Fluorescence Microscopy Sequential Scanning for Fast Blinking
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional high-resolution microscopy methods, such as SOFI, are limited by the blinking frequency of fluorophores, restricting the use of fluorophores and requiring lengthy acquisition times, which prevents the imaging of samples with fast-changing structures or those using fluorophores with high blinking rates.
Innovation Solution
A microscopy method that uses a spatially resolving detector to image a small part of the sample, moving the image field to cover the entire sample, allowing for faster frame acquisition and processing, and combining images to achieve high-resolution imaging beyond the optical diffraction limit, using a cumulant function to evaluate intensity fluctuations caused by fluorophore blinking.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of time
If the entire sample is imaged simultaneously using conventional widefield microscopy, then the spatial coverage is complete, but the acquisition time becomes excessively long due to the need for many frames to capture fluorophore blinking
Solution Approach 1:
The patent divides the sample into multiple smaller regions of interest (ROIs) that are imaged sequentially. By segmenting the large sample area into smaller zones, the system can acquire images of each region quickly without requiring the entire sample to be captured in a single long acquisition, thus reducing total acquisition time while maintaining complete spatial coverage through sequential scanning of all regions.
Solution Approach 2:
The patent transitions from capturing the entire sample in a single 2D plane to a sequential scanning approach that moves through different spatial regions over time. This dimensional transformation allows the system to achieve complete sample coverage by combining multiple quick acquisitions of smaller regions, effectively trading spatial dimension for temporal efficiency.
2Measurement precision
If fluorophores with high blinking frequencies are used, then the resolution improvement is enhanced, but the camera cannot capture the blinking fast enough due to temporal response limitations
Solution Approach 1:
The patent segments the detection task by focusing on specific regions of interest where fluorophores are located, rather than attempting to capture the entire sample simultaneously. This allows the camera to concentrate its temporal sampling on smaller areas, effectively increasing the frame rate for each region and enabling detection of faster blinking frequencies that would be lost in a full-sample simultaneous capture.
Solution Approach 2:
The patent implements dynamic scanning that adapts the imaging process to the blinking characteristics of fluorophores. By moving through different sample regions sequentially and adjusting the acquisition timing, the system can synchronize with the blinking frequency of fluorophores, enabling accurate capture of high-frequency blinking events that static or slow acquisition methods cannot resolve.
3Measurement precision
If the frame acquisition frequency is increased to capture fast blinking, then the blinking detection accuracy improves, but the total number of frames required increases excessively
Solution Approach 1:
The patent segments the acquisition process into multiple short sequences, each focused on a specific region of interest. By dividing the total number of required frames into smaller batches corresponding to different spatial regions, the system achieves high blinking detection accuracy within each segment while reducing the total acquisition time, as each segment can be captured quickly without requiring all frames to be acquired simultaneously.
Solution Approach 2:
The patent employs periodic scanning through different regions of the sample, creating a rhythmic acquisition pattern. This periodic action allows the system to capture blinking events at high frequency within each period while the overall cycle time is reduced through efficient region hopping, achieving both high detection accuracy and reduced total acquisition time through the repetitive nature of the scanning process.
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 the use of a wider range of fluorophores and significantly accelerates image acquisition, reducing total acquisition time from minutes to less than 100 milliseconds, allowing for the imaging of samples that change rapidly or use fluorophores with high blinking frequencies.
Implementation Method 1
providing the sample with a marker comprising fluorophores which, upon excitation, emit statistically blinking fluorescence radiation
Data Source
AI summary
A microscopy method for producing a high-resolution image of a 2-dimensional sample. The method includes exciting statistically blinking fluorophores in a sample by irradiating the sample with illumination radiation, repeatedly imaging the sample onto a spatially resolving detector in an image field that covers only a part of the sample to thereby obtain a frame sequence, generating an image from the frame sequence, the image having a spatial resolution increased beyond the optical resolution limit using a cumulant function, moving the position of the image field on the sample at least once and repeating the imaging and generating steps to obtain one image for each position of the image field, and combining the resultantly obtained images to form a complete image of the sample.