Day 4 - BDInflux™ Installation

Day 4 - Influx installation complete!

Again, I got in early to repeat the start up procedure and, as it should, she started up well and lasers required minimal alignment even with a tip change. This truly is the easiest instrument in the BD range to set-up, align and even teach others about Flow Cytometry (providing you have great engineers setting you up of course). Nikki with only 8 months of Flow under her belt has found the instrument to be very easy to setup and spent some time swapping nozzles over, moving the stream about and still has very little to do alignment wise. Its a beautiful thing aligning a stream to fixed objectives rather than the other way around (Vantage) it makes so much sense and is proving to be a joy to start up in the mornings.

After a morning of trouble shooting the 6 way separation, we made a few slight adjustments to the deflection plates and saw that the 'bowing' affect on the 6 streams was reduced significantly thanks to Brent's modifications, so, we were all set to show off our new instrument and skills to visitors later that day.
Centenary Institute's Dr Adrian Smith and Robert Salomon arrived in the early afternoon and we showed  sorting beads using the 6 way collection tubes, they were also interested in changing PMT detector blocks and some features of the Influx like sorting on Time as a parameter and also Index sorting. Everything went according to plan (or course) and they left satisfied. BD were great in allowing me to show them the instrument and I really appreciated Darren's offer to do the demo. Brent was there to support the higher end questions that were asked and together I feel we pulled it off. Later that afternoon, I began to shutdown so I started to pack up and head home. A pat on the back from Mark and a comment about running on fumes sealed the deal and I headed off.

Brent - I can't thank you enough for everything you have done for us with the build of the instrument, the quality of the installation was of extremely high standards and you have provided us with an amazing platform for our researchers to utilise. Mark - you have been a rock this week for us and your help, guidance, information and conversations about the instrument have been put so simply that I feel you have given us the confidence we need to really run with this so thank you so much. Darren, your knowledge of the instrument is so thorough, your experience with sorting from your old days, your a wealth of information and I am really looking forward to our training that begins in a couple of weeks so thank you and we are keen to run some panels and get used to the workflow of FACS Sortware™. Craig, having you here has been a great addition and I hope you have enjoyed your time in Sydney, I look forward to bouncing ideas with you and calling on your expertise from time to time, thank you. Also, thanks to all at BD especially Christine Bligh & Marlene Daalmeyer for their help, support, professionalism and planning to get us to this point. Thank you. Adam your help and mate-ship is much appreciated as always, we'll see you soon for a demo.

Finally to Ger, thank you, again, your ideas and expertise behind building the Influx are so greatly appreciated, from someone like myself and Nikki that run Vantage everyday, we really see the differences you have made to Flow Cytometry as a whole with this instrument and I can't thank you enough for such a beautiful piece of engineering brilliance. I look forward to seeing you soon in Seattle for Advanced hardware training with Brent (an amazing opportunity that I am very grateful for).

I would also like to thank Dr Stuart Tangye and Dr Robert Brink for the opportunity to run such an amazing instrument in such an incredible institute, our facility is proving to be a real hub for research and I am so grateful to be a part of it all. Thank you both so much

On that note, I will sign off from the Influx installation blogs, and until the LSR7 is installed, stay tuned. Thanks for reading.

(from left back row clockwise, Craig Kelsey, Sladjan Milanovic, Mark Yoneyama, Brent Harrison, Darren Ellemore, Nikki Alling and me)

Best wishes,

Chris

ps, Marlene, we named the instrument the DMC-12 after the beloved DeLorean car from the Back To The Future movies - with its Flux capacitor how could we resist.

Day 3 - BD Influx™ Installation

Day 3 BD Influx™ Installation; Software, Six way Cell Sorting and Celebrations                       

Day 3 began again early, in an attempt to get some ‘me –time’ with the instrument I arrived at a silly 7am again, changed the compressor over to house air, turned the laser keys one – by – one and fired her up. After some minor sheath tank troubleshooting the pressure gauge soon read a steady 60psi and I began playing around with the stream. It wasn’t long before I ran into trouble and my saviour Darren walked through the lab doors dot-on 8am.

“Questions” was my first word (after good morning of course) so, we began the correct stream start up procedures and away we went, off on the 86um tip again we aligned the instrument a 488nm first, and began the process of fine-tuning each consecutive laser. Thanks to the scope, the timing of each laser is incredibly simple to get your head around, each pulse peaks in 2 channels depending on which PMT’s your interested in (simple click on a dot-plot will select which 2 channels to look at) X and Y come up on the scope, and underneath you have buckets where each pulse is to be detected, by moving the timing of the laser in the software the buckets shift left or right, align a bucket under a pulse and bingo you have a signal. Amazingly, the majority of the alignment is done by looking at beams through pinholes on the pinhole camera, focal points within those pinholes help to peak the signals, its only at each final point that we sweep a laser and watch the signal on the digital acquisition templates. Once I get a few more hours on her I know this will become very quick.

I change my first filter and move my first detector block out of the way to do so, simply, it works and no light is ever able to enter the casing that contains the filters, we install the 70um and amend our settings (the usual suspects; timing delay, sheath pressure and frequency). The cells are due to arrive at 3pm and we spend an hour ensuring each stream hits the 6 collection tube perfectly (all is done by moving the stage), samples are run and single colour controls are acquired, gates are set and we steadily chip away at the Sortware™ gating positive/negative population and dropping them into the compensation layout matrix. It has a FlowJo feel and the matrix is set smoothly and quickly, overlaps are minimal (except for a few controls that didn’t work – which ended up creating 117% overlap, which happens) and we soon begin sorting our populations. We both sigh as the sort button is clicked and purity checks reveal that all 6 were successfully collected. Workflow is still a little clumsy as get where we need to be, but perhaps not in the most effective way but are soon rewarded with dots in the respective gates on purity check.

As we head to dinner, the sun sets behind the cityscape and we sip Italian beer while feasting on carpaccio, zuchinni flowers, sorbet pallet cleansers pork bellies and fish-in-a-bag. Crème brulee and profiterolls see us waddle out at 10:30pm and I jump a train home.

First day setting up from scratch and sorting 6 populations J I am amazed at the speed in which this has all been possible. Joe, the software is perfect, Ger your instrument is elegant, rich and complex yet simple, seamless and solid. Thank you BD!! day 3 was amazing.

Chris 

Day 2 - BD Influx™ Installation

Day 2 - Lasers, Alignment, Testing and Sorting

Video link - http://tbit.ly/eVhMW0

Another big day began at an early 4:37am with an excited core facility manager eager to return to the lab and continue building the BD Influx™. 7:21am I arrived at the lab and began clearing space for the day ahead, Mark and Brent arrived shortly after and we launched into it again.

In the wise words of an old friend 'Fluidics First' is exactly how today began by working on getting the jet up and running straight away. Mark worked on the compressor and Brent began on the optics. Once the compressor was in place, the power supply for the UV laser was hooked up with large power cables thick enough to swing from. Labels were printed for the 532 and 561 lasers as the sheath tanks were filled up and the fluidics was primed. The all-too-familiar hum of a mildly deafening compressor began vibrating away which was soon drowned out by the jibbering vacuum pump, it wasn't long before we began making alternative arrangements to run both vacuum and air off the house which makes a significant difference to noise reduction (note to potential customers; run house air and more importantly house vacuum). As we stepped down the pressure from the wall to the tip, each point being 20 psi less than its predecessor the sheath pressure valves soon read 90 psi and the vacuum guage read a steady 5. We began tweaking the pressure setttings on the instrument at the sample differential points and discussed nozzle tip options (70um/86um/100um/140um/200um).

In Influx fashion; simple and seamless, we boot up computers and begin firing up lasers. 7 pinholes, 7 lasers, each laser is assigned a respective pinhole which is obvious in the software, first comes the 488nm, then the 640nm, 405nm follows soon after and then comes 355nm. Brent warns us before the UV is lit-up and with good warning as a business card quickly begins to smoke as its dipped into the light path. 532nm and 561nm sit smartly side-by-side and follow each other through the pinholes to the intercept point on the stream, 532nm light is rejected using a 530/40 rejection band with a width of 2nm and we soon learn about alternative options for optimal FITC detection.

We install the 86um tip, running at 37psi, 59kHz, and set up a stream to sort the drop at a delay of roughly 40.1, the camera sits steady at 231, the sorter is ready. 6 way stream separation is made possible with clever modifications and plate zapping is eliminated with the use of conductive tape, the stream is steady as a rock and the droplet formation looks solid, consistent and clean. 6 streams sit in test mode as Brent aligns the collection tubes using the motor driver (unlike old Vantage manual stream movements). 6 peak beads are sorted into each collection tube and each pass above 99% purity with ease, installation is complete.

So many similarities to our old Vantage sorter, so many modifications and so many improvements makes this transition from Vantage sorting to Influx even sweeter, the day ends with a quick lesson in wet and dry shutdowns and we look forward to running samples tomorrow, keen to see 6 way sorting of cells and work out sort modes - excited and ready for day 3.

Chris

Day 1 - BD Influx™ Installation

Day 1  - Unboxing, Building and Boosting

Video link - http://bit.ly/eVhMW0

The morning began at 9am sharp sipping coffee on the Garvan balcony with Sladjan and Mark. Shortly soon after Craig arrived off a flight up from Melbourne, Brent pulled up in a cab from the airport, fresh off a flight from Seattle and we all eagerly awaiting the trucks arrival. After a mix up on loading docks, the truck finally reversed into the loading dock and we began unloading.

BD worked swiftly and seamlessly, unpacking boxes, arranging the components and it wasn't long before the facility closely resembled a lounge room on christmas morning. The BD Influx™left San Jose only a week ago and arrived in perfect condition only soon to be dismantled and rebuilt after a slight customised addition was fitted: 6 way sorting capabilities.
Brent and Mark raised the instrument by 140mm by fitting new rails onto a new marble plate that would allow for the 6 streams to effectively separate into each 5ml collection tube. The 'boost' took an hour after the Image block was assembled, in order to confirm the placement of the stream camera a quick connect to the screen images would reveal how the piece travelled, this however, turned out to be slightly more difficult than expected so the dismantling and rebuilding began. The Image block or 'heart of the instrument' contains a 20x lense with an NA (numerical aperture) of 0.6 which aids in providing the high end sensitivity for which the Influx is renowned, pieces are pulled apart and removed like arms off a voltron toy, each panel revealing electronic chips, tubes, plugs, cables and ports. Surprisingly, each component looks simply designed and they piece together in a natural, simplistic way, almost as if it were a leg bone connecting to the knee bone; the pre-amp connects to the sort acquisition rack - the electronics connect to the ..etc

A control board is unpacked revealing 24 ports and each cable is carefully connected to its matching partner port on the instrument, 3 data cables extend from the box, the first for ports 1-8, the second 9-16 and 17-24, our system currently uses up 21 of the ports with 7 lasers with hopes to soon fill the last 3 spots with additional PMT's for a 594nm laser line. Almost Medusa like, cables spill out from behind the instrument, umbilicals of leads connect hardware components and we soon learn briefly about the computer system UTOPEX and its remote abilities to be accessed, information extracted and data accrued, the IEC connectors provide easy access for powering up laser lines and are next connected to the power-boards on the legs of the frame. The sample control box clicks into place and the regulator tubing is untangled from pressure console as we listen to tales of the so called 'crack pipe' which turns out to be a sample tube on the end of a weigh funnel, hilarious.

Meanwhile, Sladjan and Craig work hard on the rack mount housing the monitors, video screens and oscilloscope. Sheath regulators and filters are then connected above the tank weigh station and we run through a quick lesson in nozzle assembly, piezo charging and droplet formation - again with a brilliantly simplistic design. The nozzle manifold shoe sits snugly to the bottom left of its housing and the Image relay blocks are mounted. We have a quick look at the herring bone style design of the optical bench, a look into filters, splitters and detection components. The instrument soon regains its shape as its rebuilt into a newly modified version of itself, with the ability to sort more streams, a new black protection door, and an overall seriousness that is the BD Influx™.

We eagerly await tomorrow and look forward to laser installation, alignment and new software.


Yours truly,

Chris

Update

Flowers,

Due to 514nm laser testing our BD LSR7 awaits validation in the San Jose BD HQ due for shipment on December 13 and arrival mid Jan pending customs.

As for our BD Influx, she has left the US shores and by now is most likely sitting in customs at Melbourne awaiting transfer to Sydney for a Monday morning start.

With our new Flip Mino III we look forward to providing you with an inside view of what it is like to receive, install and align a 7 laser Influx very soon..