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This BYOC was designed as to achieve one of my goals of I had set upon joining the ISAC SRL EL program. The purpose of the BYOC is to act as an additional aid to spark interest when teaching cytometry to students. It has been designed to be affordable (<$500$500-$1500), 3D printable and fun to build. I have included the build list, instructions, notes and highlight there are many opportunities for improvement/modification (I can attest to this with my experience over the last 6 years).
The BYOC Workshop was first held @ ACS 2016 in Sydney, Australia, @ ACS 2018 in Adelaide, Australia and conjointly with the Garvan Flow Facility @ ACS 2019, Melbourne, Australia. This page was updated with the plan to host the workshop @ ACS2022 in Melbourne.
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Overview
The cytometer uses a <$50 adjustable 5-25mW laser (interchangeable) that is directed onto a jet in air stream. The stream is ejected throughout a 100um nozzle that is assembled using luer locks, cogs and HPLC tubing to allow injection of sample in a laminar flow. The laser is focused onto the stream and signals are detected using a 25mm, 25mm focal lens that directs the emitted/scattered light onto the FSC/SSC/FL detectors. A diagram of the light path from excitation is shown in the diagram below. The signals are detected with a APD/Photodiode or PMT and then converted to a voltage output using a pre-amp. The signals can be viewed using an oscilloscope or can be displayed on a bivariate dot plot using a ADC connected to a computer.
3D Printable Parts (2022 updates)
Steering laser mirror mount - https://www.thingiverse.com/thing:3523131
Flow cell - https://www.thingiverse.com/thing:3902321
Detector - https://www.thingiverse.com/thing:3902266
Mounting - https://www.thingiverse.com/thing:3902335
Sample tube mount - https://www.thingiverse.com/thing:3902328
Flow control - https://www.thingiverse.com/thing:3902432
Laser mount - https://www.thingiverse.com/thing:3902366
Notes
- The laser interception point as of 2022 has been fixed at 127mm from the base. This 'fixing' allows for a simpler setup and easier modifications.
- Using the relatively affordable 'blackridge' air regulator from Supercheap auto, with a 0.1um PES filter in line, dial 3 full rotations and then dial back a 1/2 rotation of the air pressure regulator valve for this project. This provides approximately 2-3psi that is hard to observe on the blackridge air regulator.
- When focusing the laser it is important to focus it so that there are no striations seen when centered. Striations indicate a focal point either behind or infront of the stream. An optimal focus will have striations briefly when aligning the laser stream and it is crossing the edges but will result in no striations when centered.
- The microscope used in this project is a modified kids toy without any optical filters. To view the core stream interception point ensure the microscope is angled so that stray laser speckle does not overwhelm the scope.
- The testing rig LED can be run from an external pulse generator. >1000 pulses per second work well for the preamplifier circuit design. Try increasing the speed of the flash if there is a negative pulse.
- Photodiode - 9x9V for Photodiodes or none!
- APD - 9x9V + Polulu fine control. 86V saturates APD, 83V just detects, 85 less.
Notes - other references
mmHg to PSI
**Divide by 51.715 to obtain PSI**
| mmHg | PSI |
| 0 | 0 |
| 20 | 0.386735 |
| 40 | 0.77347 |
| 60 | 1.160205 |
| 80 | 1.54694 |
| 100 | 1.933675 |
| 120 | 2.32041 |
| 140 | 2.707145 |
| 160 | 3.09388 |
| 180 | 3.480615 |
| 200 | 3.86735 |
| 220 | 4.254085 |
| 240 | 4.64082 |
| 260 | 5.027555 |
| 280 | 5.41429 |
| 300 | 5.801025 |
Bill of materials - 3D Printed Version (acrylic assists can be interchanged)
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| Item | Qty | Purpose | Sources | Cost ($AUD) | Notes | Image |
|---|---|---|---|---|---|---|
| 600x600 tile | 1 | base | Local tile supplier or Bunnings (requires a packet) | $0-$40 Free (tell them what you are making) | Aim for a free marble tile |  |
| 600x600 steel sheet | 1 | build platform | Bunnings (Sydney) | $26 | Ensure it is magnetic. Thickness does not matter, thinner normally means cheaper. I recommend sign steel. Have some fun and wobble it ;p | |
| 0.3M7TIA gear | 2 | holds HPLC in the centre of luer lock | Aliexpress | <$5 for 20 | https://www.aliexpress.com/item/33000449925.html?spm=a2g0s.9042311.0.0.86a84c4dqSRqUr |  |
| 450 extension springs | 19 (2 detectors), additional 6 per detector | provides pulling force to mirror, laser, lenses | Bolts & Industrial (Sydney) Century Springs (US) | $4 | We need to buy it from a reseller. Spring Catalogue: https://www.centurysprings.com.au/products/prod_catalogue.htm |
Tools/Equipment
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Common University library resource
Shapeways - online printer
Can also buy about 10x cheaper from ebay. |
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| M3 socket screw | bag | used as screws to put the whole thing together | RS components or ebay | |||
| M3 nut | bag | Ensure outside dimensions are 5.5mmx5.5mm | RS components | $13.80 | RS PRO, M3 5.5mm Steel Square Nuts, Bright Zinc Plated Finish 837-262 |
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| 25mmx25mm optics | 2-3 | focus light onto detector from intercept point | Edmund optic | $52.80 | Stock #32-490 https://www.edmundoptics.com.au/p/25mm-dia-x-25mm-fl-mgfsub2sub-coated-double-convex-lens/2375/ | |
| 5mmx25mm optics | 1 | focus laser light onto stream | Edmund optic | $61.50 | Stock No. #87-913 https://www.edmundoptics.com.au/p/50mm-dia-x-250mm-fl-mgfsub2sub-coated-plano-convex-lens/30185/ | |
| 100um nozzle | 1 | eject stream | Intertronics or FISNAR | 38 pounds | https://www.intertronics.co.uk/product/preci-tip-precision-dispensing-tips/ IDMSPREC-M100-NS | |
| Luer lock connectors and 1/4inch BSP to barb fittings | 1 | to connect to nozzle | Bunnings & Kinesis Australia & USA KENT | I will need to separate this column a bit more - information will be provided during the workshop. |  | |
| 30mm x 10mmx 3mm | 20 | mount items | Ebay | $18 | 20pcs N50 Strong Block Cuboid Rare Earth Neodymium Magnets 30mm x 10mm x 3mm | |
| Circular magnet | To mount APD instead of photodiode |  | ||||
| 20L Drum | 2 | used to create a vacuum | recycle bin | free | to make see instructions | |
| Regulator | 2 | adjust pressures | supercheap auto | $30 each or $30 for 2 | https://www.supercheapauto.com.au/p/blackridge-air-regulator/340078.html or | |
| APD or Photodiode | 1 minimum | convert photons to current | many sources including old instruments | $15 each | APDs used are AD500-8 TO52 package or BNC photodiode package | |
| Laser 532nm (or equivalent) | 1 | AIXIZ lasers | $38.50USD | https://www.aixiz.store/product-page/532nm-5-25mw-adjustable-power-3vdc-line-cross-attachable-ad-532-25adj | ||
| Electronics shopping list (per detector) | ||||||
| Prototyping board | 1 | $8.19 | ||||
| RJ45 breakout | 2 | $1.33 | https://core-electronics.com.au/breakout-board-for-rj45.html |  | ||
| LT1364CN8#PBF-ND | 2 | opamp used for I>V and for amplification | DIGIKEY | #13.73 each | Now obsolete, any high speed high slew op amp with low noise, rail to rail should work. I will look to include a specific part soon. | |
| Cat7 RJ45 cable | 1 | $8 | Amazon/Ebay | |||
| Adjustable 10^5 25 turn resistor | <$1 | Jaycar (generic electronics parts store) | ||||
| Adjustable 10^3 25 turn resistor | <$1 | Jaycar (generic electronics parts store) | ||||
| 1N4148 diodes | 4 | <$1 | Jaycar (generic electronics parts store) | |||
| 10pF ceramic capacitors | 2 | <$1 | Jaycar (generic electronics parts store) | |||
| 10K resistor (1% tolerance) | 3 | <$1 | Jaycar (generic electronics parts store) | |||
| 100K resistor | 2 | <$1 | Jaycar (generic electronics parts store) | |||
| 1K resistor | 1 | <$1 | Jaycar (generic electronics parts store) | |||
| 22K resistor | 1 | <$1 | Jaycar (generic electronics parts store) | |||
| 0.22uF ceramic cap | 1 | <$1 | Jaycar (generic electronics parts store) | |||
| 1uF ceramic cap | 1 | <$1 | Jaycar (generic electronics parts store) | |||
| 2 pin molex pcb header 2.54mm pitch | 1 | $0.20 | https://www.jaycar.com.au/2-pin-0-1-straight-locking-header-2-54-pitch-single/p/HM3412 | |||
| 3pin molex header female 2.54mm pitch | 1 | used to connect to APD | $0.55 | https://www.jaycar.com.au/3-pin-0-1-header-with-crimp-pins-2-54-pitch/p/HM3403 |  | |
| hookup wire and crimpers | $0 | Spares in every household | ||||
| 9V battery connectors | 4 | buy different types of connectors to ensure flat 80-90V power supply | $1.40 | Jaycar | ||
| 8 pin IC housing | 2 | mount for IC chips | $1.25 | https://www.jaycar.com.au/8-pin-gold-insert-machined-pin-ic-socket/p/PI6452 | ||
| 4 or 6 way DIP switch | 1 | used to turn power on / off | $1.65 | https://www.altronics.com.au/p/s3050-4-way-dip-switch/ | ||
| shielded RJ45 port | 2 | needed to suppress noise | $1.75 | https://www.altronics.com.au/p/p1538-oupiin-8p8c-rj45-modular-socket-shielded-pcb-mount/ | ||
| Differential pressure regulation | ||||||
| pressure transducer - 24PCAFA6D *only required for digital pressure readout | 1 | accurate pressure differential between sample/flow cell | Element14 | $61.89 | 0-1psi - 0-45mV - 2002508.pdf |  |
| OPAMP - breakout for pressure transducer *only required for digital pressure readout | 1 | simple 2 stage amplification of 100 | Core electronics | $9.90 | BOB-09816 100x gain |  |
| LCD keypad *only required for digital pressure readout | 1 | pressure output monitor | Core electronics | $16.50 | https://core-electronics.com.au/lcd-keypad-shield-for-arduino.html DFR0009 |  |
| arduino and case *only required for digital pressure readout | 1 | arduino uno | Core electronics | $39.95 + $4.70 | CE05634 A000066 | |
| Aluminum laser mount and heat sink | 1 | holds and cools the laser | Aixiz laser supplies | USD$3.95 | 12X30mm aluminum mount for AixiZ 12X30mm units[AIX-ALM-MT-1] |  |
| 6mm nuts | many | mounts with 3mm threads | RS components | $12.23 pkt of 100 | RS PRO, M3 5.5mm Steel Square Nuts, Bright Zinc Plated Finish 837-262 |
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| Molex 2 way connector and crimp | 1x for laser power source | Allowing the disconnection renders the laser and power supply modular. | RS components |
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| Cable ties | many | Need I say more | 100mm x 2.5mm |
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| Polulu fine control stepup/down voltage regluator | 1 per APD detector | 9V batteries are coarse adjustment - this gets you finer control | core electronics | $27.15 | https://www.pololu.com/product/2869 The output voltage of the regulator is controlled with a 12-turn precision potentiometer. Turning the potentiometer clockwise increases the output voltage, and it can be measured using a multimeter. |  |
| 2xD battery holder | 2 | Used to power the laser and vacuum pump | Jaycar | $2.75 | https://www.jaycar.com.au/2-x-d-cell-side-by-side-battery-holder/p/PH9220 CAT.NO: PH9220 | |
| Vacuum pump | 1 | Used to generate a vacuum | Core Electronics | $10.15 | https://core-electronics.com.au/370-mini-vacuum-pump.html SKU: FIT0801 Brand: DFRobot Runs for over 24 hours using 2xD batteries. Current measured during use @ 3V = ~250mA. |  |
Instructions
Getting started - making a base!
- Obtain a 600mm x 600mm tile (marble preferred but ceramic is more economical)
- Place it on the left hand side of a table that is longer than the tile (we need the space on the right for the electronics).
- Place a 600mm x 600mm steel sheet over the tile.
- Secure with clips as shown.
Air, sheath and vacuum reservoirs
- Place magnets, nuts and screws into the 3D printed bottle holders.
- Place the 3 x bottle holders at the rear of the build as shown.
Let's vacuum!
The instrument uses a vacuum to draw away excess liquid. There are a few variants to the vacuum (including a gravity version that I absolutely love, and not only because I designed it, but because it uses gravity lasts ~10 minutes).
In 2022 we'll be setting up an battery operated electric vacuum pump.
- Obtain a vacuum pump (listed above).
- Solder the terminals to a 2xD size battery holder.
- Obtain a wine bottle with a aluminum lid, preferably a top shelf one
. Utilise wine thoughtfully. - To drill the holes we start with a guide drill bit ~1-2mm, drill our holes while the lid is on the bottle and then follow through with a hole size that is about ~2mm smaller than the tubing. I've found a 4mm drill bit works well for the ~6mm Tygon tubing obtained for this project. The wine bottle lid requires 2 holes, 1 to draw out the vacuum and 1 to lead the liquid waste in.
- Drill guide holes followed by larger holes (4mm for 6mm tubing)
- Insert tubing ends that have been cut on an angle to assist pulling tubing through. Lubrication with water of the tubing outer face may help. Ensure the tubing to be connected to the vacuum pump is cut close (within 1 inch to the bottle cap) to prevent liquid being sucked in to the hydrophobic filter.
- Connect connectors including a hydrophobic filter to prevent liquid entering the vacuum pump.
Setting up the laser
Most standard cytometers use a laser as a light source. The BYO cytometer is no different. Read on to mount your laser!
- Insert the laser of choice into the aluminum heat sink and hand tighten the screw to lock the laser in.
- Here are 2 compatible lasers (left is a 405nm, right is a 532nm laser available from aixiz.com.
- Insert 2x5.5mm square nuts into the laser mount nut sockets and attach the laser mount using 2xM3 10mm socket screws
- Insert 2 rectangular magnets into each base to secure the laser to the steel base.
- Attach a 2.5mm ~100mm cable tie to secure the laser power cables. This is to prevent them falling off with too much movement.
- Cut off the excess on all cable ties going forward
- Crimp on a 2 way molex connector using a crimper
- Attach the connector and use a scapel to expose the laser control potentiometer (the yellow heat shrink is what you need to cut back).
- The laser is now ready!
Making a flow cell...
- obtain male to male luer lock (transparent is great), and 2 small 0.3M7TIA gears
- Setup the luer lock to hold the gears - this will be used to hold the PEEK tubing.
- Insert the precut peek tubing
- Connect the remaining luer lock connectors followed by a luer lock to barb connector as shown
- Using 2 Y connectors, tubing and luer lock to bar connectors create the following Y connector that will join the stiff main flow cell.
- Pass the peek tubing up through the female luer lock connector.
- Create a sheath bleed point and transition to silicon tubing as shown.