Home

Flow Cytometer Laser Alignment System

sponsored by

http://www.bdbiosciences.com/home.jsp

Team 10: Carlos Acuña, Justin Chen, Kevin Chen, Daniel Hwang

Sponsor: Paul Chen

UCSD MAE-156B Spring 2014

Mechanical Engineering Senior Design Project

Department of Mechanical and Aerospace Engineering

Professor: Dr. Jerry Tustaniswkyj

Fig. 1. Laser Alignment Microscope Camera System

Project Background 

    In Biotechnology and medical research, the importance of flow cytometry is unparalleled. The ability of facilitated cell counting, cell sorting, biomarker detection and protein engineering through flow cytometry provides the capability of diagnosing health disorders and additionally provides valuable information for basic and clinical research. Becton Dickenson is a world leader in bringing innovative diagnostic and research tools to life science researchers, clinical researchers, laboratory professionals and clinicians who are involved in basic research, drug discovery and development, biopharmaceutical production and disease management. The BD Biosciences segment is focused on continually advancing the science and applications associated with cellular analysis which products include: Fluorescence-activated cell sorters and analyzers, monoclonal antibodies and kits for cell analysis, reagent systems for life science research, cell imaging systems, and cell culture media and supplements for biopharmaceutical manufacturing.

    BD Biosciences currently has a fleet of flow cytometers that utilize many laser configurations requiring constant performance evaluations from day to day operations. Over extended periods of time, the laser alignment of each system will slowly drift from optimal and degrade data quality. While newer designs include options such mechanized optics stages and auto-alignment software, older designs and cost-sensitive designs continue to use stationary optics. For the stationary optic designs, field service engineers re-align the lasers of the system periodically; they utilize an objective lens microscope to view the intersection point (known as the flow cell) between the lasers and the sample flow area while adjusting x-y lead screws to ensure the lasers are well aligned in the center of their respective intersection areas. This current method of re-aligning the lasers in the flow cell is rather crude, and requires some engineering thought on how to make this re-alignment procedure safer and more efficient.

Project Objective

    The  objective of the Flow Cytometer Laser Imaging Alignment System project is to fit a novel laser alignment solution to BD Biosciences’ fleet of flow cytometers that require constant performance evaluations from day to day operations. To achieve this goal, our team has developed a more efficient and safer technology that will optimize the overall ease of use. A smaller form function for the alignment microscope will provide maximized portability and elimination of crude procedures. Overall system redesign will provide safer laser alignment procedure and a reduction of imprecision. Our system is capable of providing linear motion in the X and Z directions while providing constant visualization of the laser alignment view field. 

Review of Existing Solutions

    Currently, BD Biosciences utilizes a simple 63X magnification microscope to observe live high powered lasers for visual alignment. This presents many safety hazards for the technicians. Not only can a stray laser beam reflect back into the microscope, the optics of the microscope can concentrate the laser beam to increase the potential for permanent eye damage as seen in Fig 1. In addition to safety hazards; portability is very limited. The microscope measures about 7 inches in length, not including the mounting bracket. This proves to be cumbersome for the technician who has to carry the microscope with them in addition to the various tools required for regular maintenance. Lastly, the adaptability of the microscope mounting bracket is very unorthodox. The bracket requires multiple operators to quickly and effectively mount the mounting bracket onto the flow cytometer. Different configurations, such as flipping the entire system upside down is one of the problems that the current solution has from one product to another, adding additional inconvenience for the operator. A unified, compact system that a field technician can easily operate at ease, without risking personal safety is the ideal solution.

Fig 2. Current Solution with microscope (rig. Red arrow depicts a stray laser potentially damaging the viewer

Statement of Requirements

Primary Requirements

• Design and fabricate a laser imaging alignment system that can be easily operated by a single field service technician

• Black anodized aluminum for entire system which includes mount and microscope

• A mounting system that is capable to be utilized on all product families 

• A camera connected to the microscope with a quick refresh rate that facilitates live viewing

• System Compatibility with FACSAria and FACSVerse product families

• Under 10lb (4.5kg) weight requirement

• Within 5"x3"x3" (12.7cm x 7.62cm x 7.62cm) dimension requirement 

• Pinhole back light illumination system 

Secondary Requirements

• System Compatibility with FACSCanto and LSRFortessa product families

• Software and its configuration within the system that ensures the laser is correctly oriented in the flow cell

Deliverables

The engineering student team will be delivering to BD Biosciences the following:

• A single fully functioning laser viewing alignment system with possibility of multiple mounts

• A CAD model of product and CAD animation.

• An instruction manual to operate the apparatus, including the detailed explanation of how each component of how each component functions.

• All information, specification sheets, and documentations for all parts and materials purchased, including a step-by-step assembly guide of the prototype for mass production.

• User-friendly programming interface/ software that can be easily modified by further development teams

Final Design

Fig. 3. Annotated Diagram of Final Design

Description of Final Design

1. The final design of the Flow Cytometer Laser Alignment System consists of an integration of four primary key components. The completed system comprises of an integrated USB microscope and camera system, AD7013MTL Dino-lite Premier, attached to a custom fabricated mounting bracket. An additional LED illumination system was designed and fabricated to assist in pinhole visibility.  Internal software was developed to utilize data acquired from the USB microscope and camera and provide laser and pinhole tracking capabilities. 

2. The Flow Cytometer Alignment System uses an off the shelf AD7013MTL Dino-lite Premier to provide accurate and live imaging of flow cytometer lasers and pinholes. The 5.0 MP camera provides a platform that is safe and efficient with a constant refresh rate of 30 frames per second. The attached microscope offers up to 90x magnification. This viewing platform reduces potential eye damage by removing the need of a direct line of sight to the lasers.

3. The Flow Cytometer Alignment System’s mounting bracket consists of three easily detachable and reassembled parts. An off the shelf MicroscopeNet Attachable Large X-Y Mechanical Stage for Compound Microscopes (SKU: A113) was adapted to a custom fabricated mounting bracket made of black anodized aluminum. Additionally, a custom microscope holding bracket is attached to the top face of the Z-axes linear slider. This modular mounting bracket provides movement in the Y and Z axes (73mm x 30mm) for facilitated specimen focus and multiple cytometer model adaptability. Careful mounting bracket design also prevents laser path interference and easy access to system mounting holes.

4.  The Flow Cytometer Alignment System also comes with a custom fabricated pinhole illumination system, designed to be easily mounted within a pre-existing filter. This LED illumination system further optimizes the alignment procedure by providing increased pinhole visibility.

5.  The software developed for the Flow Cytometer Alignment System is able to track location and orientation of cytometer lasers and pinholes. This capability reduces human error and inconsistency and additionally provides a higher resolution viewing platform during laser re-alignment. 

System Performance

Fig. 4. System Mounting Performance on FACSCanto, FACSAria, FACSVerse, and FACSAria Fusion 

The final bracket assembly performed far better than expected. Considering that much of the testing was done with the acrylic and ABS plastic parts, the machined aluminum bracket exceeded our expectations. Thanks to the precise dimensions and tight tolerances, the bracket cleared the cuvette very easily along with all of the surrounding hoses and lines needed to run the flow cytometer. With the machine on and the lasers shining through, the data collected did not show any discrepancy with the expected results and this proved that the bracket did not interfere with the lasers in any way.

The mechanical stage performed just as expected as well. With the camera in place and the system secured properly onto the flow cytometer, the sliders remained in their respective positions. In other words, the weight of the camera was not enough to move the stage in the vertical direction, which was exactly what was expected. The knobs were very easy to operate and the stage provided smooth and precise movements necessary for proper laser alignment.

The only hardware discrepancy was system mounting attempts on the FACSVerse. Unfortunately, due to existing hardware constraints, the system had to be mounted upside down for a secure fit. However, this issue was treated as a minor concern due to the system’s overall compatibility and successful performance with the FACSVerse. 

Image Acquisition Test Results

Primary image quality verification tests took place on site at BD Biosciences, San Diego. Unfortunately, only two flow cytometer product families were available on site for testing, the FACSAria and FACSCanto. FACSVerse and LSRFortessa image quality tests have yet to be more carefully analyzed. However, completed test results give a high level of confidence to overall system performance.

Fig. 5. FACSCanto Full Forward Lighting (Left) and No Forward Lighting (Right)

Figure 5 (left) shows the image quality on the FACSCanto with the AD7013MTL Dino-lite Premier’s full forward lighting applied. It was noted that the cuvette and pinhole visibility was enhanced. However, the intensity of the forward lighting drowned out the visibility of the laser traces. Figure 5 (right) shows the image quality on the FACSCanto no forward lighting applied. With this change in settings, the laser traces became easily visible. The Pinhole Illumination System contributed to pinhole visibility which gave illumination that was easily picked up by the AD7013MTL Dino-lite Premier. However, it must be noted that with no forward lighting, image noise increased slightly. Image quality, however, was still was kept at an appropriate level on the FACSCanto.

Fig. 6. FACSAria Full Forward Lighting (Left) and No Forward Lighting (Right)

Figure 6 (left) shows the image quality on the FACSCanto with the AD7013MTL Dino-lite Premier’s full forward lighting applied. It was noted that the cuvette and pinhole visibility was enhanced. However, the intensity of the forward lighting again drowned out the visibility of the laser traces. It can be noted here that the laser intensity is higher on the FACSCanto as can be seen below. With full forward lighting, laser traces are still slightly visible. Figure 6 (right) shows the image quality on the FACSAria with no forward lighting applied. With this change in settings, the laser traces became easily visible. The Pinhole Illumination System contributed to pinhole visibility which gave illumination that was easily picked up by the AD7013MTL Dino-lite Premier. However, it must be noted that with no forward lighting, image noise increased quite significantly on the FACSAria due to increased laser intensity. Future changes, as will be further mentioned, are necessary.

Software Performance

The software performed very well in test environments. The software was capable of the center of the pinhole and mark with appropriate accuracy. The pinhole detection was evaluated using a roundness test to determine that detection of the pinhole gave an average roundness of 80%. The laser traces were fairly difficult to pick up, but proved to be somewhat accurate, but introduction of noise into the system may have impact on overall system accuracy. The software was tested on both the FACSCanto and FACSAria.

Fig. 7. Left image shows laser tracking on the blue laser while the right shows red laser of FACSCanto

Overall, performance of the software on the FACSCanto was good. Even with a noisy or dim image acquired from the different cameras, the software was able to pick up the different colors fairly distinctively. However, because the lasers of the systems tested on could not be adjusted, live laser tracking tests could not be conducted.

When tests were done on the FACSAria, images became more difficult to process due to the overall brightness and introduction of noise into the image from increased laser intensity and additional lasers. Overall, the image experiences large amounts of noise throughout. While the red and green channels tracked comparably as the FACSCanto, the blue and violet lasers became more difficult to detect. One of the largest problems with tracking the two lasers was the mixing in the blue color channels. For example, parts of the violet laser would appear as blue and vice versa, distorting the detection of the laser. A different method besides color detection will be necessary rather than observing RGB color channels to be able to detect the blue and violet lasers distinctively.