A Programmable Illumination System to Enhance Contrast in Microscopic Imaging

Overview

In the context of optical imaging, “active illumination” refers to the controlled illumination of a specimen, characterized by the spatial illumination pattern, the angular spectrum, and the wavelength spectrum of the light source. The following describes the hardware of a low-cost microscope-mounted active illumination system to control the color and incident angle of illumination. This represents the evolution of a system over two years. The current system integrates the concept of a programmable light emitting diode (LED) light source with an off-the-shelf stereomicroscope. It has a long-term goal of improved microscopic imaging for low-contrast specimens embedded in a non-homogeneous background medium. The illumination system consists of an Arduino controlling a 24 Red Green Blue (RGB) Neopixel array arranged in an 85.9mm diameter ring mounted around the stereomicroscope objective. The Neopixel array is suspended above the microscope stage with a 3D-printed adapter. Two adapters were designed for mounting the LED array, either the same distance from the specimen as the microscope lens, or about 4cm closer to the specimen. Images are captured with a Canon Rebel T5 DSLR camera mounted to the camera port of a Nikon stereomicroscope. The system is operated either in multi-spectral imaging mode, where a series of frames with different source colors are recorded, or in a structured illumination mode, where the angle of the incident light is changed among frames. Brightness and color of each LED is independently controlled with the Arduino microcontroller via a serial connection. The system was evaluated with two types of test specimens. The first type contained specimens with several different colors designed to test the ability of the system to separate color information. The second specimen contained a variety of structured surfaces to demonstrate the response of the system to surface texture to illumination with a changing incident angle. The results obtained with the first model showed that the contrast of colored specimens can be controlled by selecting the color of the illumination system. This permits the selective highlighting of specimen details. Images obtained with the second type of test specimen showed a brightness change as a function of the incident angle of the illumination, which can be used to extract information about the surface gradient of the specimen.