SPIM Microscopy with Pulsed Laser Light Sheet
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Solution Overview
Problem
Current microscopy devices for fluorescence lifetime imaging are time-consuming, prone to fluorophore bleaching, and struggle with discriminating between excitation and autofluorescence light, leading to inaccurate measurements and increased measurement times, especially in multiexponential decay processes.
Innovation Solution
A microscopy device utilizing the SPIM technique with a pulsed laser generating a light sheet for illumination and a spatially and temporally resolved detector to achieve high-speed optical sectioning with reduced sample bleaching and efficient discrimination of signal components, allowing for time-resolved acquisition of fluorescence decay curves.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If widefield microscopy methods with gated image intensifiers are used, then time resolution in the range of 100 ps can be achieved, but at least three image recordings are required to determine the phase shift, resulting in increased measurement time
Solution Approach 1:
The patent segments the illumination in time by using pulsed laser excitation with specific pulse widths (e.g., 100 ps to 10 ns), allowing single-shot fluorescence lifetime measurement. The temporal segmentation of excitation pulses enables direct time-domain measurement without requiring multiple sequential image recordings, thus resolving the contradiction between time resolution and measurement time.
Solution Approach 2:
The patent employs periodic pulsed laser excitation to stimulate fluorescence emission, where the pulse repetition rate is synchronized with the detector gating. This periodic action allows the system to capture fluorescence decay information within each pulse cycle, achieving both high time resolution and reduced measurement time by eliminating the need for multiple separate recordings.
2Measurement precision
If multiple image recordings are performed to determine phase shift or multiexponential decay, then measurement precision improves, but fluorophore bleaching increases
Solution Approach 1:
The patent performs preliminary temporal gating of the detector to capture only the relevant fluorescence decay signal within a specific time window after excitation. By pre-configuring the detection time window to match the expected fluorescence lifetime, the system obtains sufficient measurement precision from a single or minimal number of recordings, thereby reducing cumulative exposure and fluorophore bleaching.
Solution Approach 2:
The patent changes the temporal parameters of excitation and detection by using ultrafast pulsed lasers with controllable pulse widths and repetition rates. By optimizing these parameters, the system achieves high measurement precision with reduced total excitation energy delivery, thus minimizing fluorophore bleaching while maintaining accurate fluorescence lifetime measurement.
3Measurement precision
If time window-controlled image intensifiers are used for fluorescence lifetime imaging, then time resolution can be achieved, but the quantity of required image recordings increases for multiexponential decay processes
Solution Approach 1:
The patent implements continuous pulsed excitation with synchronized detector gating that operates throughout the measurement period. The detector continuously captures fluorescence photons within the gated time window for each excitation pulse, allowing the system to build up sufficient signal statistics for multiexponential decay analysis without requiring discrete sequential recordings, thus reducing the complexity of the measurement protocol.
Solution Approach 2:
The patent replaces the mechanical/sequential approach of acquiring multiple discrete image recordings with an electronic/time-domain approach using pulsed excitation and gated detection. The continuous time-correlated single photon counting or intensified camera operation during pulsed excitation cycles substitutes for multiple mechanical recordings, simplifying the measurement process while maintaining time resolution capability.
4Illumination intensity
If conventional microscopy illumination is used, then the entire sample is illuminated, but this causes excessive bleaching and prevents efficient optical sectioning
Solution Approach 1:
The patent applies local quality by using selective plane illumination microscopy (SPIM) where only a specific focal plane of the sample is illuminated by a thin light sheet, while other regions remain dark. This localized illumination provides sufficient signal from the plane of interest without exposing the entire sample to excitation light, thus reducing overall fluorophore bleaching while maintaining adequate illumination intensity for the targeted region.
Solution Approach 2:
The patent introduces a spatial dimension constraint by using a thin light sheet perpendicular to the detection axis, creating optical sectioning in the thickness dimension. This dimensional approach illuminates only a thin plane (e.g., 1-10 µm thick) rather than the entire sample volume, achieving both adequate illumination for the plane of interest and reduced bleaching by limiting excitation to a small volume.
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
Enables rapid image recording with minimized sample stress and effective separation of signal components, enhancing the accuracy and efficiency of fluorescence lifetime imaging by reducing bleaching and autofluorescence interference.
Implementation Method 1
illumination means (1) having a pulsed laser
Implementation Method 2
the fluorescence intensity in particular can be detected. Further, it is possible to use the individual fluorescence lifetimes of fluorophores to generate contrasts
Implementation Method 3
A time-correlated photon counting can be used in this case, for example
Data Source
AI summary
A microscopy device, particularly for use in an imaging fluorescence lifetime microscopy method is provided. The microscopy device comprises an illumination means for generating an illumination beam, an imaging detector for spatially resolved acquisition of an emission radiation emitted by an object to be examined, an illumination beam path between the illumination means and the object to be examined, and a detection beam path between the object to be examined and the detector. The illumination beam path comprises illumination optics which are designed to generate a light sheet of illumination radiation extending transverse to the axis of the illumination beam path, wherein the axis of the detection beam path is oriented substantially perpendicular to a section plane of the light sheet and of the object to be examined. The illumination means comprise a pulsed laser.