Image: (A) The development of an illuminator and its calibration process. A blood-coated image sensor was used to determine the 3D light source positions. (B) The illuminator was used for Fourier ptychography microscopy (first row), 3D tomographic imaging (middle row) and on-chip microscopy (bottom row). see more
Photo credit: Pengming Song et al.
To expand the possibilities of programmable illumination, a group of researchers at the University of Connecticut developed a strategy for building and calibrating free-form illuminators that offer greater flexibility for computational microscopy. Their calibration method uses a blood-coated sensor to reconstruct light source positions. They demonstrated the use of calibrated free-form illuminators for ptychographic Fourier microscopy, 3D tomographic imaging and on-chip microscopy, and used a calibrated free-form illuminator in an experiment to track bacterial growth.
The group’s research was published in Intelligent Computing, a Science Partner journal, on February 20.
New possibilities for the experimental setup with free-form lighting allow not only more flexibility, but also more efficiency: “With this platform, we can start to transfer Petri dish-based experiments from the traditional labor-intensive process to an automated and streamlined process,” according to the research paper it.
Programmable light sources simplify the work of illuminating samples in different types of microscopy contexts, but traditional programmable arrays consist of a flat, fixed grid of individual lights. An array of lights that can be placed anywhere in three dimensions gives experimenters the ability to reduce the size of the illuminator, place the lights closer to the sample, and increase the density of the lights and adjust the angle of illumination to suit their specific needs.
Freeform lighting can be further improved in the future with better calibration methods, denser lighting arrays, lights with a greater variety of wavelengths, and adjusted position estimates.
The authors designed and built four different free-form illuminators: an inclined flat surface, a triangular pyramid, a dome, and a Mobius strip. The pyramid illuminator had several advantages. It was angled toward the sample, it had the highest number of lights at the point farthest from the sample, and it could be placed very close to the sample to deliver light efficiently.
The authors calibrated the illuminators with a blood-coated sensor. By coating the sensor with a thin but dense layer of particles, the authors were able to calculate the position of each lighting element using ray tracing. The advantage of using human blood from a finger prick is that no complicated or expensive tools are required. However, other substances with similar properties can be used in its place. The authors checked their calibration method and found that the recovered positions matched the actual positions with only minor deviations.
The authors demonstrated the use of their free-form illuminators in several experiments. All four illuminators have been successfully used for Fourier ptychography microscopy, a computational microscopy technique that combines different images produced by illumination at different angles.
The pyramid illuminator was used to capture separate images illuminated with red, green, and blue light, which combined to create a full-color image. It has also been used for 3D tomography, a technique that creates a 3D image by stacking cross-sectional images of a 3D sample.
Pyramid illumination was used for on-chip microscopy, a setup where the sample sits directly on the sensor, just like the blood smear in the calibration step. Combining the images created with separate lights in the free-form illuminator results in a final image with a higher resolution than either of the raw images alone, which is why this technique is called super-resolution microscopy. One experiment produced a still image of blood cells and another produced a time-lapse series of the growth of a colony of E. coli bacteria on an agar block.
Guoan Zheng conceived the idea and designed the experiments. Pengming Song and Tianbo Wang conducted the experiments. Your co-authors on this article are Shaowei Jiang, Chengfei Guo, Ruihai Wang, Liming Yang, and You Zhou.
subject of research
Free-form illuminator for computer microscopy
Article publication date
January 5, 2023
The authors declare that they have no competing interests.
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