研究目的
To construct a low-cost microscope slide scanner for digital imaging of newborn piglet brain tissue and to quantify fluorescent microspheres in tissue for estimating regional cerebral blood flow.
研究成果
A low-cost slide scanner was successfully constructed and validated for imaging and quantifying fluorescent microspheres in tissue, showing reasonable agreement with manual counting. It offers a versatile alternative for digital slide imaging with a large FOV and acceptable resolution, though with limitations in high-resolution applications and tissue compartment analysis.
研究不足
The slide scanner cannot compete with professional confocal or high magnification microscopes; image quality is limited by the camera sensor and light source; underestimation of microsphere counts may occur due to tissue slice thickness; regional estimation in different tissue compartments was not feasible in the piglet model due to small white matter thickness; resolution and FOV are constrained by equipment used.
1:Experimental Design and Method Selection:
The study involved designing a custom slide scanner using a DSLR camera and UV light sources for fluorescent imaging, with algorithms developed in MATLAB for microsphere detection and rCBF calculation. Bright-field imaging was also tested with light diffuser film.
2:Sample Selection and Data Sources:
Newborn piglet brain tissue slides were used, specifically for fluorescent microsphere detection in rCBF studies.
3:List of Experimental Equipment and Materials:
Included a sturdy frame, DSLR camera (EOS 1100D, Canon), UV light bulbs (Omnilux), optical filters (shortpass 400 nm cutoff from Edmund Optics), frosted glass plate, light diffuser film, lens extension tubes (MeiKe), microscope objective lenses (Zeiss and Olympus), and computer with MATLAB software.
4:Experimental Procedures and Operational Workflow:
The slide scanner was assembled, camera settings were optimized through factorial designs, images were captured, and algorithms processed the images to detect and count microspheres, with validation against manual counts.
5:Data Analysis Methods:
Statistical comparison of manual and automatic microsphere counts, calculation of mean differences and relative errors, and spectral analysis using a monochromator system.
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DSLR camera
EOS 1100D
Canon
Used for capturing digital images of tissue slides in fluorescent and bright-field modes.
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Microscope objective lens
EC Plan-Neofluar 40×/0.75
Zeiss
Used for high-magnification bright-field imaging with the DSLR camera.
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Microscope objective lens
LCAch P&P 20×/0.4
Olympus
Used for bright-field imaging with the DSLR camera.
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Double monochromator system
SPECTRO 320
Instrument Systems
Used to characterize the spectral power distribution of the light source.
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UV light bulbs
Omnilux
Provides ultraviolet light for exciting fluorescent microspheres in the slide scanner setup.
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Shortpass cutoff glass filter
400 nm cutoff
Edmund Optics
Filters light to remove wavelengths above 400 nm, improving contrast for fluorescent imaging.
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Lens extension tubes
MK-C-AF1-A
MeiKe
Increases magnification and resolution of the camera for imaging.
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Light diffuser film
Creates homogeneous lighting for bright-field imaging by diffusing UV light.
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Computer
Intel I7-2600
Intel
Runs MATLAB software for image processing and algorithm execution.
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Software
MATLAB
MathWorks
Developed and used for algorithms to detect fluorescent microspheres and calculate rCBF.
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