Segmented Illumination in Flow Cytometry for Fluorescence and Scatter
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Solution Overview
Problem
Existing optical flow cytometers face mechanical design challenges due to the need for high NA optics for both fluorescence and scattering measurements, leading to space constraints and increased costs, especially when using non-coherent light sources like LEDs.
Innovation Solution
A beam truncation device that segregates the illumination beam into two parts with different divergences for fluorescence and scattering measurements, allowing for separate optical paths and reducing the need for high NA optics, particularly for forward scatter measurement.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high NA optics are used for both fluorescence and scattering measurements, then measurement sensitivity is improved, but mechanical space around the flowcell becomes insufficient and device complexity increases
Solution Approach 1:
The illumination beam is segmented into two distinct parts using a beam truncation device: a first part with low divergence for scattering measurements and a second part with high divergence for fluorescence measurements. This segmentation allows each measurement type to use optimized optics independently, reducing the total space required around the flowcell while maintaining high measurement sensitivity for both modes.
2Measurement precision
If high NA optics are used for fluorescence measurement, then fluorescence light collection is maximized, but the same optics cannot be used for scattering measurement due to beam divergence constraints
Solution Approach 1:
The illumination beam is divided into two parts with different divergence characteristics. The high divergence second part is dedicated to fluorescence excitation where high NA optics are used to maximize light collection. The low divergence first part is dedicated to scattering measurements where the same high NA optics can be effectively used without being limited by beam divergence, thus achieving both specialized optimization and multi-functionality.
3Ease of manufacture
If a single non-coherent light source is used, then device cost is reduced, but the highly divergent beam requires high NA optics for both illumination and collection
Solution Approach 1:
The single non-coherent light source generates a highly divergent beam that is then segmented by a beam truncation device. The first part with reduced divergence allows scattering measurements with smaller optics, while the second part maintains high divergence for fluorescence excitation. This segmentation enables the use of a cost-effective single light source while reducing the size requirements for optical components through selective beam manipulation.
4Measurement precision
If scattered light is collected at small angles, then size measurement accuracy is improved, but illumination beam divergence limits the minimum collection angle
Solution Approach 1:
The illumination beam is segmented to create a first part with low divergence specifically optimized for small angle scattering measurements. This low divergence beam enables the collection of scattered light at very small angles relative to the optical axis, significantly improving size measurement accuracy. The beam truncation device creates a clean, well-defined low divergence beam that eliminates the angular spread constraints of the original highly divergent source.
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 accurate and cost-effective simultaneous fluorescence and scattering measurements by decoupling the illumination beams, improving measurement accuracy and reducing mechanical constraints around the flowcell.
Implementation Method 1
use LED source in an optical flow cytometer
Implementation Method 2
a beam truncation device comprising: a first passage to let pass a first part of the illumination beam with a first divergence, a second passage to let pass a second part of the illumination beam with a second divergence
Implementation Method 3
at least a focalization lens to focus the first part and the second part of the illumination beam on a flowcell
Implementation Method 4
Light intensity scattered by a single cell rapidly fades with increase of scattering angle
Implementation Method 5
A dye binds to a specific cell characteristic and emits fluorescence light when illuminated at appropriate wavelength
Implementation Method 6
Fluorescence light emission is typically very faint and requires high sensitivity photodetector like photomultiplier (PMT), Silicon PhotoMultiplier (SiPM) or avalanche photodiodes (APD)
Data Source
AI summary
An optical flow cytometer for fluorescence and scattering measurement includes a non-coherent light source generating an illumination beam; and a beam truncation device having: a first passage accommodating a first part of the illumination beam with a first divergence, the first part performing scattering measurements; a second passage accommodating a second part of the illumination beam with a second divergence, the second part being dedicated to fluorescence measurements; the second divergence being greater than the first divergence; the first and second passages separated by a beam truncation device area that stops illumination beam; a focalization lens focusing the first part and the second part of the illumination beam on a flowcell having an interrogation zone; a scattering detector receiving light scattered from the first illumination beam part as a particle crosses the interrogation zone; and a fluorescent detector receiving fluorescent light emitted by the particle crossing the interrogation zone.