Open-Stage Near-TIRF Microscope for Live Cell Imaging
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Solution Overview
Problem
Traditional total internal reflection fluorescence (TIRF) microscopes are limited by the need for a prism to be pressed down on the sample, which restricts the conditions for studying living cells, such as cardiomyocytes and neurons, and prevents physical access or control over the sample environment, leading to unsatisfactory fluorescence microscopy for observing fine details of electrically active cells.
Innovation Solution
A near-TIRF microscope design where all optical components are positioned underneath the sample, allowing side illumination and maintaining an open stage configuration, enabling physical access and control over the sample environment, and utilizing optogenetic proteins like QuasAr2 and CheRiff for imaging and activating cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If a prism is pressed down onto the sample in traditional TIRF microscopy, then background fluorescence is reduced, but physical access to the sample is prevented and sample environment control is limited
Solution Approach 1:
The patent inverts the traditional TIRF microscope configuration by placing the objective lens below the sample and illuminating from the bottom, rather than placing the prism above the sample. This inversion allows the sample stage to remain open and accessible from above while still achieving TIRF illumination through the bottom surface of the sample chamber.
Solution Approach 2:
The patent changes the spatial dimension of illumination by illuminating the sample from the bottom through the substrate rather than from the top through a prism. This dimensional change allows simultaneous access to the sample from above and TIRF illumination from below, resolving the contradiction between background reduction and physical access.
2Object-affected harmful factors
If a prism is pressed down onto the sample in traditional TIRF microscopy, then background fluorescence is reduced, but sample environment control is prevented
Solution Approach 1:
By inverting the illumination configuration to illuminate from below through the substrate, the patent maintains an open sample stage that allows full environmental control and manipulation from above, while still achieving the background reduction benefits of TIRF microscopy.
Solution Approach 2:
The patent separates the illumination function (performed by the bottom-illuminating objective) from the sample manipulation function (performed from above), allowing both background reduction and environmental control to occur simultaneously without interference.
3Illumination intensity
If illumination is provided through the objective lens, then fluorescence is collected, but illumination intensity is limited and field of view is reduced
Solution Approach 1:
The patent inverts the conventional fluorescence microscopy configuration by placing the objective lens below the sample and illuminating from the bottom. This allows the full numerical aperture of the objective to be used for illumination while collecting fluorescence from the same objective, increasing illumination intensity and field of view without proportionally increasing complexity.
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 configuration allows for intense illumination and a larger field of view, reducing background fluorescence and enabling the study of living cells with high spatial and temporal resolution, facilitating the observation of electrical activity in cells like neurons and cardiomyocytes, which can aid in understanding diseases such as Alzheimer's and heart disease.
Implementation Method 1
illuminating the sample with an illumination light via a prism disposed underneath the transparent bottom portion, whereby the prism imparts near total internal reflection on the illumination light
Implementation Method 2
an imaging subsystem including an objective lens unit disposed beneath the sample dish and an image capture device
Data Source
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AI summary
The invention provides an open-stage near-TIRF microscope in which all of the optical components are positioned underneath the sample, allowing for physical access to, and control over the environment of, the sample. The microscope can be used to image cells expressing fluorescent voltage indicators. Since the TIRF components do not interfere with the sample, living cells can be studied using a microscope of the invention. Where a sample includes electrically active cells expressing fluorescent voltage indicators, the microscope can be used to view voltage changes in, and thus the electrical activity of, those cells.