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Fig. 1. Laser Alignment Microscope Camera System
Design of Key Components
The functional requirement was to design and fabricate a flow cytometer laser and pinhole viewing system that facilitates the laser alignment procedure performed by field service technicians. The design considerations were to offer a magnified live video feed of the pinhole and laser specimen as well as offer the ability to move the mounted system in the Y and Z directions in a consistent and repeatable manner for facilitated specimen focus and multiple cytometer model adaptability. Additional design considerations include a laser and pinhole tracking software to facilitate the laser alignment process by reducing human error and inconsistency by adding a higher resolution platform. Lastly, a method of pinhole illumination was required for increased pinhole visibility. These selected functional requirements provide an uncomplicated and optimized laser alignment procedure that offers ease reproduction and increased safety for the BD Bioscience's field service technicians.
Based on those design considerations, the key components of the final prototype are:
An integrated USB microscope and 5.0MP camera system that provides 30fps live image viewing and up to 90X magnification
A black anodized aluminum custom mounting bracket equipped with Y and Z direction linear sliders that easily attaches the viewing system to the flow cytometer mounting points
A pinhole illumination LED platform that is easily adapted and fit into pre-existing data acquisition filters that feeds light through pre-existing connected fiber optics back to pinhole viewing area
A pinhole and laser tracking software that acts as a visual aid to assist with the high resolution calibration and documentation of laser positions
A single unit design which carries every component of the laser alignment microscope camera system in safe and reliable manner
Description of Key Components
USB Microscope & Camera System
The AD7013MTL Dino-lite Premier was ultimately selected as the best choice for the Flow Cytometer Alignment System. One of the biggest advantages of this microscope and camera system was its already integrated design.
Microscope
Out of the box, the AD7013MTL Dino-lite Premier comes equipped with a variable magnification microscope which ranges from 20x to 90x on its front end. This high quality microscope offers consistent optical zoom and internal glass lenses for increased optical clarity. Further, the AD7013MTL Dino-lite Premier offers long working distance capabilities with a minimum working distance of 56.1 mm at 90x magnification. The field of view offered at this magnification lies at 4.4 mm x 3.3 mm. Both these functions far exceed the necessary functional requirements requested by BD Biosciences.
Camera
On the rear end of the AD7013MTL Dino-lite Premier is an already attached digital camera that has a 5 megapixel color CMOS sensor capable of providing max resolutions of 2592 x 1933 pixels. The integrated camera comes equipped with a USB 2.0 interface providing imaging and video compatibly with both PC (Windows 8,7,Vista,XP) and MAC (OS 10.4 or later) systems.
Forward Lighting
Also on the front end of the unit are 8 white variable intensity LEDs with the capability of object illumination. Total system dimensions are 10.5 cm (length) x 3.2 cm (width) and have a total weight of 140 grams. Total cost of the unit came out to be $975.00.
External Housing
To further adapt the chosen AD7013MTL Dino-lite Premier, an external housing was designed and fabricated to meet the functional requirements of being enclosed in black anodized aluminum. This simple casing was easily fabricated with minimal material costs and minimal fabrication times.
Mounting Bracket
The USB microscope and camera system must be able to be mounted to four different BD flow cytometer product families: FACSAria, FACSVerse, FACSCanto and LSRFortessa. All four product families have identical mounting hole locations but vary between M3 and 4-40 hole sizes as well as varied free space that is not occupied by tubing, brackets, sensors, instrumentation, etc. The mounting bracket system must be able to move the microscope and camera system in the Z and Y axes with a minimum length of travel of 38mm x 25mm. Y axis movement is crucial to move the microscope and camera system to appropriate heights depending on the flow cytometer model. Z axis movement is necessary for microscope and camera system focus.
Linear Slider
To meet these functional requirements, the 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. This off-the-shelf linear slider provides a moving range of 73mm x 30mm with a movement accuracy resolution of 0.1mm for both axes.
Mounting Bracket
In order to provide ease of assembly, a compact form function, and portability, the mounting bracket system was designed to consist of three separate parts that are easily detachable and reassembled. The custom mounting bracket is fastened to the linear stage by a simple thumb screw. The microscope and camera system attachment bracket is also easily detachable from each other. A final feature of the mounting bracket is its curved attachment arms. The careful design prevents laser path interference and allows the field service technician to easily access the mounting holes when first attaching the entire system to the cytometer.
Pinhole Illumination System
For accurate alignment of the flow cytometer lasers, pinhole visibility is of utmost importance. A lighting system is necessary to feed light through the pre-existing fiber optic cables from the filter wheel to the pinhole location and illuminate the pinholes at an appropriate intensity. LED wavelength selection depended on the prevention of possible interference with flow cytometer laser wavelengths.
Electrical Hardware
To meet these functional requirements, a simple circuit was constructed consisting of an LED, resistors, a battery, and a switch. All electronics were housed in a custom plastic case that easily snapped into the pre-existing BD Bioscience's band pass filters. Insertion of this custom filter into the filter wheel provided necessary illumination to distinguish the pinhole during laser alignment sessions.
Material & Fabrication
The pinhole illumination system was fabricated from these CAD models using a 3D printer. When choosing the material, resolution and robustness were the main focus, with resolution being the ultimate deciding factor. For the highest resolution, the acrylic material was chosen, despite the longer turnaround time and the slightly lower material strength.
Tracking Software
The software acts as a visual aid to assist with calibration of laser positions. Although software has been attempted in the current solution, it is not being used due to its unreliability. The software developed for this project aims to create an entirely new package to be used in conjunction with the microscope and camera system. After discussion with field service engineers about implementing software, they expressed great interest, especially with lasers near the violet/ultraviolet range. Lasers within that spectrum are very difficult to view, especially in contrast with other high power lasers, so implementation of software would greatly assist alignment of those lasers in particular.
Methodology
The software utilizes machine vision to determine and display critical information on a live video stream. Ideally, the lasers would pass through the center point of the pinhole for best alignment. To aid the technician with this process, software will detect and mark the center point of the pinhole such that the technician can easily visualize the target position. In addition, information such as the angle of the laser beam and its position with respect to alignment will be displayed on the screen to indicate how far the laser is from ideal alignment.
Additional Features
Another feature requested by BD Biosciences is the ability to save settings and data from previous alignments to make measurement consistent with previous sessions. This feature would be able to save the laser and pinhole positions following the end of an alignment session, such that alignments for the machine further down the line can be replicated without extending into the entire process.
Image Processing
MATLAB was used for the software design of this project, most notably the Imaging Toolbox. A web camera in a box was used to simulate the pinhole and flow cytometer environment as displayed in Figure X. Other software options such as OpenCV has been considered, but after discovering MATLAB compiler, it was found unnecessary to attempt to learn OpenCV. The different components of the software include centroid detection, laser detection and GUI.
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