Single Objective SPIM Microscopy System
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Solution Overview
Problem
The SPIM technique requires two microscope objectives, leading to mechanical complexity and limitations on numerical aperture, making it difficult to implement and use standard microscopes for edge illumination microscopy of thick samples.
Innovation Solution
A microscopy system using a single microscope objective for both illumination and detection, with integral deflection means and focusing mechanisms to control the light sheet's position and focus, allowing for edge illumination without the need for two independent channels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If two microscope objectives are used for SPIM (one for illumination, one for detection), then the light sheet can be focused onto the sample, but the mechanical complexity increases significantly
Solution Approach 1:
The patent combines the illumination and detection functions into a single microscope objective. The illumination beam is directed through the objective's back focal plane, allowing the same optical element to perform both illumination and detection without requiring a second objective, thereby reducing mechanical complexity while maintaining focusing precision
Solution Approach 2:
The single microscope objective serves multiple functions: it acts as both the illumination objective to focus the light sheet and the detection objective to collect fluorescence signal. This multi-functional design eliminates the need for separate illumination and detection paths, simplifying the overall system architecture
2Adaptability or versatility
If two microscope objectives are used for SPIM, then independent illumination and detection paths are created, but the numerical aperture is limited
Solution Approach 1:
By merging the illumination and detection paths through a single objective, the system allows the detection path to utilize the full numerical aperture of the objective without the constraints imposed by having a separate illumination objective with potentially lower NA
Solution Approach 2:
The illumination beam is introduced through the back focal plane of the objective, utilizing a different spatial dimension (pupil plane) rather than requiring a separate physical path. This allows the detection path to maintain full access to the objective's numerical aperture while still providing independent illumination control
3Device complexity
If a single microscope objective is used for both illumination and detection, then mechanical complexity is reduced, but the light sheet alignment with the object imaging surface becomes difficult to maintain
Solution Approach 1:
The system employs feedback mechanisms where the position of the illumination beam in the back focal plane is dynamically adjusted based on the desired focal plane within the sample. This ensures that the light sheet remains properly aligned with the object imaging surface even when the objective or sample position changes, maintaining alignment precision without requiring complex mechanical structures
Solution Approach 2:
The illumination system is designed to be dynamic, allowing real-time adjustment of the illumination beam's position and focus. This dynamic control enables the system to maintain proper alignment between the light sheet and the object imaging surface throughout the imaging volume, compensating for any positional drift or changes in sample thickness
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 simplifies the mechanical complexity and allows for effective edge illumination microscopy, enabling the use of standard microscopes and improving image quality by maintaining the light sheet's alignment with the object imaging surface, even with relative positional changes along the optical axis.
Implementation Method 1
a sample illumination path including at least one source for emitting an illumination beam, means for forming an illumination surface from the illumination beam, said microscope objective
Implementation Method 2
deflection means attached to the sample support for deflecting the illumination surface into the object space at the output of the microscope objective, to form a transverse illumination surface, located in a plane substantially perpendicular to the optical axis
Implementation Method 3
means for scanning the illumination beam for moving the transverse illumination surface along the optical axis of the microscope objective
Implementation Method 4
focusing means for superimposing the object imaging surface and the transverse illumination surface, including means separate from the means for relative axial displacement of the microscope objective and the sample support
Data Source
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AI summary
According to one aspect, the invention concerns a method for microscopy of a thick sample arranged on a sample support, with edge-illumination of the sample. The method comprises, in particular, emitting at least one illumination beam (1), forming, from the illumination beam, an illumination surface, focusing the illumination surface in the sample by means of a microscope lens (120) and deflecting the illumination surface originating from the microscope lens, in order to form a transverse illumination surface, located in a plane substantially perpendicular to the optical axis of the microscope lens. The method further comprises forming, by means of said microscope lens (120), the image of an area of the sample illuminated by the transverse illumination surface on a detection surface (131) of a detection device (130), scanning the illumination beam, allowing the transverse illumination surface to move along the optical axis of the microscope lens, and superimposing the object imaging surface and the transverse illumination surface, by focusing means comprising means separate from the means for the relative axial movement of the microscope lens and the sample.