Microscope Fiducial Pattern for Flow Cell Surface Focusing
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Solution Overview
Problem
Existing microscopes face challenges in focusing on structures at partially reflective interfaces due to photobleaching of fluorescent labels during the focusing process, which reduces the signal-to-noise ratio and makes it difficult to image fluorescently labeled nucleotides in fluidic channels.
Innovation Solution
A microscope system using a fiducial pattern and beamsplitter configuration that allows for precise focusing on the inner surface of a flow cell by forming a partially reflective interface, reducing photobleaching through low-intensity fiducial illumination and enabling high-intensity imaging of fluorescent labels with improved resolution and contrast.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the microscope is focused by using fluorescent labels as the focus target, then the microscope can be focused on the inner surface of the flow cell, but the fluorescent labels are exposed to photobleaching energy during the focusing interval, reducing the signal to noise ratio
Solution Approach 1:
The patent introduces a fiducial pattern as an intermediary focusing target instead of using fluorescent labels directly. The fiducial pattern is illuminated separately and used for focusing the microscope on the inner surface of the flow cell. After focusing is achieved using the fiducial pattern, the fluorescent labels are then illuminated for imaging without being exposed to high-intensity light during the focusing process, thus preserving their fluorescence intensity and maintaining a high signal-to-noise ratio.
2Speed
If high-intensity light is used to illuminate fluorescent labels during focusing, then the microscope can be focused quickly, but photobleaching occurs, reducing the optical energy available for imaging
Solution Approach 1:
The fiducial pattern serves as a mediator that allows focusing to be performed without directly illuminating the fluorescent labels with high-intensity light. The fiducial pattern can be illuminated with sufficient intensity for rapid focusing, and since it is not the fluorescent label itself, this illumination does not cause photobleaching of the labels. This separates the focusing function from the fluorescent label illumination function.
Solution Approach 2:
The patent performs focusing as a preliminary action using the fiducial pattern before illuminating the fluorescent labels for imaging. By completing the focusing operation first using the fiducial pattern, the system establishes the correct focal plane in advance, allowing subsequent fluorescent label imaging to be performed at optimal focus without requiring additional high-intensity illumination during the focusing interval.
3Measurement precision
If the fluorescent labels are used as the focus target, then the focusing can be performed on the correct plane, but the small size and large magnification result in a short range of microscope image focus, making it difficult to locate the labels
Solution Approach 1:
The fiducial pattern acts as an intermediary target that is easier to detect and locate than the small fluorescent labels. The fiducial pattern can be designed with larger features and higher contrast, making it simpler to identify and use for focusing. Once the fiducial pattern is located and used to establish focus, the fluorescent labels are then visible at the correct focal plane without having been difficult to locate in the first place.
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
The system effectively reduces photobleaching and enhances focus accuracy, allowing for precise imaging and measurement of fluorescently labeled nucleotides by minimizing exposure to high-intensity light during focusing, thereby improving the signal-to-noise ratio and resolution.
Implementation Method 1
position the fiducial pattern onto a change in refractive index of the flow cell sufficient to form a partially reflective interface
Implementation Method 2
positioned a fiducial pattern a fiducial lens focal length from a fiducial lens, the optical energy from the fiducial lens directed to a beamsplitter
Implementation Method 3
the objective lens on the optical axis of a detector lens, the detector lens receiving optical energy which passes through the beamsplitter and focuses the optical energy to a detector
Implementation Method 4
The fluorescent labels preferably fluoresce at unique wavelengths when exposed to a broadband optical source, thereby providing a method for identification of each of the subject nucleotides
Implementation Method 5
the optical energy from the fiducial lens directed to a beamsplitter and to an objective lens
Data Source
AI summary
A microscope has a fiducial mask and fiducial lens generating a collimated mask image onto a beam splitter which directs the optical image to an objective lens where it is directed to an optical discontinuity formed by the change of index of refraction of the inner surface of a fluidic channel. Reflected optical energy is directed through the objective lens, the beam splitter, and a detector lens to a detector. A focused image forms when an inner surface of the fluidic channel is a focal distance from the objective lens, providing for imaging of fluorescent labels at the inner surface of the fluidic channel.