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Prior Work
Prior Work
The current LED team was passed down a prototype that needed to be updated to produce a fluorescing image with LED lighting (Fig. 1). The team decided to keep the same mechanical set up proposed by previous teams with a linear shaft. Imaging from the microscope had up to this point been done only in bright-field.
Figure 1: Deconstructed microscope passed down to the current team from previous team.
Design Approach
Design Approach
Microscope was designed with linear shaft to achieve focus by moving both the sample and the microscope tube without having to overcome gravity (Fig 2a) . Parts were 3D printed to allow for customization with the approximate dimensions of 12"x4"x4" (Fig. 2b). The specimen slides and long-pass filter can easily be removed for troubleshooting and easy cleaning. The set-up for use requires a monitor for visualizing microscope images, a keyboard and mouse for navigating the Raspberry Pi UI, and the microscope (Fig. 3). Not pictured, but necessary for imaging, is an LED light source; this is addressed in the following section.
Figure 2 Horizontal set-up: (a) Use of horizontal optical set-up. (b) 3D CAD configuration of microscope.
Figure 4: Theoretical set-up, minus LED light source.
Testing
Testing
Figure 4: Final set-up of the microscope, including secure base and LED light source without (left) and with (right) flash of camera.
The microscope needed a stable base to make image acquisition and focusing reliable. Additionally, the LED light source needs to be at a specific angle to fluoresce the fluorophores in the microbial sample.
Final set-up requires access to a keyboard, visual display, external power for Raspberry Pi and display, and adjustable LED light source (Fig. 4) Final design will be closed such that Raspberry Pi is not exposed and the device in its entirety is easily transportable.
Results / Conclusions
Results / Conclusions
Results
Results
The optical set-up with longpass filter and adjustable LED light permits detection of fluorescing acid-fast bacilli.
Fluorescing M. avium, an acid-fast bacillus similar to the TB-inducing agent M. tuberculosis, was successfully imaged using blue LED light coupled with a longpass filter.
Figure 5: Fluorescing images of Mycobacterium avium. The above images were taken from the prototype with the setup pictured in Figure 4. Green spots are M. avium, indicating a functional LED fluorescence microscope.
Conclusions
Conclusions
Utilizing a long-pass filter enables acquisition of fluorescence images equivalent to a traditional dichroic mirror based optical systems at a lower cost.
Fluorescence is visible in images acquired by both this prototype and a traditional LED fluorescent microscope.
Microscope permits more efficient detection of MTB by laboratory personnel with less training, at a higher rate of throughput in the clinical lab setting, and at ¼ of the cost of a commercially available LED fluorescence microscope.
Future work
Future work
Develop software to allow for automatic focusing, interactive user-interface, and analysis of a positive TB sample from a patient (Fig. 6).
Test with Mycobacterium tuberculosis at the Virginia Department of Health Reference Lab
Publish an Instructions for Use, Maintenance, and Repair Manual
Transport to Malawi for field testing
Figure 6: Flow chart of automation conducted using a Raspberry Pi. Flow chart of automation conducted using a Raspberry Pi: TB detection and identification algorithms will be installed on the Pi, the goal is to ensure simple operation at the touch of a button. All code being written in a combination of Raspberry Pi command line and Python.
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