At the primary care setting, defined by the World Health Organization (WHO) as an area with often no or minimal laboratories available, medical devices must be simple and easy to operate by users with a variety of skill levels. Because of these requirements, fluorescence microscopy has become a standard tool used in both high and low resource laboratories owing to its ability to identify fluorescently stained cells and sub-microscopic cellular components. An alternative design that could simplify the fluorescent microscope involves the use of ultraviolet (UV) illumination. While there is little documentation on fluorescence in the short range UV wavelengths, many fluorescent dyes that are excitable in the visible region are also excitable by UV. Based on this idea, we developed a simple fluorescence microscope built out of commercially available components which uses UV illumination and can image any fluorescent sample (given that the fluorophore can be excited by UV). Because UV is not typically visible on camera detectors and is absorbed by glass components, the separation of excitation light from emitted fluorescence may be easily incorporated into in the design of the microscope, eliminating the need for excitation, emission, and dichroic filters. The simplicity of the designed fluorescent microscope may allow for a more compact and easy to use microscope for the primary care setting as well as decrease the overall cost of manufacturing the device. For biological validation, we imaged whole blood stained with acridine orange (AO) and performed a two-part white blood cell (WBC) differential count.
An achromatization method optimized for dual-channel imaging is developed. Dichroic mirrors are employed to split and recombine narrowband signals, and separation between catoptric components is used to minimize the longitudinal chromatic shift. An achromatic system based on this principle could be built from singlet lenses, since refractive element properties such as dispersion and power are not utilized to optimize wavelength-dependent performance. To demonstrate the validity of the proposed solution, a prototype miniature fluorescence microscope optimized for two emission lines of acridine orange (525 and 650 nm) is built. To reduce the cost and accelerate assembly, the system is built from commercially available optical components. The optical train consisted of two plastic singlet lenses combined with a pair of dichroic mirrors. Optical performance of the prototype is evaluated by imaging a bar line target at both design wavelengths. To demonstrate the potential of the proposed design strategy, the achromatic system prototype is used to measure a two-part white blood cells differential count on a venous blood sample. Data from the prototype fluorescence microscope are compared against results from a commercially available blood analyzer, and the difference between both instruments is within 20%.
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