TM MicCell - GeSiM mbH

In the Lab
Laser Manipulation of Cells
A so-called "optical tweezer" can be employed to manipulate
suspended cells. The necessary optical fibers can be arranged
perpendicular to the flow cell, but also parallel, i.e. in the
channel plane. In this latter case, the single-mode optical
fibers are exactly positioned by a narrow channel in the PDMS
(see picture on the right). The fibers are fed into this duct
along a mechanical guide by turning a knurled thumb screw
(picture below).
More Lab Applications
Our customers and collaborators have realized the following
projects (selection):
� Study of multi-color fluorescing particles (e.g. bacteria) in
a focused stream using fluorescence correlation
spectroscopy (FCS; bottom left picture)
� Generation of lateral concentration gradients in microchannels
with a honeycomb-like gradient mixer (bottom
right picture; channel width 50 µm)
� Nanostructuring through manipulation of single biopolymers
(e.g. DNA and motor proteins) in a directed flow
field (pictures on the right)
� Surface plasmon resonance (SPR)
Further applications are only limited by your imagination.
This arrangement of optical fibers in the PDMS channel plate
was used in a microfluidic "optical stretcher", a novel optical
trap that catches a suspended cell between two divergent,
counterpropagating laser beams (top right picture) that are
aligned to better than 3 µm. By raising the laser power, the
trapped cell is stretched by the two beams. The deformability
of a cell correlates with its developmental stage: undifferentiated
cells are softer than differentiated ones.
This is a quick method to detect cancer or stem cells. Aside
from marker-free diagnosis, sorting and thus quick enrichment
of cells from tissues may become feasible (joint project
with the University of Leipzig).
�-DNA gets attached with one end (circles) to the coverslip
surface and is stretched by applying a flow of 0.5 µl/s
(arrow). Upon flow reversal, the DNA molecules switch their
orientation within one second. This technique can be used in
nanotechnology, e.g. to produce nanowires. Pictures courtesy
S. Diez, MPI-CBG, Dresden. White bar: 10 µm.
Literature
Gast, F.-U. et al. (2006) The microscopy cell (MicCell), a
versatile modular flowthrough system for cell biology, biomaterial
research, and nanotechnology. Microfluidics and
Nanofluidics 2, 21-36
Gesellschaft für Silizium-Mikrosysteme mbH
Rossendorfer Technologiezentrum
Bautzner Landstrasse 45
01454 Grosserkmannsdorf, Germany
Phone +49 (0)351 - 2695 322
Fax +49 (0)351 - 2695 320
info@gesim.de
www.gesim.de Subject to change without notice
GeSiM
Gesellschaft für Silizium-Mikrosysteme mbH
MicCell
TM
Modular Microperfusion System for the Microscope
www.gesim.de

TM
The MicCell
Possible Applications
The study of biomolecules or cells often includes their immobilization
on surfaces. Microfluidic flowthrough systems are only
suited if the channels can be opened and closed reversibly, if
possible without tinkering with adhesive tape. Highly desirable
would be a modular fluidic system that can be easily adapted to
diverse analytical methods (e.g. electrochemical or optical) and
also efficiently cleaned.
For this purpose, GeSiM has developed microperfusion chambers
with removable coverslip for the microscope. By using a novel,
patented setup, these flow cells are mounted in seconds and can
be applied in cell imaging or single-molecule fluorescence experiments.
If needed, electrical signals can be processed or optical
fibers mounted. The system is completed by a computer-controlled
macrofluidic environment.
Layout and assembly of the system can be modified in a wide
range. So a bunch of different materials can be used and microvalves
and microelectrodes can be integrated. Due to the easy
and modular setup, even the most complicated method in
screening, diagnostics, and nanotechnology can be realized in a
customer-specific way.
Semi-automatic drug screening using adherent cells or tissue slices in
the microscope
Interaction of cells with immobilized proteins, oligosaccharides, lipids,
and other ligands
Viability testing (e.g. for cancer cells) and other cell-based tests
Cell adhesion tests with prepared surfaces
Laser manipulation of cells in the flow: e.g. with the "optical stretcher"
(for cancer diagnosis and to isolate stem cells)
DNA transfection in the flow
Measurement of the homogeneity of microbeads and size sorting
Single-molecule detection unsing fluorescent biomolecules (fluorescence
correlation spectroscopy in the flow, receptor-ligand binding studies,
motor protein kinetics, hybridization kinetics on microarrays, etc.)
Capillary electrophoresis (CE)
Application of hydrodynamic flow fields (e.g. to align macromolecules,
nanotechnology)
Generation of lateral concentration gradients in a microchannel
Activity measurment of electrically active cells on microelectrode arrays
Rapid prototyping of microsystems (e.g. for "stopped flow" or for
chemical synthesis) by use of PDMS
Study of non-transparent substrates in the flow with the fully revolvable
"sample carrier", etc.
Microelectrodes
Microelectrodes for heating and sensing are microstructured
in our cleanroom and those regions that must not come in
contact with the medium are passivated by vapor deposition
of SiO .
2
In the assembled MicCell, electrode pads are contacted by
spring contacts and the signals are then conducted to a
socket via a printed circuit board (shown in green). If
coverslip and channel plate are from glass, electrodes can be
present at both the top and the bottom of the channels.
Examples shown were
performed in cooperation
with Fraunhofer-IBMT,
Berlin, and Max-Planck
Institute for Polymer
Research, Mainz
Sample Carrier for Opaque Objects
The MicCell channel is normally translucent and thus allows
sample illumination from the rear side. Opaque objects can
be introduced into the channel with a pivotable sample carrier
(orange in the schematic drawing below) and observed in the
flow, e.g. for material testing.
GeSiM offers such a flowthrough cell that can hold objects up
to a size of 2.5x2.5 mm. As sample and sample holder are
intransparent, the sample must be illuminated from the
objective side, i.e. along the optical path. Its specialty: the
specimen can be rotated a full 360° around the optical axis of
the microscope, such that objects can be exposed to the flow
at an arbitrary angle.
Optional Design
Microelectrode Arrays (MEAs)
To measure signals of muscle or nerve cells, these cells can
be grown on coverslips containing microelectrode arrays (see
bottom picture) that are then mounted into a flow channel.
The flow system shown can process electrical potentials while
the cells are being inspected under a microscope.
Potential (mV)
1.0
0.5
0.0
-0.5
0 50 100 150 200
t (msec)

Accessories
Hydrogel Microvalve
To add liquids to the main channel, e.g. to start and stop
reactions, a dead volume-free microvalve must be placed in
the flow channel. The patented GeSiM hydrogel valve contains
hydrogel particles that dramatically swell by hydration
when the temperature drops below 34 °C, hence blocking the
channel. Warming above this transition temperature opens
the valve immediately, which allows the aspiration of
substances into the main channel.
Several versions of this valve exist (see separate brochure),
but in the MicCell only those are used where the fluid passes
vertically through holes etched into the heated actuator
chamber. This very small device can be built into a standard
UNF 1/4-28 fitting that can be screwed into a MicCell inlet
hole.
Actuator Specifications
Scanning EM picture
of an opened actuator
chamber with
dehydrated hydrogel
particles
� Hydrogel polymerized from purified N-isopropylacrylamide
(PNIPAAm)
� Standard transition temperature 34 °C (can be adjusted),
more than tenfold volume increase upon cooling ("normally
closed valve")
� Heating power max. 250 mW at 3.5 - 5 V, heating and
temperature measurement via sputtered platinum electrodes
� Switching period ca. 1 - 3 seconds; if Peltier cooling is
used, closing about as fast
5
� Watertight up to a pressure of ~ 6·10 Pa (6 bar)
� Needs aqueous solutions, but tolerates
< 15 % methanol, ethanol, acetone
< 5 % 1-propanol
Hydrogel valve PV7 without
printed circuit board (thus
autoclavable) in the MicCell
Sample injection into T and K-channel. The inlet and outlet of
the main channel are marked green. After blocking the main
inlet, samples can be sucked into the channel via the red
hydrogel valve. The K-channel (right panel) has an extra
outlet (white circle) to flush the dead volume between microvalve
and T-junction.
Microvalve filled with hydrogel particles, before (left) and
after (right) packaging
Hydrogel valve
Electrical
connection
Standard fitting
(to microstructure)
Hydrogel particle actuator with small dead volume (PV6) in
standard UNF fitting. Left: injector for manual sample addition
via a microfunnel, right: automatic sample injection
through capillary (inner diameter 25 - 762 µm).
Hydrodynamic flow chamber with eight hydrogel valves
(arrows) to generate flow in various directions (in collaboration
with the University of Technology Dresden and Namos
GmbH, Dresden)
Assembly
The MicCell microsystem consists of
� the channel plate from PDMS or glass that contains the
microstructured channel system
� the coverslip
The microchannel is covered by a coverslip that is firmly
pressed onto the channel plate as it is mounted between the
lid (polycarbonate, on the top) and support (metal, on the
bottom). Feeding and draining of the fluid is achieved by
openings in the channel plate.
The easy fluidic connection of the microchannel via standard
fittings in the MicCell lid is groundbreaking. The flow is generated
by external syringe pumps ("macrofluidics") that are
much more reliable than integrated microfluidic pumps.
A light microscope is not included in the shipment.
Components
� Working plate: metal adapter to hold the MicCell support in
practically all inverted and many upright and stereomicroscopes
(required)
� Fluid processor: computer-controlled flow control box containing
one or more syringe pumps, multi-port distribution
valve, 2/2-way valve, and controller for hydrogel valve
(recommended)
� Tubing with small inner diameter (0.3 mm, 0.5 mm etc.),
UNF fittings (1/4-28), O-rings
� Hydrogel microvalves (see below)
� Windows control software (see below) for interactive operation
and process automation with the fluid processor
(flow ramps, valve control, temperature control)
� Optional integrated devices: micromixer, thin film heater
and thermosensor, calorimetric flow sensor (GeSiM specialty,
see extra brochure), pressure sensor, microelectrode
array, impedance sensor, "sample carrier"
MicCell Control Software
For rinsing, precincuation, and the timely starting and stopping
of reactions, all devices (syringe pumps, valves), plus
the velocity and direction of the flow, can be interactively
controlled via a Windows software. In addition, complex
procedures from several single functions (pumps and valves,
delay time, repeats) can be automated using an easy
scripting language (example in the bottom right figure).
Of course, price-concious researchers can use their own fluidic
periphery, but will then lose all the comfort of a graphic
user interface and programmable operation.
Principle
Zuleitungen
Channel plate
with microchannel
Coverslip
Metal support
MicCell assembly. One can see a channel plate of glass with a
polymer channel structured on it; normally it consists of
molded PDMS. Fastening elements and the working plate
(adapter to the microscope) are not shown.
Control software for two syringe
pumps, 6/1 distribution valve ,
and hydrogel valve. On the right:
programming example
Lid
MicCell in an inverted
microscope with fluid
processor (on the left),
controlled by a notebook.
Standard fluid processor
with syringe pump (left
side), hydrogel valve
controller and 2/2 valve
(middle), and 4/1 distribution
valve (right). In
front of the fluid processor,
a MicCell, complete
with hydrogel valve and
blue metal support, is
mounted in the black
working adapter plate.

PDMS Flow Cell
PDMS Channel Plate
Although the channel plate can be made of various materials
(glass, silicon, ceramics), a good and easy-to-handle material
is PDMS (polydimethylsiloxane), a two-component silicone.
The PDMS mixture is poured onto a master tool of silicon and
cured to form a soft polymer block that seals tightly against
the coverslip.
PDMS molding is a well established lab technique, however it
is often rather bricolage than anything else. GeSiM has
developed a professional casting station in which the inlet
and outlet holes are kept open by "channel spacers" (see
picture on the right). The specialty: the lid not only defines
the PDMS thickness during molding, but is also the lid in the
fully assembled flow cell; it presses together coverslip and
microchannel and contains all fluidic connections. So all you
need to do after curing is to replace the master with the
coverslip, screw in the right fittings, and start your experiments.
The channel plate can be used several times, by the
way.
Instead of purchasing the casting station, you can let GeSiM
manufacture the PDMS channel plate, even hundred units per
month. The lids will be reused.
Due to its easy handling and flexible use, the PDMS channel
plate can be an ideal tool for rapid prototyping: to make a
new channel, only a new master is required.
Casting stations for coverslips of size 22 x 22 and 22 x 50
mm, with metal base plate, silicon master (black), Teflon
case (white), polycarbonate lid (transparent), and channel
spacer (brown)
Advantages of PDMS
� Transparent, with no background fluorescence
� Easy to cast
� Sealing without problems
� Biocompatible (at least after plasma treatment)
� Covalent bonding by plasma treatment
Disadvantages of PDMS
� Faint background in transmitted light
� Permeable for small molecules such as gases (O , CO ),
2 2
alcohols, etc. (actually an advantage in cell biology)
Principle
Inlet
Lid
Channel spacers Master
Silicon molding
master
Casting of the
PDMS channel
plate (top) and
use as flow cell
(bottom)
PDMS
Coverslip
PDMS molding in the
casting station. The
silicon master is under
the transparent lid.
Please note the channel
spacers (brown
fittings).
Demolded PDMS
channel plate with
coverslip. The channel
spacers were replaced
with green fittings.
PDMS channel plate,
mounted with coverslip
on a metal support,
with hydrogel valve
microinjector
Customized Solutions
GeSiM's great specialty is to care for individual needs of
customers. Aside from the numerous possible channel geometries
and materials, the entire setup can also vary in a wide
range. Solutions have been devised for many problems,
among them flowthrough cells that are used without microscope,
e.g. with heating plate for microarray hybrization or to
measure surface plasmon resonance (SPR). Variants for other
microscopes (such as stereomicroscopes) and the integration
of microelectrodes are also possible.
It is therefore a rather rare event that two MicCells are absolutely
identical when they leave the factory.
With polymer walls and microelectrodes...
For SPR...
Flow Cells of Different Size
For channel systems that are larger than a classical coverslip
(22 x 22 mm), up to the size of a slide, the arrangement of
the MicCell is somewhat different, as shown in these examples.
Standard PDMS MicCells with coverslips of size 22 x 50 mm
(top) and 22 x 22 mm (bottom)
Variations on a Theme...
With optical fibers...
In a stereomicroscope (see
also below)...
With glass flow cell
and electrodes on
both sides of the
channel...
With glass T-channel and
hydrogel valve injector
(arrow)...
To heat slides...
Coverslip 50
x 22 mm on
a polymer
channel
structured on
a slide.
Sealing is
achieved by
a layer of
silicone
rubber that
was screenprinted
onto
the channel
walls.
Large PDMS
channel plate
used in a
stereomicroscope.
The
channel is
covered by a
slide.