Microscope Setup for Braille Actuated

Microfluidics (MS-BAM)

Final Design Review

April 25th, 2005

Group Members: Wei Gu

Nik Kazmers Yibo Ling Royi Razi

Bryan Sack Celimar Valentin

Group Leader:

Thomas Withrow

Customers: Dr. Futai Mr. Song

Professor Takayama

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Contents Page 1 Problem Statement 2 2 Motivation 2 3 Market Needs/Competing Technology 4 4 Design Specifications 7 5 Principle of Operation 14 6 Detailed Design Description 16 7 System Validation 30 8 Recommendations 41 9 Budget 43 10 Timeline 45 11 Acknowledgements 49 12 References 50 13 Appendices 51

Figure 1: Completed MS-BAM

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1 Problem Statement The goal is to design a scaffold system for allowing the viewing of cultured cells with a Nikon TE2000-U inverted microscope subjected to PARTCELL1 or other experiments requiring real-time monitoring in a poly(dimethylsiloxane) (PDMS) microfluidic system powered by a Braille pumping/valving system. The system must allow for X-Y translation of the viewing area and be designed to house a Braille Cell Actuator, a microfluidic chip, and a heating system. 2 Motivation 2.1 Microscope Viewing of Microfluidic System Using Braille Actuators Microfluidics involves the control of fluid flow in miniscule channels ranging from 1-1000 um for a wide range of applications, including cell sorting, localized biochemical treatment of cells using laminar flow, lab-on-a-chip analysis, and high throughput screening of cultured cells. Recently, Braille displays have been used to valve, pump, and regulate fluid movement inside micro-scale channels (or pipes) within chips. A grid of small pins on the Braille display selectively pushes against rubber-enclosed channels near the chip surface in order to squeeze shut certain regions of the channels. This control requires that the pins be aligned with respect to the channels and that the chip stays on the Braille display during the course of experiments. Currently, Mr. Song, Dr. Futai, and Professor Takayama use a setup where chips are clamped to the Braille actuators (the active component of the Braille display), and chip contents are monitored with a stereoscope. However, pictures obtained with the stereoscope are limited in resolution and optical features crucial to certain experiments, and therefore we are reconstructing the configuration Braille actuators to be compatible with an inverted microscope. The goal is to be able to perform microfluidic experiments for a variety of applications (some listed above) on an inverted microscope with real-time monitoring. 2.2 Microscope We will design and fit the proposed scaffold system onto a standard Nikon TE2000-U inverted microscope (Figure 2.1) located in Rm. 1110 of the Institute of Science and Technology building, 2200 Bonisteel Blvd, Ann Arbor, MI. This microscope is currently outfitted with a 20X viewing objective, a T-SP Plain Stage, and a T-SAM Attachable Mechanical Stage that is compatible with the T-SP Stage. All components and information are available from Nikon USA (http://www.nikonusa.com).

1 PARTCELL: partial treatment of cells using laminar flows. A method of bathing a cell with two parallel streams under laminar flow.

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Figure 2.1: Standard Nikon TE2000-U Microscope 2.3 Braille Actuator and Controller Circuitry We will use the Braille Actuator SC-9 (Figure 2.2 and Appendix I), manufactured by KGS-America (http://www.kgs-america.com). A given circuit board, given by the customer, receives power and control via USB and transmits it to the SC-9 unit. Software drivers to communicate via USB are provided in C# .NET. In order to achieve consistent spacing between each Braille cell (which are loosely attached), we need to send a threaded rod through all of the Braille cells (through two see-thru holes) and sandwich the cells together with two nuts.

Figure 2.2: SC-9 Unit with all circuitry included for functional use.

SC-9

USB Power + Control

Circuits

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3 Market Needs/Competing Technology Microfluidics is a new field of study. As such, technology like Braille actuated fluid pumping/valving that’s designed to utilize microfluidics, is in its infancy. Because Brailile actuated pumping/valving was only recently developed in our customer’s lab, there is no existing commercial setup designed for the sole purpose of viewing cell culture in such systems. In fact, the only relevant ‘competition’ in the realm of microfluidics actuation similar to the Braille pumping/valving system is the designed by Fluidigm (Figure 3.1), an off-shoot of the Stephen Quake’s lab at Stanford.

Figure 3.1: The mechanism for actuating one valve using pneumatic pressure. (top) A control channel connected to pressure lines is fabricated at a right angle above a fluidic channel with an arch shape cross-section. (bottom) When high pressures are applied to the control channel via the pressure lines, the thin membrane made of silicone between the two channels deflects down into the fluidic channel, closing that portion of the fluidic channel. (Unger et al.)

Product Description: Rather than using Braille displays to actuate on-chip valves, this device uses pneumatically driven valves and pumps that consist of a membrane that pinches shut parts of channels with a certain amount of pressure (Unger et al.). The membrane consists of two layers of elastomeric rubber, which is placed on top of the microfluidic channels. The company’s flagship product, Topaz, screens hundreds of conditions for crystallizing a protein using a few microliters of the protein sample. Work in the Zare lab at Stanford makes use of pneumatic valves to treat non-adherent single cells with a stream of fluid (Hansen et al.).

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Product Similarities and Differences: The strategy presented for actuating valves is analogous to Braille display actuation. Both involve deflecting silicone rubber to seal off microfluidic channels. Unlike Braille display chips, pneumatic chips are not actuated from the bottom by a Braille pin mechanism, but require pneumatic tubes to be attached to the chip. Interesting Design Aspects: Chips are placed into a hydrating buffer to prevent evaporation. While a handful of pneumatic lines are used (20), the number of valves is a magnitude more (hundreds) since each pneumatic line regulates several valves that actuate together.

The Micronics Corporation developed a system of syringe-pump systems for actuating microfluidics. As shown in Figure 3.2, the disadvantage of such systems is that they are rather burdensome compared to Braille pumping/valving systems.

Figure 3.2: Setup for a typical Micronics chip. Syringes and feed ports (above) drive the mechanisms within the chip (below).

Product Description: The device uses three highly accurate syringe pumps and pneumatic or fluidic lines to drive chip processes. The chip is made of hard plastic with 4 fluidic ports and 8 pneumatic ports. The chips utilize t-sensors and h-filters in conjunction with an inverted microscope to analyze inputs such as blood. The t-sensor uses diffusion to quantify solutes and the h filter sorts small and large particles away from each other. Similarities and Differences: There are not many similarities. Micronics uses passively driven mechanisms (gravity) and precision syringe pumps to actuate its fluid. Much of the fluidic control is regulated by preset channel architecture.

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The core of our project is the development of a microscope scaffold upon which cells cultured in Braille actuated microfluidic chips can be viewed (under magnification). The Franklin Mechanical and Control Inc., perhaps our most direct competitor, designs custom microscope stages (Figures 3.3 & 3.4). Product Description: Franklin Mechanical’s transmitting light microscope stage, Model #4060-T, provides simple viewing of an object. With the ability to move the stage 6” in the x-direction and 4” in the y-direction, Franklin Mechanical offers a simple and effective inverted XY-microscope stage. Similarities and Differences: For economical reasons, the manual control of the microscope stage is an aspect that we envision in our future design. The viewing area of this piece may be too small for the microfluidic circuit that we will be viewing. Interesting Design Aspects: The location of the viewing area at the edge of the stage would provide easier positioning of the Braille cell actuator. Also note that Franklin Mechanical custom builds microscope stages for their customers. This resource may be used in the future.

Figure 3.3: Franklin Mechanical, Inc. Model #4060-T

Figure 3.4: Example of a custom microscope stage designed for a customer

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At approximately $1300, Franklin’s custom microscope stages are somewhat more expensive than the stage we designed and manufactured. Furthermore, they would not have been able to provide a housing unit for the Braille Cell Actuator. In sum, there are no ‘direct’ competitors to the system that we have developed. 4 Design Specifications In order to compile a list of critical design specifications, a functional decomposition was done (Appendix II). To accomplish all required functions, the following design requirements and device subsystems are proposed and listed in Table 4.1.

Table 4.1: Critical Design Parameters System Component Specific Design Requirement Stage Useful as ‘foundation’ of scaffold, can

attach to microscope, can attach to XY stage

Stage Block Props stage to appropriate height XY Stage Fits microscope, can be attached to stage,

can attach to Extender Extender Proper length (free side can rest on stage),

can be attached to XY stage, can attach to housing

Braille Cell Housing Unit Support Braille cell actuator and components, secure microfluidic chip to Braille cell actuator, can be attached to Extender

4.1 Main Stage

Figure 4.1: A 3-D drawing of the position of the main stage relative to other components of the system.

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The stage is designed to firmly fit a Nikon TE 2000-U microscope (Figure 4.1). In order to view different locations on the microfluidic chip, an XY stage is attached to the main stage to provide movement in the XY plane. This allows for alignment of the chip over the objective in order to produce the desired image. We determined that the current Nikon stage was close to what we needed. There was however, no space upon which some kind of support could be attached for the Braille actuator and microfluidics chip. Thus, our design philosophy was to mimic the screw-hole type and position in order to guarantee compatibility with both the Nikon TE 2000-U microscope infrastructure and its XY stage. Aluminum was used to ensure the main stage is both strong and rigid enough to support viewing. To accommodate the Braille actuator and chip, we designed a large opening to the right end of the Main Stage. Thus the Main Stage looks like a ‘C’. The XY stage is attached on the left and can extend to the right end with a ledge that stretches across the bottom portion of the stage. The two ends of the ‘C’ extend out 4 centimeters further than the current Nikon stage in order to provide support and protect the Braille Actuator and Extender. 4.2 Stage Blocks

Figure 4.2: A 3-D drawing of the position of the stage blocks relative to other components of the system

The stage blocks (Figure 4.2) are solid aluminum blocks attached to the bottom of the stage that allow for attachment of the stage to the microscope and which raise the stage to a height appropriate for viewing of the microfluidic channels. The stage blocks follow

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the general design of the original stage blocks (the blocks underneath the Nikon stage) with the difference being that new blocks are higher to account for the distance that the Braille Actuator hangs from the plane of the Extender. Two identical pieces were manufactured, one for each end of the Main Stage support. Small grooves at the bottom of the blocks mimic the design of the original blocks to account for cylindrical protrusions from the main microscope infrastructure. Outer holes are used for screws that go into the main microscope infrastructure, and inner holes (closer to the center) are used for screws that go into the Main Stage. Aluminum (as opposed to steel) was used to ensure sufficient rigidity. 4.3 XY Stage Figure 4.3: A 3-D drawing of the position of the XY Stage relative

to other components of the system The XY Stage (Figure 4.3) from Nikon is taken from the existing microscope setup; although it isn’t a part of our design, our system was designed to utilize it and so a brief description follows: in short, the system is designed with an easily accessible knob that extends from the left-front end of the system which can be rotated to adjust x-y movement. The rotating knob allows separate pieces of the x-y translation system to rotate relative each other (moving gears in a standard mechanical system) thereby inducing movement in the x- and the y-directions. It should be noted that the XY Stage is directly attached upon the main stage (the foundation) and is the unit upon which the Extender is attached. As such, both the main stage and the extender are designed to fit the XY Stage.

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4.4 Extender

Figure 4.4: A 3-D drawing of the position of the Extender relative

to other components of the system The Extender (Figure 4.4) is a roughly rectangular-shaped rigid steel piece which attaches to the left side of the XY Stage. In addition to attaching directly (via screws) to the XY Stage, the Extender also rests just above the Main Stage on the opposite side; under extremely heavy loads (that have no relevance to conditions of actual use), it bends slightly and rests directly on the Main Stage. Screw holes located in the middle of the Extender allow for attachment of the Braille Cell Housing Unit. The arc in the middle portion of the piece allows for easy access to the chips resting on the Braille actuators (for possible reagent delivery). 4.5 Braille Cell Actuator and Microfluidics Chip The Braille Cell Actuator is fixed to the microscope scaffold (via the Braille Cell Housing Unit) and the modular microfluidics chip is firmly secured to the Braille Cell Actuator. Both pieces are supplied by the customer. The Braille Cell Housing Unit is designed to allow for effortless fastening and interchanging of the microfluidic chip from the Braille cell actuator.

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4.6 Braille Cell Housing Unit

Figure 4.5: A 3-D drawing depicting most relevant components of the Braille Cell Housing Unit: Side Pieces (dark green), Chip Retention Device (brown and blue), Microscope Slide Bracket (light green), Microscope Slide (pink) with the heater (red), and the Microfluidic Chip (aqua). The Braille Cell Actuator and Plexiglas Braille Cell Covering are not depicted.

Easily the most complicated component, the Braille Cell Housing Unit (Figure 4.5) fulfills several vital functions. 1) It provides a structural framework to support the SC-9 Braille cell actuator (Appendix I) and couple it to the Extender bracket, 2) it contains components that are capable of attaching the microfluidics chip to the Braille cell so that the pins can actuate or valve fluid within the channels; this subsystem secures the microfluidics chip to the Braille cell with evenly-distributed pressure: the proper amount of pressure is determined qualitatively as the amount required to secure the chip in place relative to the Braille pins but not physically deform the microfluidics chip, 3) it allows sufficient overlap between the Braille pins and chip; although the microfluidics chips will be tailored specifically to fit this device, this subsystem allows for a minimum of 2 cm of overlap needed for the Braille pins to interact with the microchannels for pumping and valving purposes, 4) since the microfluidics chip is often replaced, the user must be able to easily and quickly swap chips, preferably without the use of any tools; this entails allowing the user to view the channels and Braille pins so that they can be properly aligned during setup, 5) it incorporates a replaceable glass microscope slide to which the heaters are adhered just below the microfluidic chip to simultaneously warm the cells and prevent the chip from bending; the need for disposability of the glass slide stems from the possibility of it breaking or becoming scratched to the extent of degrading the image obtained with the microscope. 4.6.1 Side Pieces The Side Pieces are roughly L-shaped pieces of aluminum which attach the Braille Cell Actuator to the Extender. The long arm of the ‘L’ are attached to each side of the Braille Cell Actuator while the short arm of the ‘L’ is attached to the Extender.

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4.6.2 Chip Retention Device The Chip Retention Device is a piece of Plexiglas which slides over the microfluidics chip and fixes it in place on top of the Braille Cell Actuator and heater. Two holes on either side of the piece allow for it to slide down metal rods. The piece is in turn sandwiched by springs below (which allows for easy removal of the microfluidics chip) and wingnuts above (the wingnuts can be turned to lower the chip retention device). 4.6.3 Microscope Slide Bracket The Microscope Slide Bracket is an angle iron trimmed to fit the width of the Braille Cell Actuator. It is attached to the front of the Braille Cell Actuator at either end of the Side Pieces (via screws) and provides an extra space at the front of the Braille Cell Housing Unit to allow for the Microscope Slide to attach. 4.6.4 Microscope Slide The Microscope Slide is a glass slide below which the Heater is attached via a thin layer of PDMS. It is attached to the front end of the Braille Cell Actuator (via a angle iron attached to the front) and affixed by alligator clips on either side. It performs the simultaneous functions of allowing the microfluidics chip a place to ‘rest’ during viewing (to minimize bending of the chip and disrupt microscope focusing) as well as heating the microfluidics chip to assist in cell culture experiments. 4.6.5 Plexiglas Braille Cell Covering The Plexiglas Braille Cell Covering consists of three rectangular pieces epoxied together which slide over the Braille Cell Actuator like a sleeve to protect the circuitry of the device from possible accidents such as fluid spills. 4.7 Heater The heating system maintains a mammalian cell culture environment of 37 oC ± 1.0 oC. It is placed beneath the microfluidics chip in order to provide proper heating, and since it is comprised of filaments of metal wiring, does not impede the viewing of the cell culture. The heater itself is physically attached with poly(dimethyl)siloxane to the underside of the 75x50mm2 glass slide placed underneath the PDMS chip. The temperature controller resides beneath and attached to the Extender via Velcro strips. Thermal couples are used to measure the temperature of the fluid channel and nearby areas while an independent voltage source powers the heater.

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4.8 Software The software, provided by the customer, is flexible and user-friendly. It allows for spatio-temporal control of the pumping/valving action of the Braille cell actuator through a custom-designed circuit control box. In short, the laptop computer on which the software resides attaches to the circuit control box via USB while the circuit control box attaches to the Braille Cell Actuator through a custom interface provided by the customer. 4.9 Safety The relevant safety issues with the design involve the electrical and mechanical components. Since we are working with an electrically powered heater and Braille Cell Actuator, electrical shortages are very possible. If an electrical shortage is to occur, not only will the piece of equipment no longer be functional, a fire could also initiate. By housing the Braille Cell Actuator, the design includes the proper precautionary measures to prevent an electrical shortage in this device. Also, specific heaters have been chosen that will work properly when in contact with minimal amounts of fluid, which is likely during the pumping of fluids in the microfluidic setup. The circuitry of the heater which maintains the PDMS chip at a steady temperature of 37.0 ± 1.0°C also poses some risk. The circuitry has been sequestered in a box to minimize the risk of liquids spilling and to avoid any electric shock to the user. There is however, one issue that a user should be aware of—the connection between the Braille device and its circuit control box is exposed; any direct contact between the skin and this exposed connection poses the risk of an uncomfortable but fairly harmless electrical shock. The user should further be mindful that the design is made of sharp-edged metals. The user should take care to use the equipment properly and carefully in order to prevent any physical damage. Furthermore, certain screws are exposed and used for tightening and positioning the chip retention device and the housing unit for the Braille Cell Actuator. Although the mechanical and electrical hazards do pose potential hazards, measures (such as the housing unit enclosure) have been taken to reduce risks. In addition, it should be noted that the danger of injury from sharp metallic edges with our design is not appreciably greater than that posed by microscope stages in general. As such, it should pose very little risk to the conscientious user. 4.10 Quality In order to assure a high level of quality, a large amount of time has gone in to this design to ensure that the measurements and designs are very accurate and specific. A number of tests for validating and confirming that the final product meets the aforementioned design requirements are described in Section 7.

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5 Principle of Operation

Figure 5.1: Block diagram of principle of operation of system. Our system consists of twelve main components (Figure 5.1), eight of which are engineering or modified ourselves. A brief description follows. The microscope setup will fit on a Nikon TE2000-U inverted microscope to which the planar stage will be physically attached. The XY stage and Extender will be coupled to each other, and will be attached to the planar stage via screws from the XY stage to the main stage. The Braille Cell Housing Unit is fixed to the Extender by means of clamps and contains the Braille Cell Actuator, the Microfluidics Chip, and the Heater. The Chip Retention Bar allows the user to easily replace the Microfluidics Chip and also provides fixes the chip to the Braille cell actuator. A glass slide will provide the interface between the heaters and the PDMS chip. The Braille cell actuator will connect to the customer-provided circuitry that will be controlled by means of software.

User Microscope Extender XY stage

Circuitry Software

PDMS Chip

Braille Cell Actuator

Housing

Planar stage

Chip retention

bar

Heater

Cell Culture Image

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Figure 5.2: Block diagram of system operation procedure. As Figure 5.2 shows, two parallel pathways must be followed during system assembly: 1) the system must be physically assembled piece-by-piece (in terms of XY Stage, Extender, Main Stage, etc.), 2) the modular Microfluidics Chip must be aligned and fastened to the Braille Cell Housing Unit. The second pathway is the one that the user will need to follow on a daily basis in order to proceed from experiment to experiment and swap out Microfluidics Chips. The lower portion of Figure 5.2 depicts the procedure to follow after the reagents and cells are loaded into the Microfluidics Chip. Note that data collection from experiments can only be performed after the microscope is focused and the image acquisition software (SimplePCI) is opened.

Screw Extender Bracket into X-Y Stage Arm

Screw X-Y Stage to the Scaffold

Load Microfluidic Chip with Cells and Media/Reagents

Align Channels in the Chip with Braille Pins

Fasten the Chip to Braille Cell Housing with the Clamp

Turn on Heater

Attach Braille Cell Housing with Attached Chip to Extender bracket

Turn on the Microscope

Adjust X-Y Stage to Bring Cells into Viewing Area (Above Objective)

Focus Microscope on Cells

Open Software that Controls Braille Actuation

Enter Flow Rates and Valving Patterns into Software

Connect Braille Cell Electrical Terminal to Computer Interface Module

Connect Computer Interface Module and Video Camera on Microscope to Computer

Microscopy and Control of Braille Actuator

Start the Software Controlling Braille Cells, and Software for Real-Time Digital Image Aquisition

Start Both Software Programs When Ready to Start Experiment

Run Experiment and Collect Data

Remove X-Y Stage from Nikon Scaffold

Remove Nikon Scaffold from Microscope

Screw Scaffold to Microscope

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Main Stage

Objective

Support Brackets & Housing Unit

Extender Glass Slide w/ Heater

PDMS Chip

XY-Stage

Chip Retention Device

6 Detailed Design Description

6.1 Conceptual Overview The overall design can be broken down into two sets of components. One set, including the Main Stage, Stage Blocks, XY Stage, and Extender, stays on the microscope setup for multiple experiments using the Braille Actuator (until the other stage is required for different experiments). The other set (The Braille Cell Housing Unit) is removable from the prior, stationary set. It includes the Braille Cell Actuator, Side Pieces, Chip Retention Device, Microscope Slide Bracket, Microscope Slide, Heater, and the assorted circuitry. The position of the Microfluidics Chip relative to the viewing objective (Figure 6.1) is coordinated through this pathway: objective, microscope, Main Stage, XY Stage, Extender, Braille housing, clamp to the chip, and finally the chip. Lateral movement of the chip (parallel to the static stage) relative to the objective is accomplished through the movement of the XY stage to Extender connection relative to the XY stage’s fixation on the static stage. Vertical movement between the chip and the objective is performed by a focusing unit on the objective, i.e. raising or lowering the objective. Note that this movement is restricted to only a few centimeters and the chip needs to be placed relatively close (< 1 centimeter) to the objective and within the focus range.

Figure 6.1: This image contains all aspects of a functioning Microscope Setup for Braille Actuated Microfluidics

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6.2 Detailed Design Specifications 6.2.1 Main Stage

Figure 6.2: 3-D drawing of the Main Stage

The newly designed Main Stage (Figure 6.2) replaces the existing Nikon version. The main reason for this replacement is due to the fact that the Nikon version has a viewing area in the middle, and would not allow access to the Braille Cell Actuator and the Microfluidics Chip. The Nikon XY Stage that connects to this newly designed Main Stage. The location and size of all the holes in the main stage (threaded or non-threaded) were determined based on need for it to attach onto the Nikon microscope and the Nikon XY Stage. The U-shape of the Mainstage accommodates easy and accessible viewing of the objective. It is made out of 8 mm aluminum plate to maximize the strength reduce the weight (all dimensions are given in Appendix III). The long arms provide extra support for the XY stage and the Extender in order to prevent deformation in a case of excessive force application by the user. Velcro strips are attached below one arm of the stage to allow for the adherence of a black sealed box that contains the circuitry needed to actuate the Braille Cell Actuator. Its placement there provides easy access in terms of cable connections to the Braille Cell Actuator.

Holes for Stage Block Attachment (Identical on Opposite Side)

Holes for X-Y Stage Attachment

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6.2.2 Stage Blocks

Figure 6.3: 3-D drawing of the Stage Blocks The height of the Nikon Stage Blocks is 20 mm. The designed Stage Blocks (Figure 6.3) boost the Main Stage somewhat higher since the distance between the bottom of the chip (or top of the Braille actuators) and the interface between the Extender and the Braille actuator housing is 29 mm. As such, Stage Blocks height is 49±1 mm (20+29) (refer to Appendix IV for specific dimensions). 6.2.3 Extender

Figure 6.4: 3-D drawing of the Extender

The Extender (Figure 6.4, or Appendix V for dimensions) allows for attachment of the housing unit underneath it via two treaded rods that extend from the housing unit through two holes inset holes. The threaded rods of the Braille Cell Housing Unit are placed through the two inset holes of the Extender and are secured using two wing nuts. The location of these inset holes is such that the Microfluidics Chip attached to the Braille Cell Housing Unit is made to hang directly above the microscope lens. The Extender connects to the XY stage via two screws. This attachment allows for movement of the Braille Cell Housing Unit in the x-y plane. The Extender is made out of 4 mm aluminum

Holes for X-Y Stage Arm Attachment Holes for Braille Cell Housing

Unit Attachment

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plate (specific dimensions are given in Appendix V), and extends from one side of the Main Stage to the other. The adjacent end (that is, opposite the attachment to the XY Stage) of the Extender is fixed approximately 3 mm above the other side of the Main Stage so that this end of the Extender may rest upon that Main Stage under a heavy load (only a slight elastic bending occurs). The arc shape cutout in the mid-section of the Extender allows easier access to the Braille Cell Housing Unit (when it needs to be removed). 6.2.4 Braille Cell Housing Unit The Braille Cell Housing Unit consists of two ‘Side Pieces’, a ‘Chip Retention Device’, the ‘Microscope Slide Bracket’, the SG-9 Braille cell itself, and a Plexiglas covering for it. Through the incorporation of the Chip Retention Device and the Microscope Slide Bracket, the Braille Cell Housing Unit secures the microfluidic chip to the Braille cell, and couples the Braille cell and microfluidic chip to the extender. Since the extender is fixed to the X-Y stage, the Braille cell housing unit allows the chip and Braille cell to translate laterally in the X-Y plane above the objective lens, which makes possible the viewing of the entire chip. 6.2.4.1 Side Pieces Due to the presence of two holes that span the entire width of the SC-9 Braille cell (see Appendix I), sandwiching the Braille cell between two sheets of aluminum plate with appropriately placed holes is possible. In order to accomplish this, the aluminum plates, or Side Pieces, have holes drilled them that line up with the holes in the Braille cell. The side pieces are affixed to the sides of the Braille cell by two M2 threaded rods that pass through the assembly, and are bolted on both sides (see Figure 6.5).

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Figure 6.5: Braille Cell (White) Sandwiched between Side Pieces (Green), and Bolted Treaded Rods (Black).

The side pieces of the Braille Cell Housing Unit are shaped so they can contact the Extender, which is located above the Braille Cell Housing Unit. Attachment of the Braille Cell Housing Unit to the Extender is essential, for it allows the X-Y movement of the microfluidic chip. In order to attach the Braille Cell Housing Unit to the Extender, 2.0 cm long M4 threaded rods are inserted into the top of the side pieces to serve as attachment points between the side piece and Extender with the use of wing nuts (See Figure 6.5). Each side piece has one of these threaded rods, giving a total of connection points between the Braille Cell Housing Unit and extender. Regular M4 screws can be used instead of the threaded rods and wingnuts if the user desires, but with the prior attachment method, alignment should be easier. The side pieces also serve as a substrate to which the Microscope Slide Bracket and Chip Retention Device attach. These attachment points are clearly marked in Figure 6.5. Although only one side of the Braille cell contains protruding electrical terminals, both Side Pieces were made identically with a cutout. Although the cutout on one of the Side Pieces is unnecessary, it was significantly less expensive to machine two copies of the same part opposed to machining two different parts. This is due to the time spent in

Side Piece

Braille Pins

Electrical Connection Terminal

Holes for Chip Retention Device Attachment (M4)

Side Piece

Holes for Extender Attachment (M4)

Braille Cell

Holes for Microscope Slide Bracket Attachment (M4)

Threaded Rods and Bolts for Sandwiching (M2)

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entering the dimensions into the software system of the CNC mill. Refer to Appendix VI for the CAD drawings of the Side Pieces used for machining. The electrical terminal connector of the Braille (see Figure 6.5) is connected to a customer-provided computer interface module via a ribbon cable that attaches to the electronic terminal connector on the Braille cell (see Figure 6.6). This module is placed in a plastic electronics project box, which is attached to the bottom of the main stage with Velcro. Velcro allows the box to be easily removed during disassembly. The ribbon cable attached to the Braille cell does not inhibit its X-Y movement in any way, nor does it interfere with image acquisition. The exact dimensions of this module are 10 x 5 x 4 cm.

Figure 6.6: Braille Cell Electronics Box 6.2.4.2 – Microscope Slide Bracket To provide a support system for the glass microscope slide, the Microscope Slide Bracket (Figure 6.7) is attached to the side pieces in an orientation where the top of the glass microscope slide is even with the surface of the Braille cell. This is important so the microfluidic chip can rest flat on a plane parallel with the ground with respect to the microscope, which allows the entire chip to be in the plane of focus. The glass microscope slide prevents bending of the flexible PDMS microfluidic chip, which further aids focusing. Additionally, the glass microscope slide contains the heater for maintaining the cell culture within the chip at the proper temperature.

USB Connector (to computer)

Box Containing Computer Interface Module

Connection To Braille Cell Electrical Terminal Connector

Velcro for Attachment to Main Stage

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Figure 6.7: Microscope Slide Bracket. The Microscope Slide Bracket is composed of an angled piece of metal (at a 90º angle), often known as an “angle iron,” that connects to the front of the Braille Cell Housing Unit with two M4 screws and washers. In this configuration, part of the Microscope Slide Bracket juts out from the front of the Braille Housing Unit to provide the flat plane for the microscope slide to rest on (see Figure 6.8). Refer to Appendix VII for a CAD drawing of the microscope slide bracket that includes dimensions.

Figure 6.8: Microscope Slide Bracket (light green) attached to the Side Pieces (dark green) of the Braille Cell Housing Unit. The Braille cell is omitted for clarity.

The 75 x 50 mm glass microscope slide (1.0 mm thick) has a heater mounted to it as described in section 8.6 The microscope slide is attached to the microscope slide bracket

Side Pieces

Microscope Slide Bracket

Bolts for Attaching the Microscope Slide Bracket to the Side Pieces

1.0 mm Gap to Accommodate the Glass Microscope Slide

Front

Back (Adjacent to Braille Cell and Side Pieces)

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with miniature alligator clips (see Figure 6.9). An alligator clip is an appropriate means of attaching the microscope slide to the microscope slide bracket because it allows the slide to be moved so that the heater can be placed directly below the cell culture, which is important for adaptability of this device to microfluidic chips with different microchannel patterns. Removability of the glass slide is also critical so it can be removed if damaged. Although it is easy to remove the glass slide, the alligator clips provide sufficient force to keep the glass slide secured to the Microscope Slide Bracket even when an excess of 1.25 pounds are placed on the slide. They are also easy to use, and no tools are required.

Figure 6.9: Miniature Alligator Clips

Figure 6.10: Overall Diagram of the Braille Cell Housing Unit (Side View)

6.2.4.3 Chip Retention Device

The task of securing the microfluidic chip to the Braille cell while still allowing the quick and easy chip removal, replacement, and alignment with Braille pins is accomplished by the Chip Retention Device, which consists of the Chip Retention Bar, and a spring loaded screw-down mechanism that is operated without tools. Refer to Figure 6.11 for an illustration of the Chip Retention Device.

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Figure 6.11: Chip Retention Device (and Microscope Slide Bracket) mounted to the Side Pieces of the Braille Cell Housing Unit.

The Chip Retention Bar (Figure 6.12, or Appendix VIII for dimensions) is constructed of 4 mm thick clear Plexiglas, and can be tightened down in order to sandwich the microfluidic chip between itself and the Braille cell (see Figure 6.10 for a side view, and Figure 6.13 for a front view). The advantage of having a clear Chip Retention Bar is it does not obstruct the user’s view of the microchannels within the microfluidic chip or the Braille pins on the Braille cell, making the alignment of the two accurate and simple. The Chip Retention Bar does not cover up the fresh media reservoirs, so the media levels are easily observable, and can be replenished without having to remove the chip or Chip Retention Bar during experimentation.

Figure 6.12: Chip Retention Bar.

The spring loaded component of the Chip Retention Device consists of two M4 threaded rods that pass through the center of springs, through holes in the Chip Retention Bar, and

Side Pieces

Wing Nut of Chip Retention Device

Chip Retention Bar (actually is clear)

Spring

Microscope Slide Bracket

M4 Threaded Rod of Chip Retention Device

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through washers placed on both ends of the springs. These rods bolt into the helicoil-equipped (threaded) M4 holes in the Side Pieces (see Figure 6.11), and contain wing nuts that can be manipulated to control the amount of force applied to the chip when securing it to the Braille cell (see Figure 6.10 for a side view, Figure 6.13 for a front view, and Figure 6.14 for a top view). If the wing nuts are loosened enough, the chip can be removed and replaced with ease, for the Chip Retention Bar rises automatically because of the compressive forces provided by the springs. This allows for the user to insert and remove a microfluidic chip with one hand. In the absence of springs, the Chip Retention Bar would fall to the Braille cell due to gravity, which would cause the addition of insertion/removal of a microfluidic chip more difficult: the user would have to simultaneously lift the Chip Retention Bar up with one hand, and insert the chip with the other hand. Having two independent threaded rods passing through holes the Chip Retention Bar allows the Chip Retention Device to firmly secure any microfluidic chip even if its top and bottom surfaces are not completely parallel. Although the microfluidic chips used in this device are typically only 1 cm thick, the Chip Retention Device can accommodate microfluidic chips that are over 2.0 cm thick, and 51.2 mm wide (corresponding to the width of the Braille Cell).

Figure 6.13: Chip Retention Bar in relation to other components in the Braille Cell Housing Unit.

Microscope Slide Bracket Braille Cell

Chip Retention Bar (clear) Wing Nut for Fastening Chip

Spring and M4 Treaded Rod of the Chip Retention Device

Microfluidic Chip

Side Piece

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Figure 6.14: Top view of the Braille Cell Housing Unit. The Extender is omitted for clarity.

6.2.4.4 – Plexiglas Braille Cell Covering Since the electronics of the Braille cell are exposed, a Plexiglas covering is provided to enclose the top, bottom, and back of the Braille cell. These three Plexiglas pieces are bonded to each other with a strong epoxy. The finished Plexiglas “C-Shape” fits snugly over the electronics of the Braille Cell (Figure 6.15). See Appendix IX for CAD drawings of the Plexiglas coverings. Plexiglas coverings of these dimensions, along with the side pieces of the Braille Cell Housing Unit, ensure that the entire Braille cell is seamlessly covered for electronic protection.

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Figure 6.15: Plexiglas Covering Assembly. The labeled positions are with respect to the Braille cell pins.

6.2.5 Heater The Heater used for our setup is a Therma-Clear™ transparent heater (H6700R9.0L12(B)). It is controlled by via a HEATERSTATTM sensorless temperature controller model CT198-1003R10.4L1. Both are provided by Minco Products (Minneapolis, MN) and shown in Figure 6.16. The heater consists of thin resistor filaments (that heat in response to current flow) fixed upon a flexible plastic strip. Two wires extend from the heater (50x10mm2) to attach to the temperature controller and voltage source. The voltage input range for this heater is 8 to 26VDC with a set point range of 8.79 to 13.18Ω. Figure 6.17 depicts the heater setup. A TEMMA DC Power Supply provides the necessary input voltage to our heater. The heater’s temperature has can be controlled by either varying the resistance of the heater control box (by turning a tiny screw) or by varying the voltage/current across the heater. The use of the TEMMA DC Power Supply is advantageous in that it allows the simultaneous monitoring of input voltage and current while giving the user full control of either current or voltage through the adjustment of a knob (as opposed to turning a tiny screw). Furthermore, the TEMMA DC Power Supply can power the heater for extended periods of time without the worry of for example, batteries running out.

Figure 6.16: Top view of flexible heater and temperature controller

Top

Bottom

Back

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Note that the Therma-Clear™ transparent heater is not directly attached to the PDMS chip. It rests on the underside of a 75x51mm2 glass slide, which also provides support to PDMS chip. Figure 6.18 depicts a side view of the setup in which both the glass slide and PDMS chip rest upon an angle bracket for support. Some clearance is required between the aluminum angle bracket and the heater to prevent any electrical conductance from the heater onto the aluminum. In addition to providing support, the glass slide prevents the heater from harming the PDMS chip in case of any electrical mishaps. A thin layer of PDMS is used to fix the heater to the bottom side of the glass slide. The use of PDMS allows for easy removal of the heater from the glass slide in case the glass slide breaks or is scratched. This mode of attachment is particularly advantageous in that it allows for the heater to be reused in case of damage to the glass slide.

Figure 6.17: Heater setup with power supply

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The controller set point is set so the area near the cell culture can be kept at a constant physiological temperature of 37.0 ± 1.0 °C (standard mammalian cell culture temperature). The heater is placed directly below the cell culture area in order to provide optimal heating to the desired area (Figure 6.19). It was determined that the filaments of the heater would not impede viewing of cell culture as long as the Microfluidics Chip was placed appropriately relative to the filaments—that is, since the filaments and microchannels are both so thin, there is sufficient room to place them ‘apart’ from each other.

Figure 6.19: (A) Heater placement, (B) Top view of channels and heater placement

PDMS Chip

Braille Cell Actuator angle

bracket

heater

glass slide

Figure 6.18: Side view of heater setup

Heater element

A

B

Heater wires

Microscope Slide

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7 System Validation When validating our final product, we primarily ensured that our setup can (i) securely hold a PDMS chip, (ii) keep the chip flat or perpendicular to the microscope objective, (iii) allow proper viewing of the chip contents at the level of the channels, (iv) move the chip relative to the objective, and (v) heat the chip to 37.0±1.0 °C. Secondary validations will also be performed but will be performed after primary validation requirements (After April 25th). Secondary validation will be to establish a 3-stream laminar flow inside of a microfluidic device using software (to be modified for our specific experiment) and circuitry provided to us by the customer. A final validation will be the culture of live cells inside the channels for 6 hours under the same laminar flow. The following experimental tests demonstrate the validity of MS-BAM. 7.1 System Assembly Validation Upon receipt of all mechanical parts, system assembly (Figure 7.1) was validated. The average assembly time (after 10 trials) with a fully sub-assembled Braille Cell and stage blocks already fixed to the main stage was approximately eight minutes.

Figure 7.1: System assembly

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Since it was impossible to quantify the degree to which different parts fit together, we empirically verified that all parts fit together satisfactorily. There were no noticeable gaps between adjacent pieces and all the screws fit as expected. We further verified that the extender was indeed horizontal (Figure 7.2).

Figure 7.2: A ‘Level’ was used to verify that the extender is sufficiently parallel in relation

to the ground. Unfortunately, the Level device was too large for placement onto the Braille Cell Housing Unit to verify its horizontal positioning. This would have been useful to ensure efficacy of image acquisition and microscope focusing. However, as we show in Section 7.4, image acquisition efficacy is quite satisfactory. 7.2 Pumping/Valving Validation One of the main design requirements is to demonstrate that fluid can be pumped through a PDMS chip while being manipulated by the MS-BAM device. In order to properly demonstrate that this MS-BAM needs to be assembled onto the microscope, as well as fabrication and fitting of the PDMS chip before actuating begins. The following steps explain the detailed procedure taken to validate the pumping of the Braille Cell when a part of the MS-BAM device. Steps in Validation Experiment:

1. Chip Fabrication 1.1. Obtain channel mold from customer 1.2. Pour mixed PDMS prepolymer onto the mold 1.3. Spincoat PDMS prepolymer onto a flat substrate 1.4. Crosslink both polymer sets in the oven

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1.5. Treat both surfaces with oxygen plasma 1.6. Seal the two surfaces and load with distilled water

2. Microscope Stage Setup 2.1. Unscrew original stage from the microscope infrastructure 2.2. Screw new main stage onto the microscope infrastructure 2.3. Screw extender onto XY stage independent of the main stage 2.4. Screw XY stage + extender onto the main stage

3. Load Chip into Movable Viewing Range 3.1. Align the chip onto the Braille actuator with the aid of a stereoscope 3.2. Clamp the chip onto the Braille actuator with the retention bar 3.3. Load the Braille actuator onto the extender 3.4. Adjust height of microscope lens to bring the channels into focus 3.5. Check the XY movement of the chip for 1 cm in each direction 3.6. Ensure that the channel do not go out of focus during the XY movement.

Move chip towards and away from the objective for 18.3 mm and in the orthogonal direction for 13.7 mm. Take pictures at both extremes to see the degree of chip bending

3.7. Add weights onto the far side of the chip and ensure again that the channel does not go out of focus during XY movement

4. Experiment to Demonstrate Proper Channel Actuation 4.1. Plug the Braille actuator to the circuit board 4.2. Attach circuit board to the main Stage 4.3. Plug the circuit board to a computer 4.4. Load the software to control the Braille actuator by double clicking the

application file. 4.5. Suction out extra liquid from entering ports 4.6. Add liquid with 3 micron diameter beads 4.7. Have the software pump the beads using Type 0 pumping and filling the

speed, and the coordinates of the each pins composing the pump 4.8. Ensure flow at the intersection of the channel and record a movie

7.3 Heater Validation Calibration of the heater subsystem was accomplished by utilizing a power supply, heater controller box, a heater attached with PDMS to a glass slide, a thermocouple and a PDMS chip. Figure 7.3 shows the setup.

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The first validation test consisted of connecting the heater to the controller box and power supply. The thermometer’s thermocouple was inserted into the PDMS chip in an area above the heater and near a microchannel in order to record temperature within the area where the cells and fluids would flow through. The heater was powered and the voltage and current were varied. When the temperature was measured at 37.1°C the voltage was approximately 5V and the current was approximately 320mA. It should be noted that the voltage was set at 12V before turning on the power supply, but decreased to 5V once the power supply was turned on. After decreasing the temperature (by decreasing the current) to 36.0°C, it took 15 minutes to get the temperature back up to 37.3°C, after increasing the current. A second test was conducted after disconnecting the heater and allowing it to cool for 10 minutes. The setup was then reconnected to the power supply and turned on. Figure 7.4 shows the change in temperature in relation with the time. It must be noted that the current settings were changed at several points during the experiment. The temperature

Figure 7.3: (A) Power supply and controller box, (B) PDMS chip, glass slide, heater and thermocouple

A

B

A

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was originally set at 320mA. After 18 minutes the current was changed to 300mA, at 22 minutes to 280mA, at 29 minutes back to 300mA, at 35 minutes a little above 300mA and at 37 minutes returning to 300mA.

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

0 10 20 30 40 50

time (minutes)

degr

ees

(Cel

sius

)

The third test conducted was to see if the temperature was constant throughout the length of the chip. Three different sites of the chip were tested as demonstrated in Figure 7.5, by inserting the thermocouple into these sites. The temperature at (A) was 37.6°C, the temperature at (B) was 37.4°C and the temperature at (C) was 37.1°C. The difference in temperature could have been due to the difference in thermocouple depth.

Figure 7.4: Heater’s temperature change with time

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7.4 Image Acquisition Validation In order to properly assess the validity of the MS-BAM system, images need to be captured using the inverted Nikon TE-2000u microscope. After the setup was assembled properly, including the PDMS chip to the Braille Cell, microfluidic pumping was initiated. With the use of the image acquisition software “Simple PCI,” images were captured at different locations on the chip. The most important aspect of the MS-BAM device is to allow the user to easily and clearly view the microfluidic channels of the PDMS chip. A validation test was performed to see how out of focus the image becomes when the XY-Stage is manipulated to move the chip is completely moved in either the X or Y. When the PDMS chip

A

B

C

A

B

C

Figure 7.5: Testing for temperature differentials across the chip

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position was moved from the far left and then to the far right (approximately 2.2cm), minimum re-focusing needed to occur. Figure 7.6 are before and after images of the microfluidic channel after the microscope position was moved the length of the chip.

Left-Side (Focused) Right Side (Unfocused)

Figure 7.6: Before (left) and after (right) images after

lengthwise movement of the chip The same validation test was completed for the width of the PDMS chip. A focused image was captured at the top of the chip and an unfocused image was captured at the bottom of the chip. The distance between the locations of these two images was approximately 1.5 cms. Figure 7.7 shows the top (focused) and bottom (unfocused) images of the microfluidic channels.

Top Side (Focused) Bottom Side (Unfocused)

Figure 7.7: Before (top) and after (bottom) images after

widthwise movement of the chip

Overall the MS-BAM setup allows for proper image acquisition with minimum refocusing. Although slight refocusing is needed to clearly see different locations over the entire chip, most other microscopes also need refocusing when repositioning occurs over the smallest of distances. 7.5 Structural Integrity Validation 7.5.1 Microscope Slide One of the concerns raised during the design process was the cantilevering aspect of the glass slide under a certain amount of force. It was questioned that because of the delicate nature of a microscope slide and the breaking and deflecting when being manipulated by the user. The following test was conducted to ensure sufficient strength and minimal deflection of the glass slide.

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Once the Braille cell-housing unit was assembled, a microscope slide was selected and used for validation. Since the alligator clamps are not strong enough to counterbalance a force that is necessary to test the deflection and strength of the slide we held an angle iron firmly over the microscope slide (Figure 7.8). Once this was in place, we slowly added weights to the end of the microscope slide. The weights that were used were 1.25 and 2.5 lbs. Each time a new weight was added, 10 seconds passed to allow for confirmation of the strength of the slide. The amount of deflection was also noted.

Figure 7.8: Experimental setup for deflection and strength of Microscope Slide

The amount of deflection that was present under each set of weights was very small and not measurable with the measurement device present. It must be noted that a small deflection will cause for an increased amount of re-focusing when viewing the specimen. Although this is true, the amount of force that would be applied during experimentation would only come from the weight of the PDMS chip and the fluid present in the chip. Therefore, the amount of deflection that would occur under a larger force would be when the user is manipulating the device and not viewing it with the microscope. When the first weight of 1.25 lbs was added, no breaking occurred (Figure 7.9). Once the 2.5 lbs weight was added the microscope slide fractured in a number of different locations (Figure 7.10). Although breaking did occur, since this device is being used in a microscopic environment, it is safe to assume that this amount of force should and will not be present during experimentation.

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Figure 7.9: Microscope Slide supporting weight of 1.25lbs

Figure 7.10: Fracturing of Microscope slide by a weight of 2.5 lbs

7.5.2 Main stage The main stage was made out of an 8 mm thick aluminum, in order to give it a combination of strength and lightness. We tried to apply a force of about 100lb on the main stage while it was connected and did not observe any noticeable deflection. 7.5.3 Extender The extender was made out of 4 mm thick aluminum in order to give it enough rigidity to support the housing device. We conducted two experiments in order to validate the rigidity of the extender (Figure 7.11 and Figure 7.12); in the first we used weights starting from 1.25lb to 3.75lb with increments of 1.25lb, this force was applied at the far end of the extender with respect to the XY stage/extender connection. The experiment was also conducted when the system was completely put together, in order to simulate a real time usage. We measured the deflection with a caliper, and in order for our measured deflection to be as accurate as possible we conducted several measurements and averaged them. When the applied force was 2.5lb the extender hit the far side of the main stage (deflection of 4 mm).

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Figure 7.11: Extender with no force applied - no deflection

Figure 7.12: Extender with an applied force of 2.5lb – max deflection (contact with main stage)

The main stage has been designed especially with long arms in order to stop the extender from deflecting more than 4 mm. By preventing the extender from further deflecting we avert the XY stage’s arm from deforming.The graph below in Figure 7.13 approximately depicts the experiment’s results.

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Force Vs. Deflection

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.5 1 1.5 2 2.5 3 3.5 4

F(lb)

Def

lect

ion(

mm

)

Force Vs. Deflection

Figure 7.13: A graph of force applied versus Extender deflection. In the second experiment we tested the extender separately in order to assure it is rigid enough and could withstand the force applied on it by the housing unit or by the user. We applied a force of 6.25lb on the far end of the extender, without any noticeable deflection (Figure 7.14). From that experiment we deduced that the part is definitely rigid enough.

Figure 7.14: Testing Rigidity of Extender

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7.5.4 Stage Blocks The stage blocks were designed exactly with the same length and width dimensions as the original stage blocks, the only difference was the height, which we had to elevate in order to allow movement of the housing device without hitting the microscope lens. The dimensions of the stage blocks are: width: 20 mm, length: 99 mm, and height: 49 mm. the blocks were made out of aluminum, and because of their large thickness, they are extremely rigid. We could not really quantify their rigidity, due to the fact that none of our group members managed to apply enough force on them to even slightly bend them. 7.5.5 Side Pieces Those two parts were made out of 8 mm thick aluminum, and turned out to be very rigid, especially in the current setting in which they sandwich the Braille cell actuator. 7.5.6 XY Stage The XY Stage was manufactured by Nikon and could not be completely assessed for rigidity. As mentioned previously in the extender’s deflection measurements, the maximum possible deflection of the extender was 4 mm, which occurs at the far end of the extender. When the XY Stage is deflected a maximum of 4mm, permanent deformation does not occur. 8 Recommendations 8.1 Current Design Flaws Now that the first prototype has been designed and manufactured, there are certain design flaws that have been noticed after assembly. One of the most difficult problems that we had to overcome during the creation of MS-BAM was the creation of a secure connection between the main stage and the XY-Stage. After shortening the main stage because of an incorrect measurement, the XY-Stage did not properly fit onto the main stage. The reason why this occurred is because the helicoils, which are the threaded inserts for a screw hole, were not properly placed at the edge of the main stage. This did not allow for the screws that were already attached to the XY-Stage to connect to the stage. After buying a helicoil replacement kit, the helicoil was fixed. It is recommended that the helicoils of the main stage and of the housing unit be watched carefully and replaced when non-functional. Another recommendation would be to research a more efficient way to secure the microscope slide to the microscope slide bracket. Initially it was proposed to use mini-C-Clamps, but because they were too large it was thought of using either binder clips or the current solution of mini-alligator clips. Although the alligator clips provide enough force to secure the glass slide to the bracket, the sharp teeth present on the clip have the potential of scratching and/or breaking the glass slide. The scratching that has been observed has been minor, but an improvement might be needed.

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8.2 Improvements/Concerns based on Validation Tests One of the major concerns that was discovered during validation was the viewing of the specimen through the PDMS secured heater. Although the user is still able to clearly see the microfluidic channels in the PDMS chip, there is a small amount of debris that can be seen when using the custom designed microscope slide when compared to a clean unused microscope slide. Since heating is a very important aspect of the design, it is recommended that this both sides of the custom designed microscope slide be cleaned well and often. Another concern that was noticed during pumping validation is the slight deflection of the chip retention bar when placed over the PDMS chip. There is a small chance that when the PDMS chip is secured with too large of a force that the retention bar will fracture. After securing the PDMS chip a number of times, this force should be well outside of the range that would be applied by the user, but it is still a possibility and a concern that should be taken into account. After testing the structural integrity of the system, there was only one aspect of the device that caused a deflection under a certain amount of force. When enough force is applied to the extender, approximately two to three pounds, a small deflection occurs on the XY stage, causing for the extender to come in contact with the main stage. When the force is released the extender and the XY-Stage return to their normal position, no deformation occurs and the image is still clear and easily focused. It is recommended to continually monitor the deflection of the XY-Stage and make sure that no permanent deformation occurs. Although the current heater properly maintains the cellular environment at 37oC, the heater cannot accurately maintain the temperature outside of the heater area. Previous designs have proposed for two different heaters to be lined up side by side in order to more accurately regulate the temperature of the area between the two heaters. Unfortunately, because of the inability to contact the Minco, the heater company, and the lack of time one heater was used to regulate the temperature. Once getting in contact with the heater company we were informed that wiring two heaters together in series or in parallel is possible. Since Minco two different heaters that were available for this type of wiring setup were out of stock, one heater was still used for temperature regulation. It is recommended that if two heaters setup is desired to order heater model CT198-1006R20.8L1 for two heaters wired in series or heater model CT198-1000R5.2L1 for two heaters wired in parallel. 8.3 The Next Step…

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Now that real time monitoring of microfluidic experiments can be monitored and kept at proper temperature, other improvements can be made to better mimic the actual cellular environment. One of our customers’ desires is to provide the proper carbon dioxide concentrations within the microfluidic channels. A solution for this could include a small enclosing over the Braille cell actuator, with an inlet for carbon dioxide. Since MS-BAM has been designed specifically for the Nikon TE-2000u, in the future the user may want to use this device on a stereoscope or some other microscope. By redesigning the stage blocks and acquiring the proper screws, MS-BAM should be easily attached to other microscopes. This may only be possible as long as the Braille cell housing device does not interfere with any of the microscope equipment that could potentially be present around the device. MS-BAM does fulfill the most basic and important requirements of real-time monitoring, but other capable improvements are only a few small steps away. 9 Project Budget The final budget of the project was somewhat below the projected budget outlined at the beginning of the project. Note that a Bill of Materials can be found in Appendix X. 9.1 Final Project Budget (April 24th, 2005) Existing Start-Up Equipment Cost

• Microfluidic Equipment

o Chip fabrication equipment and materials …………. $80.00 o Braille Actuation Device (SC-9 by KGS Corporation) $730.00 o Braille Actuation Device hardware interface……….. $500.00 o Personal Computer………………………………….. $1000.00 o LabView Software Package………………………… $3000.00 o Electrical Heating System…………………………… $200.00

• Microscopes and Related Devices

o Nikon TE2000-U……………………………………. $15000.00 o Nikon XY Stage, Model #91121……………………. $600.00 o Simple PCI Software ………………………………... $750.00 o Hamamatsu CCD Camera and Acquisition Hardware $1500.00

Required Resources: (listed by subsystem)

• Stage and Extender o Aluminum Material and Machining.………………… $270.00 o Stage Blocks…………………………………………. $80.00

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• Housing Unit o Side Pieces

8 mm Aluminum Material….…………… $79.00 Machining Cost (1.5 hrs at $100/hr.)……. $150.00

o Top, Bottom and End Pieces Plexiglas Material ………………………. $10.00

• Chip Retention Device

o Plexiglas Retention Bar………………………….. $10.00 o 3mm Threaded Rod, 20 cm long x 2…………….. $2.00 o Spring x 2………………………………………… $3.00 o Wing nuts for 3mm Threaded Rod x 4 (2 extra)…. $4.00

• Microscope Slide Brackets

o Alligator Clips……….. …………………...……. $2.00 o 75x50mm Microscope Slides x 72 ……………… $3.95 o Angle Aluminum Side Bracket x 2……………… $20.00

• Heater

o Additional Heater (if 1 does not suffice) ………… $41.00 o Misc. electrical components (wire, resistors, etc)… $15.00 o Mounting Hardware………………………………. $25.00 o Thermocouple…………………………………….. $89.90

• Software-Hardware Interfacing

o Misc. electrical components (wires, resistors, etc…) $50.00

• Miscellaneous o Stainless Steel Hex Nuts x 10…………………….. $1.25 o Extra Screws, Bolts…………………………….… $30.00

Final Total……………………………………………………… $886.10 Budget Given………………………………………………………… $1500.00

Budget Notes: The final cost of the project fits within the original estimate of between $700 and $1190. The main source of our minimization comes from the large the amount of machine work provided by our contacts in Israel at a great discount. The main stage in particular, was much less expensive than originally envisioned. There were however, some unexpected cost additions as well (such as the additional thermocouple that costs ~$90) which evened out the cost to match our projected budget. 9.2 Original Projected Budget (January 24th, 2005)

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Required Resources: (listed by subsystem) Cost Scaffold System:

● Steel or aluminum for custom stage…………………………………$0-75 ● Material for linkage between Braille cell-chip and XY stage……… $120 ● Plexiglas for retention of Braille cell to chip………………………. $10 ● Material for housing/interchanging of the chips…………………… $50 ● Machining of miscellaneous metal parts & shipping costs………... $60-700 ● Powdercoat finishing of all custom metal parts.…………………….$120-150

Heating System: ● Miscellaneous electrical components (wire, resistors, etc)…………$15

● Additional heater (if 1 does not suffice)…………………………… $200 ● Mounting hardware………………………………………………… $30

Software-Hardware Interfacing: ● Miscellaneous electrical components (wire, resistors, etc)………… $100

Estimated Total………………………………………………………………..$700-1190 Budget Given: $1500 10 Final Project Timeline 10.1 Estimated Proposed Project Timeline Before the Detailed Design Review, a list of tasks that were thought to be all-inclusive for the completion of the project was compiled (Figure 10.1). From this list, a timeline was created that estimated the durations, start dates, and finish dates of the tasks (Appendix XI). A Gantt chart was chosen to represent the project timeline because of the ease and clarity this type of document lends to viewing of the order, duration, and interdependency of all project-related tasks. Through this type of organization, the Gantt chart reveals which tasks can be simultaneously executed, and which tasks, if not completed in a timely fashion, may cause a delay in the commencement later steps in the design process. The timeline is based on considering all seven days of the week to be working days.

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Figure 10.1: List of Tasks and Milestones on the Estimated Project Timeline

10.2 Actual Project Timeline Figures 10.2a & b contain a list of the actual tasks and milestones involved with the design of MS-BAM. Appendix XII contains the Gantt chart form of this timeline. Over the course of this project, the timeline constantly evolved. Common deviations from the estimated project timeline (Figure 10.1) include breaking individual tasks into several more detailed tasks, addition of more important milestones, as well as delays in start and finish dates. Although delays did occur in the design process, the timeline served as a helpful tool to delineate critical paths and pinpoint tasks that must be completed as soon as possible as to avoid downstream delays. As an example, it was possible to correct misplaced holes in the main stage because of the early deadline set for its initial machining, which allowed a sufficient buffer of time for error reconciliation. Some significant tasks and milestones that were not finished on time include receiving the voltage regulator circuit from the customer, which delayed heater testing and validation by weeks. When the heater was finally tested, the digital temperature sensor

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provided by the customer turned out to be grossly inaccurate, which led to the unanticipated purchase of a new one. Once the proper instrumentation was attained, heater testing revealed that the heater and heater controller circuit provided by the customer were not compatible. A new controller box was ordered at the last minute, and just in time for the final deadline: the Final Design Review. Thus, heater calibration proved to be a critical path and a potential threat to completing the project even though it was regarded as a trivial task on the estimated project timeline. Through planning ahead with the process of machining, including completion and submission of CAD plans to machinists, significant problems were avoided. The main stage had two holes that were improperly placed, but because this part was received months before prototype assembly, they were re-machined in time to avoid any downstream delays in the timeline.

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Figure 10.2a - List of Tasks and Milestones on the Actual Project Timeline (Part 1 of 2)

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Figure 10.2b - List of Tasks and Milestones on the Actual Project Timeline (Part 2 of 2)

11 Acknowledgements We would like to thank Dr. Rachael Schmedlen for her constructive comments and feedback, and for her desire to help us through the design process. We also thank Mr. Tom Withrow for attending the many hours of group meetings, and for all of his help and patience in the Orthopedics Research Laboratory machine shop. Without his interest in our project and his second-guessing of our preliminary design ideas, our device would

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not be in the state that it is. Finally, we would like to thank C-Cut for their help in arranging the machining our main stage, extender, stage blocks, and side pieces. 11 References

1. Unger, M. A.; Chou, H. P.; Thorsen, T.; Scherer, A.; Quake, S. R. Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 2000, 288, 113-116.

2. Wheeler, A. R.; Throndset, W. R.; Whelan, R. J.; Leach, A. M.; Zare, R. N.; Liao, Y. H.; Farrell, K.; Manger, I. D.; Daridon, A. Microfluidic device for single-cell analysis. Anal. Chem. 2003, 75, 3581-3586.

3. Hansen, C. L.; Skordalakes, E.; Berger, J. M.; Quake, S. R. A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion. Proc. Natl. Acad. Sci. USA, 2002, 99, 16531-16536.

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12 Appendices

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Appendix I – SC-9 Braille Cell Schematics Note: All measurements are in millimeters (mm)

● Top View (The Side with Electrical Connections is at the Bottom)

● Side View of SC-9 Braille Cell

Holes spanning the width of the Braille cell

This knob in the plastic casing will be cut off

Braille Pins

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Appendix I (Continued) – SC-9 Braille Cell Schematics

Actual Photograph of the SC-9 Braille Actuator

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Appendix II – Functional Decomposition

1. Viewing of culture

1.1 Design System to View with Nikon TE2000-U

1.1.1 Design scaffold to fit the Nikon TE2000-U microscope 1.1.1.1 The scaffold, composed of the stage, the X-Y translation

system, an Extender, and a housing on which the Braille chip, microfluidics chip, and heater must be designed to fit properly into the space available on the TE2000-U microscope

1.1.1.2 Beyond the scaffold simply fitting into the available space, some room to allow manual adjustment by hand must be designed

1.1.1.3 The ‘stage’ of the entire scaffold should be designed to fit via screws onto the screw holes which are currently on the Nikon TE2000-U microscope

1.1.2 Design transparent area for cell culture viewing

1.2 Design X-Y and Z translation capability

1.2.1 Design X-Y translation for proper viewing of cell culture area 1.2.2 Design Z-Axis translation ability

1.2.3 Design a limit to Z-Axis movement 1.2.3.1 The movement range in the Z-axis should be limited to a

meaningful range for viewing 1.2.3.1 If meaningful range for viewing goes outside of practical

boundaries and parts of the scaffold would run into parts of the microscope, then the Z-axis should be limited to only the range in which there such accidents could not occur

1.2.4 Focusing designed to be controlled by TE2000-U’s existing mechanism

2. Cell Culture

2.1 Design Rubber Silicone Culturing Device 2.1.1 Ensure material is capable of cell culture

2.1.1.1 Cells should be able to adhere to and grow on the material (PDMS) 2.1.1.2 The material shouldn’t degrade and release chemicals that would kill the cells

2.1.2 Ensure material is transparent 2.1.2.1 Ensure material is transparent so that the cell culture can be viewed under the microscope

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2.1.2.2 Ensure the refraction index of the material is such so as to allow for the microscope viewing to work properly

2.1.3 Ensure material is durable—useful over longer culturing times 2.1.4 Design silicone culturing device to prevents contamination of

culture 2.1.5 Ensure silicone culturing device microchannels

2.1.5.1 Confirm that the microchannels allow fluid flow so as to provide the cell culture with nutrients 2.1.5.2 Confirm that the microchannels are configured in such a way so as to be useful for running relevant experiments

2.1.6 Design to allow laminar flow 2.1.6.1 There should be multiple ‘input’ channels which would allow for different lamina of fluid to flow adjacent to one another 2.1.6.2 The scale of the channels should be conducive to inducing laminar flow to allow for ‘PartCell’ experiments

2.2 Verify Efficacy of Pumping/Valving 2.2.1 Verify use of Braille chip

2.2.1.1 Verify that the pump/valve system utilizes peristaltic pumping action via a Braille cell 2.2.1.2 Verify that the microfluidic chip ‘fits’ the specific Braille chip

2.2.2 Verify pumping mechanism works 2.2.3 Verify valving mechanism works 2.2.4 Verify reliability of Braille pump/valve

2.2.4.1 The pumping/valving device should be able to pump and valve in a consistent manner 2.2.4.2 The pumping/valving device should not easily break and have to be replaced, it should be capable of being used for long periods of time

2.2.5 Design the system so that the pumping/valving system does not obstruct viewing

2.3 Verify efficacy of heating

2.3.1 Verify system capable of heating at 37C 2.3.2 Verify system capable of heating at other useful biological

temperatures 2.3.3 Verify system reliability

2.3.3.1 The device should be able to keep the able to keep the heat at a consistent 37C without much variation 2.3.3.2 The device should be able to provide heat over extended periods of time 2.3.3.3 The device should not malfunction easily

2.3.4 Verify heating system does not obstruct viewing

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2.3.5 Verify heating system is user-friendly 2.3.5.1 The device should be easy to operate 2.3.5.1 Trouble

2.3.6 Design the placement of the heating device such that it is easily removable

2.4 Design Computer Control (Software)

2.4.2 Design the Braille system’s pumping mechanism to be controlled

via the computer software 2.4.3 Design the Braille system’s valving mechanism to be controlled

via the computer software 2.4.4 Design the heating element to be controlled via the computer

software 2.4.5 Design the capacity for automation

2.4.5.1 The software should be able to actuate particular pumps at desired times while the user is absent 2.4.5.2 The software should be able to actuate particular valves at desired times while the user is absent

2.4.6 Design the system to be robust 2.4.6.1 The software should be adaptable to different microchannel patterns 2.4.6.2 The software should be capable of adding pumps and valves ‘on-the-fly’ in the middle of a cell culture experiment

2.4.8 Design the software to be user-friendly 2.4.8.1 The software should be easy to operate 2.4.8.2 The operation of the software should be intuitive

3. Structural Support (Scaffold)

3.1 Design the stage

3.1.1 Design to stage to support X-Y translation system 3.1.2 Design stage to support one end of Extender 3.1.3 Ensure stage material is durable

3.1.3.1 Should be capable of supporting weights far above relevant ‘use’ range to prevent damage from user error (i.e. accidentally leaning on the stage)

3.1.3.2 Should be capable of supporting many load cycles without material failure

3.1.4 Ensure the stage material is rigid 3.1.4.1 Should have high modulus of elasticity and toughness

3.1.4.2 Should not be susceptible to plastic deformations 3.1.5 Ensure the stage material is light in weight 3.1.5.1 Reduced stress to other components

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3.1.5.2 Easy for user to handle when removed 3.1.6 Ensure the stage material is relatively inexpensive 3.1.7 Design the stage to fit TE2000-U microscope

3.2 Design X-Y translation system

3.2.1 Design the system with the capacity for X-Y translation 3.2.1.1 X-Y translation should be capable of translation in 2x2

viewing area; at the same time it should ‘fit’ into its area 3.2.1.2 Easy X-Y translation via mechanically actuated rotating

lever extending from the translation system 3.2.3 Design the system to be attachable onto the stage 3.2.4 Design the system to be capable of supporting Extender 3.2.5 Design the system to be removable 3.2.6 Ensure translation system’s material is durable

3.2.6.1 Should be capable of supporting weights far above relevant ‘use’ range to prevent user error

3.2.6.2 Should be capable of supporting many load cycles without material failure

3.2.7 Ensure translation system’s material is rigid 3.2.7.1 Should have high modulus of elasticity and toughness 3.2.7.2 Should not be susceptible to plastic deformations

3.2.8 Ensure translation system’s material is light in weight 3.2.9 Design translation system to fit within rest of scaffold 3.2.10 Ensure the translation system’s material is relatively inexpensive

3.3 Design the Extender

3.3.1 Design Extender to be capable of attaching onto X-Y translation system

3.3.2 Design Extender to be capable of resting on stage 3.3.3 Design a lowered (notched) middle portion for placement of

housing 3.3.4 Design Extender to be removable 3.3.5 Design Extender to be durable

3.3.5.1 Should be capable of supporting weights far above relevant ‘use’ range to prevent user error

3.3.5.2 Should be capable of supporting many load cycles without material failure

3.3.6 Ensure Extender material is rigid 3.3.6.1 Should have high modulus of elasticity and toughness 3.3.6.2 Should not be susceptible to plastic deformations

3.3.7 Ensure Extender material is light 3.3.7.1 Reduced stress to other components 3.3.7.2 Easy for user to handle when removed 3.3.8 Design Extender to fit within rest of scaffold

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3.3.9 Ensure Extender material is relatively inexpensive

3.4 Design Housing

3.4.1 Design indentation on which Braille Chip fits 3.4.1.1 Indentation should fit Braille chip snugly but should not so

be tight as to create difficulty in placement of Braille chip 3.4.1.2 The depth of the indentation should be such that the PDMS

chip can be flush with both the surface of the Braille chip and the transparent viewing area

3.4.2 Design placement for PDMS Chip 3.4.2.1 Should have a spot on which the PDMS chip can fit

3.4.2.2 Should allow PDMS chip to lie flat over both the Braille chip and the transparent viewing area

3.4.3 Design placement for heating system 3.4.3.1 A proper space should exist for placement of the heating

system 3.4.3.2 The heating system should be easily removable

3.4.4 Design space for wiring 3.4.4.1 Should provide sufficient room for placement of wiring 3.4.4.2 Wiring should be easily accessible/removable

3.4.5 Ensure material is durable 3.4.5.1 Should be capable of supporting weights far above relevant

‘use’ range to prevent user error 3.4.5.2 Should be capable of supporting many load cycles without

material failure 3.4.6 Ensure material is rigid

3.4.6.1 Should have high modulus of elasticity and toughness 3.4.6.2 Should not be susceptible to plastic deformations

3.4.7 Ensure material is light weight 3.4.7.1 Reduced stress to other components 3.4.7.2 Easy for user to handle when removed

3.4.8 Design housing to fit within rest of scaffold 3.4.9 Ensure material is relatively inexpensive

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Appendix III - Main Stage CAD Plans Note: The main stage is 8.0 mm thick, and this is a top view. All holes are threaded with helicoils (4mm diameter holes with M4, 5mm diameter holes with M5). All Fillets have a radius of 5mm. All dimensions are in millimeters.

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Appendix IV - CAD drawing of the Stage Blocks, with dimensions(dimensions in mm).

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Appendix V - Extender CAD Plans Note: The extender is 4.0 mm thick, and this is a top view. All holes are through holes (non-threaded). Fillets are 5mm in diameter. All dimensions are in millimeters.

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Appendix VI - Side Piece CAD Plans All views needed to machine the side pieces are included below. All dimensions are in millimeters.

(Top View)

(Top View)

(Front View)

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Appendix VII - Microscope Slide Bracket CAD Plans

● Overall View:

● Front View (Dimensions are in millimeters):

Front

Back (Adjacent to Braille Cell and Side Pieces)

12.7 mm

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Appendix VIII - Chip Retention Bar The Chip Retention bar is constructed out of 4.0 mm thick clear Plexiglas. All dimensions are in millimeters. ● Top View

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Appendix IX - Plexiglas Covering Pieces CAD Drawings

All dimensions are in millimeters. Piece a). goes on the back of the Braille cell, b). goes on the top, and c) goes on the bottom.

a).

b).

c).

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Appendix X – Bill of Materials

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Appendix XI - Estimated Project Timeline Gantt Chart (Part 1 of 1)

Gantt Chart Key Milestone General Project Task Software-Related Task Mechanical Related Task Heater Related Task

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Appendix XII - Actual Project Timeline Gantt Chart (Part 1 of 3)

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Appendix XII (Continued) - Actual Project Timeline Gantt Chart (Part 2 of 3)

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Appendix XII (Continued) - Actual Project Timeline Gantt Chart (Part 3 of 3)