Hardware: Transformer Mark 3

• 1: Laboratory Stand
• 2: Camera and focus
• 3: Filter wheel and optics
• 4: TIRF LED and temperature control assemblies
• 5: Main body

Introduction

Our Sequencing instrument, “Transformer Mark 3.0”, is a custom-built total internal reflection fluorescence (TIRF) imaging system, specifically designed to support the innovative Lightning Terminators™ (LT) one pot sequencing. This system combines advanced optical engineering with the versatile control offered by a Raspberry Pi 4, augmented with stackable HAT (Hardware Attached on Top) boards for enhanced functionality.

Key functionalities

Temperature control and monitoring

The Transformer Mark 3.0 ensures optimal environmental conditions for sequencing reactions. Precision temperature control is crucial for accurate and reliable sequencing results.

Waveguide-based TIRF imaging module

At the heart of the system is the TIRF imaging module. Utilizing a waveguide approach, this module excites the fluorescent molecules linked to incorporated LTs a few hundreds of nanometers from the sequencing substrate surface. This module also performs effective UV cleavage of the terminating groups for continuous sequencing.

Motorized imaging filter switching and focus adjustment

To adapt to various imaging requirements, the Transformer Mark 3.0 includes a motorized filter wheel and focus adjustment mechanism. This allows for rapid and precise changes between different fluorescence channels and focus levels, ensuring sharp images and accurate data capture.

Procedure

The Transformer Mark 3.0 is assembled in four main sections. The process is detailed in the subpages below — we recommend assembling in this order (generally from bottom to top):

1 - Laboratory Stand

We use a simple laboratory stand that the Transformer is assembled on without skins or covers.

Parts

The following 3D printed and/or machined parts are available in our hardware repository .

Procedure

Tap the 3D printed base with M5 tap for the 4 flat head screws.  Attach the adhesive-back bumpers to the underside of the base and attached the lab stand plate with 4 flat head screws.  The assembled system is then attached to the lab stand with the two button head hex drive screws.

2 - Camera and focus

Parts

The following 3D printed parts are available in our hardware repository .

Procedure

Start by attaching the Positioning Table (60935K71) with two M1.6 socket head screws (91292A264) and thin hex nuts (90710A145) to the camera mount.  Then attach the focus motor mount to the other side of the positioning table with 4 zinc plated flat head screws (91263A862).  Then mount the stepper motor (STEPPERONLINE Micro Step Motor Nema 8 Bipolar Stepper Motor 0.6A 5.7oz.in/4Ncm DIY Robot, stepperonline.com) with 4 flat head screws (92125A052).  Attach the small pulley to the motor and the large pulley to the adjustment knob and tighten the pulley with one M2 socket head screw (91292A833).

Connect the two pulleys a 2x22 Buna-N O-ring (9262K183). Mount the camera (XSenIMX183) to the camera mount with 4 Socket Head Screws (91292A831).  Set the subassembly aside.

3 - Filter wheel and optics

Parts

The following 3D printed and/or machined parts are available in our hardware repository .

Start assembly by attaching the filter housing bottom to the camera and focus assembly with 6 flat head screws (92125A086).  Place the assembly on the laboratory stand and attach the assembly with two button head hex drive screws (92095A212 – from laboratory stand hardware list).

Attach the filter wheel hub to the filter wheel with three hex drive flat head screws (93395A198) and insert a set screw to lock the filter wheel once ready to assemble (91390A092).  Insert the filters into the wheel in the order shown below and set the complete filter wheel assembly aside.

Attach the stepper motor (STEPPERONLINE Micro Step Motor Nema 8 Bipolar Stepper Motor 0.6A 5.7oz.in/4Ncm DIY Robot, stepperonline.com) to the filter housing lid with 4 flat head screws (92125A052).

Optics (From top to bottom): Screw the 5x long working distance LM Plan achromatic objective lens (Bolioptics MT05073231) into a RMS to SM1 adapter (Thorlabs SM1A3), an adjustable lens tube (Thorlabs SM1V05), a stackable lens tube (Thorlabs SM1A10), and then finally into the filter housing lid. 

Attach the three aluminum legs with three Phillips flat head screws (92010A424) to the filter housing lid and slide the alignment brace over the three legs and tighten the set screws (92015A104) to lock the brace in place.  The thumb screws (92545A119) are adjusted to brace and align the optical stack.

Attach the fully assembled filter wheel to the shaft of the stepper motor and tighten the set screw. Carefully place the subassembly on the filter housing bottom ensuring that the filter wheel stop is not hitting the stop pin in the filter housing bottom.  Secure the assembly with three button head screws (92095A185).

4 - TIRF LED and temperature control assemblies

Introduction

The TIRF LEDs and heaters are mounted directly to heat sinks, which do double duty: they both dissipate heat from the high-power LEDs and maintain a consistent temperature when the sequencing reservoir is inserted into the main body.

You will need to assemble four LED boards per unit:

• Two for UV cleaving
• One for the red and orange LEDs
• One for the green and blue LEDs

Parts List

Optical Imaging

• 1x each Edmund bandpass filters: 675/50nm (#86954), 625/50nm (#86953), 575/50nm (#86952), 525/50nm (#86951)
• 1x filter holder (3D printed, see CAD file)
• 1x Long working distance 5x LM Plan Achromatic Objective lens (BoliOptics, MT05073231)
• 1x SONY IMX183 CMOS camera (Vision Components)
• 1x Thorlabs Objective adapters (SM1A3)
• 1x Thorlabs 1” adjustable lens tubes (SM1V05)
• 1x Thorlabs 1” lens tubes (SM1L10)

LED

• 4x MK 3 Heatsinks (See CAD file)
• 2x visible LEDs boards (See pictures)
• 2x UV LED boards (See pictures)
• 1x each four color visible LEDs (LUXEON Rubix Color Line LEDs, MFG P/N: L1RX-L1RX-RED1000000000, L1RX-PCA1000000000, L1RX-GRN1000000000, L1RX-BLU1000000000)
• 16x UV LEDs (Light Avenue: LA UY20WP1)
• 4x 33Ohm UV LED current limit resistor (DigiKey: ERJ-P08J330V)
• 2x 1 Ohm 3/4W SMD 1206 visible LED current limit resistor (DigiKey: SR1206FR-7T1RL)
• 1x 1.27 Ohm 3/4W SMD 1206 visible LED current limit resistor (DigiKey: CRCW12061R27FKEAHP)
• 1x 1.8 Ohm 3/4W SMD 1206 visible LED current limit resistor (DigiKey: CRCW12061R80FKEAHP)
• 26 Gauge Enamel Wire
• 30 AWG flexible silicone tinned copper wire (Fermerry)
• 4x Molex FFC/FPC Jumber cables (Mouser P/N: 538-98267-0711)
• 8349TFM Thermally Conductive Adhesive (One syringe of 8349TFM-A and one syringe of 8349TFM-B)

Other Mechanical Parts

• 4x 4.7 Ohm 16W 1% 2512 SMD Resistor (Mouser P/N: 754-CPA2512Q4R70FST)
• 1x NTC Thermistors (Vishay BCcomponents: NTCLE413)
• 4.8mm diameter clear 3:1 heat shrink tubing 10mm long
• 1x 10K Precision Epoxy Thermistor - 3950 NTC (Adafruit 372)

Assembly Instructions

LED Board Soldering

1. The LED Board Soldering picture shows the PCB for visible LEDs. First, clean the board and apply a thin layer of SAC305 solder paste (Mouser #910-SMD291SNL250T5). Using a stencil is recommended.

2. Place the LEDs (Red with PC Amber, Green with Blue) and the current limiting resistors (Red: 1.8 Ohm, PC Amber: 1.27 Ohm, Green: 1 Ohm, Blue: 1 Ohm) at the location shown below

3. Heat the board to 250-270C, the solder paste will melt and the LEDs and the resistors will be well attached. Use the tweezer to adjust the position if needed.

4. The UV LEDs were connected in series (four as a set) by wire bonding.

5. Carefully solder the silicone tinned copper wires to the bottom of the PCB

LED Heatsink Preparation

6. Prepare a pea-sized volume of 8329TCM Thermally Conductive Adhesive by thoroughly mixing one half-pea-sized ball of 8329TCM-A and one half-pea-sized ball of 8329TCM-B. Spread a thin layer of the prepared 8329TCM over the Mk3 heatsinks, on the face that has the LED board seating area, in the region marked below. The layer should be roughly the thickness of a few pieces of paper

7. Place one of the visible LED boards (Either the Amber + Red board, or the Blue + Green board) onto the prepared surface. Make sure the board is well-aligned with the side fins and the center pin.

8. Press the stack together either by a press or by hand.

9. Repeat steps 1 through 8 for the rest of the visible and UV LED boards. It will take 24 hours for the 8329TCM to fully cure. Heating up to 40-50C can slightly speed up the curing process.

Heater Cable Assembly

10. Cut 2 lengths of 26 Gauge Enamel Wire roughly 50mm long. Strip one end of each wire segment, and solder the stripped end of each wire segment to one side of the underside of the 4.7 Ohm 16W 1% 2512 SMD Resistor

11. Prepare a pea-sized volume of 8329TCM Thermally Conductive Adhesive by thoroughly mixing one half-pea-sized ball of 8329TCM-A and one half-pea-sized ball of 8329TCM-B. Spread a thin layer of the prepared 8329TCM over one of the Mk3 heatsinks, on the face that does not have the center ribbon cable groove, in the region marked below. The layer should be roughly the thickness of a few pieces of paper

12. Place one of the prepared resistors, with the soldered face away from the heatsink, onto the bed of 8329TCM that was prepared. Press down on the resistor to ensure it is seated into the 8329TCM. It will take 24 hours for the 8329TCM to fully cure

13. Repeat steps 10 through 12 for the remaining 3 Mk3 heatsink

Thermistor Cable Assembly

One thermistor is used per unit to monitor the temperature of one heatsink, which is used as a proxy for the temperature of the sample.

1. Prepare a pea-sized volume of 8329TCM Thermally Conductive Adhesive by thoroughly mixing one half-pea-sized ball of 8329TCM-A and one half-pea-sized ball of 8329TCM-B, and apply it to the thermistor.
2. Dip the thermistor and adhesive into the cylindrical hole of one of the heatsinks. Historically, we have used the red/amber heatsink for this, but there is no reason to use one heatsink over another. It will take 24 hours for the 8329TCM to fully cure
3. Splice female jumper cables to the other end of the thermistor cable.

5 - Main body

Parts

The following 3D printed and/or machined parts are available in our hardware repository .

Procedure

We start by attaching the light shroud (note the orientation of the shroud with respect to the three legs) to the main body using two narrow cheese head slotted screws (97710A113).  We then attach the main body to the rest of the assembly via two socket head screws (92290A320) and one Phillips flat head screw (92010A424).  Note: Carefully align the light shroud with the optical stack when positioning the main body on the assembly.

Attach the 4 heat sink height adjusters with 8 socket head screws (91290A010) to the main body.  Start with the height adjusters placed in the middle of the extreme positions.  The heat sink is raised by moving the adjuster closer towards the center of the main body and lowered by moving the adjuster further away from the center. When placing each of the 4 LED heat sink assemblies, gauge the alignment of the LEDs with the glass substrate in an assembled reservoir and adjust so that the LEDs are centered around the glass substrate.  The assembly of the LED heat sinks is described here.

The next step is a little tricky.  The cam ring needs to be placed onto the main body such that the heat sink pins are riding in the inner grove (see image below). If one of the pins rides in the outer grove the heat sink will not actuate properly.

Next we have to assemble the reservoir insert plate.  Place one dowel pin (91585A368) in each of the 4 bearings (7804K124) and press them into the bottom slots of the reservoir insert plate.

Insert the 8 set screws (92605A024) halfway into the holes such that the bearings can freely move up and down in the space provided (see image below):

Once the Reservoir Insert plate has been attached to the main body using 4 flat head screws (92125A126) the set screws are adjusted until the pin from the heat sink no longer has “wiggle room”.  Note that it is quite easy to overtighten the set screws so carefully adjust both screws in ¼ turn increments until the pin from the heat sink no longer moves freely.

Finally, the Locking Ring with standoffs is installed with 4 shoulder screws (90265A323) and 4 compression springs (9657K604).  Turning the cam ring to the open position allows the insertion of a reservoir and once turned to the closed position pivots the LED heat sinks such that the pins set the distance of the LED to the edge of the substrate surface and locks the reservoir into position.