light
100
drops
and
the
fluctuation
50
increases,
but the AF signal
does not increase and stays at
0
the noise level of about 40. In
0
50
100
150
200
the distances of about 0 to 20
distance (pm)
urn, 50 to 70 urn and 130 to 140
urn no cells are located.
Figure 6. Intensity line scan of the AF signal of two
To our knowledge, this is the
granulocytes and several red blood cells at 505-530
first
demonstration
of using a
nm and the transmitted 488 nm laser light.
label-free,
AF-based
detection
in microstructures for living-cell sorting.
4. Conclusions
We have shown a new technique for sorting living cells based on their intrinsic AF
signal in a microfluidic structure. Detection of granulocytes between red blood cells
based on their intrinsic AF is possible. In this study with human melanoma cells it can be
concluded that the AF-signal is mainly localized from the cytosol.
Acknowledgement
Financial support from STW (project TMM 6016, “NanoSCAN”) and valuable work
by Jan van Nieuwkasteele and Floor Wolbers is gratefully acknowledged.
References
[l] R. DaCosta, B. Wilson, N. Marcon, J. Gastroenterol Hepatal., 17, S85-104,2002.
[2] H. Andersson, T. Baechi, M. Hoechl, C. Richter, J. Microscopy, 191, l-7, 1998.
[3] P. Tellernan, U.D. Larsen, J. Philip, G. Blankenstein, A. Wolff, uTAS, 39-44, 1998.
[4] A. Fu, C. Spence, A. Scherer, F. Arnold, S. Quake, Nature biotechnology,
17, 11091111,1999.
[5] P. Li, J. Harrison, Anal Chem, 69, 1564-1568, 1997.
[6] H. Andersson, A. van den Berg, Sensors and Actuators B, 92,3 15-325,2003.
[7] P. Dittrich, P. Schwille, Anal. Chem, 74,4472-4479,2002.
[8] S. Gawad, L. Schild, Ph. Renaud, Lab on a Chip, 1, 76-82, 2001.
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