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Enhanced light collection in fluorescence microscopy using self-assembled micro-reflectors.

Göröcs Z, McLeod E, Ozcan A - Sci Rep (2015)

Bottom Line: The three-dimensional shape of this micro-reflector can be tuned as a function of time, vapor temperature, and substrate contact angle, providing us optimized SNR performance for fluorescent detection.Based on these self-assembled micro-reflectors, we experimentally demonstrate ~2.5-3 fold enhancement of the fluorescent signal from 2-10 μm sized particles.A theoretical explanation of the formation rate and shapes of these micro-reflectors is presented, along with a ray tracing model of their optical performance.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical Engineering, University of California Los Angeles (UCLA), CA 90095, USA.

ABSTRACT
In fluorescence microscopy, the signal-to-noise ratio (SNR) of the optical system is directly linked to the numerical aperture (NA) of the microscope objective, which creates detection challenges for low-NA, wide-field and high-throughput imaging systems. Here we demonstrate a method to increase the light collection efficiency from micron-scale fluorescent objects using self-assembled vapor-condensed polyethylene glycol droplets, which act as micro-reflectors for fluorescent light. Around each fluorescent particle, a liquid meniscus is formed that increases the excitation efficiency and redirects part of the laterally-emitted fluorescent light towards the detector due to internal reflections at the liquid-air interface of the meniscus. The three-dimensional shape of this micro-reflector can be tuned as a function of time, vapor temperature, and substrate contact angle, providing us optimized SNR performance for fluorescent detection. Based on these self-assembled micro-reflectors, we experimentally demonstrate ~2.5-3 fold enhancement of the fluorescent signal from 2-10 μm sized particles. A theoretical explanation of the formation rate and shapes of these micro-reflectors is presented, along with a ray tracing model of their optical performance. This method can be used as a sample preparation technique for consumer electronics-based microscopy and sensing tools, thus increasing the sensitivity of low-NA systems that image fluorescent micro-objects.

No MeSH data available.


Measured fluorescent intensity enhancement during the PEG micro-reflector formation process for 10 μm, 5 μm, and 2 μm fluorescent beads, respectively.Fluorescent microscope (4×; NA = 0.13) images are shown at different time points. The width of the shaded orange region for each curve is equal to twice its standard deviation.
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f2: Measured fluorescent intensity enhancement during the PEG micro-reflector formation process for 10 μm, 5 μm, and 2 μm fluorescent beads, respectively.Fluorescent microscope (4×; NA = 0.13) images are shown at different time points. The width of the shaded orange region for each curve is equal to twice its standard deviation.

Mentions: The results of our imaging experiments are summarized in Fig. 2, where for all the bead sizes a maximum intensity increase of 2.5-3 fold with respect to the initial state (2nd column) was observed once the optimum micro-reflector shape (last column) had been achieved. Interestingly, for 10 μm and 5 μm particles, the normalized fluorescent intensity plots (Fig. 2, left column) show an initial drop in the relative intensity to 0.5 and 0.7, respectively, before it starts to increase. In the case of 2 μm particles, however, the initial drop in intensity was not observed (Fig. 2, 3rd row). As we discuss in more detail below, we attribute these initial decreases in the relative fluorescent intensity of the micro-bead to the PEG condensation that effectively smoothens the bead surface, causing light to be better trapped within the micro-particle due to the whispering gallery modes29. However, at later times, the fluorescent signal enhancement resulting from the micro-reflector dominates the loss from the smoothening effect, resulting in a net enhancement in the collected light (Fig. 2, rightmost column).


Enhanced light collection in fluorescence microscopy using self-assembled micro-reflectors.

Göröcs Z, McLeod E, Ozcan A - Sci Rep (2015)

Measured fluorescent intensity enhancement during the PEG micro-reflector formation process for 10 μm, 5 μm, and 2 μm fluorescent beads, respectively.Fluorescent microscope (4×; NA = 0.13) images are shown at different time points. The width of the shaded orange region for each curve is equal to twice its standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470325&req=5

f2: Measured fluorescent intensity enhancement during the PEG micro-reflector formation process for 10 μm, 5 μm, and 2 μm fluorescent beads, respectively.Fluorescent microscope (4×; NA = 0.13) images are shown at different time points. The width of the shaded orange region for each curve is equal to twice its standard deviation.
Mentions: The results of our imaging experiments are summarized in Fig. 2, where for all the bead sizes a maximum intensity increase of 2.5-3 fold with respect to the initial state (2nd column) was observed once the optimum micro-reflector shape (last column) had been achieved. Interestingly, for 10 μm and 5 μm particles, the normalized fluorescent intensity plots (Fig. 2, left column) show an initial drop in the relative intensity to 0.5 and 0.7, respectively, before it starts to increase. In the case of 2 μm particles, however, the initial drop in intensity was not observed (Fig. 2, 3rd row). As we discuss in more detail below, we attribute these initial decreases in the relative fluorescent intensity of the micro-bead to the PEG condensation that effectively smoothens the bead surface, causing light to be better trapped within the micro-particle due to the whispering gallery modes29. However, at later times, the fluorescent signal enhancement resulting from the micro-reflector dominates the loss from the smoothening effect, resulting in a net enhancement in the collected light (Fig. 2, rightmost column).

Bottom Line: The three-dimensional shape of this micro-reflector can be tuned as a function of time, vapor temperature, and substrate contact angle, providing us optimized SNR performance for fluorescent detection.Based on these self-assembled micro-reflectors, we experimentally demonstrate ~2.5-3 fold enhancement of the fluorescent signal from 2-10 μm sized particles.A theoretical explanation of the formation rate and shapes of these micro-reflectors is presented, along with a ray tracing model of their optical performance.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical Engineering, University of California Los Angeles (UCLA), CA 90095, USA.

ABSTRACT
In fluorescence microscopy, the signal-to-noise ratio (SNR) of the optical system is directly linked to the numerical aperture (NA) of the microscope objective, which creates detection challenges for low-NA, wide-field and high-throughput imaging systems. Here we demonstrate a method to increase the light collection efficiency from micron-scale fluorescent objects using self-assembled vapor-condensed polyethylene glycol droplets, which act as micro-reflectors for fluorescent light. Around each fluorescent particle, a liquid meniscus is formed that increases the excitation efficiency and redirects part of the laterally-emitted fluorescent light towards the detector due to internal reflections at the liquid-air interface of the meniscus. The three-dimensional shape of this micro-reflector can be tuned as a function of time, vapor temperature, and substrate contact angle, providing us optimized SNR performance for fluorescent detection. Based on these self-assembled micro-reflectors, we experimentally demonstrate ~2.5-3 fold enhancement of the fluorescent signal from 2-10 μm sized particles. A theoretical explanation of the formation rate and shapes of these micro-reflectors is presented, along with a ray tracing model of their optical performance. This method can be used as a sample preparation technique for consumer electronics-based microscopy and sensing tools, thus increasing the sensitivity of low-NA systems that image fluorescent micro-objects.

No MeSH data available.