Quantitative high dynamic range beam profiling for fluorescence microscopy
- Centre for Advanced Instrumentation and Biophysical Sciences Institute, Department of Physics, Durham University, Durham DH1 3LE (United Kingdom)
Modern developmental biology relies on optically sectioning fluorescence microscope techniques to produce non-destructive in vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3D) intensity profile of the illumination employed, the ability to directly record and analyze these profiles is of great use to the fluorescence microscopist or instrument builder. Though excitation beam profiles can be measured indirectly using a sample of fluorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly within the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam profiling device and high dynamic range flux reconstruction algorithm that together are capable of accurately reproducing quantitative 3D flux maps over a large focal volume. Performance of this beam profiling system is verified within an optical test bench and demonstrated for fluorescence microscopy by profiling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely flexible beam amplitude diagnostic tool for use within the life sciences.
- OSTI ID:
- 22305895
- Journal Information:
- Review of Scientific Instruments, Vol. 85, Issue 10; Other Information: (c) 2014 Author(s); Country of input: International Atomic Energy Agency (IAEA); ISSN 0034-6748
- Country of Publication:
- United States
- Language:
- English
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