Transillumination Autofocusing for Fluorescence Microscopy
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Solution Overview
Problem
Fluorescence imaging of cells faces challenges such as photobleaching and determining suitable exposure times, especially when finding the best focal position, which affects image quality and data reliability in automated environments.
Innovation Solution
A microscope system that uses transillumination-based autofocusing to collect a stack of bright-field images, calculate focus metrics, and determine a candidate focal position, which guides the collection of photoluminescence images with a smaller search range, reducing photobleaching and allowing for more precise exposure times.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If fluorescence imaging is performed at multiple focal positions to find the best focus, then image quality is improved, but photobleaching increases and fluorescence signal is diminished
Solution Approach 1:
The patent applies preliminary action by performing transillumination-based autofocusing before fluorescence imaging to pre-determine the optimal focal position. This allows the fluorescence imaging to be performed at a single predetermined focal position without needing to scan multiple positions, thereby preventing photobleaching while still achieving optimal image quality.
Solution Approach 2:
The patent segments the imaging process into two distinct phases: (1) transillumination phase for determining focal position, and (2) fluorescence phase for actual imaging. This segmentation allows each phase to use the most appropriate illumination method for its specific purpose, minimizing overall photobleaching.
2Measurement precision
If exposure time is extended to capture sufficient fluorescence signal, then signal detection is improved, but photobleaching increases
Solution Approach 1:
The patent uses transillumination imaging as a preliminary step to determine the optimal focal position before fluorescence imaging. This preliminary action ensures that subsequent fluorescence images are captured at the correct focus with optimal exposure times, maximizing signal detection while minimizing the total exposure duration and associated photobleaching.
3Measurement precision
If multiple fluorescence images are collected to determine appropriate exposure time, then exposure time accuracy is improved, but imaging time increases and photobleaching worsens
Solution Approach 1:
The patent performs transillumination imaging as a preliminary step to rapidly determine the optimal focal position and guide fluorescence imaging parameters. This preliminary action eliminates the need to collect multiple fluorescence images to determine exposure time, as the transillumination phase provides the necessary focus information to optimize subsequent fluorescence imaging in a single pass.
4Measurement precision
If transillumination imaging is performed over a large scan range with fine pitch, then focal position accuracy is improved, but imaging time increases
Solution Approach 1:
The patent segments the focusing process from the imaging process. Transillumination imaging is performed over a large scan range with fine pitch to accurately determine the focal position, but only once as a preliminary step. Subsequent fluorescence imaging is performed at the predetermined focal position without scanning, thereby achieving high focal position accuracy while maintaining fast imaging speed.
Solution Approach 2:
The patent performs the time-consuming transillumination scan as a preliminary action to establish the optimal focal position. Once determined, this focal position is used for all subsequent fluorescence imaging, eliminating the need for repeated scanning and thereby achieving both high accuracy and fast imaging speed.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables faster imaging, reduces photobleaching, and ensures consistent image quality by determining the best focal position before significant photobleaching occurs, allowing for accurate exposure times and improved data collection.
Implementation Method 1
light transmitted through the sample is detected for a first set of focal positions
Implementation Method 2
Photoluminescence is detected from the sample for a second set of focal positions
Data Source
Figure 1A~1B
Figure 2~3
Figure 4
AI summary
Microscope system for, and methods of, imaging a sample including biological cells. In an exemplary method, light transmitted through the sample may be detected for a first set of focal positions to collect a first stack of images. Values of a focus metric may be calculated for the first stack of images. A candidate focal position may be determined based on the values. Photoluminescence may be detected from the sample for a second set of focal positions to collect a second stack of images. The second set of focal positions may define a smaller range than the first set of focal positions. At least one focal position of the second set of focal positions may be based on the candidate focal position. In other words, the candidate focal position may serve as a guide for finding a suitable photoluminescence focal position.