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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.


Enhancement of fluorescence collection during the PEG micro-reflector formation process for 10 μm, 5 μm, and 2 μm fluorescent beads, respectively.In order to match our simulations, the emission data was normalized to the first minimum on the curve, corresponding to a smoothened fluorescent bead (through PEG deposition), and the square root of the intensity was taken to account for the increased excitation caused by the micro-reflector. Regardless of the bead size, the peak values for the enhancement of fluorescence collection are ~2.3-2.4, agreeing very well with our model results shown in Fig. 3b. For 2 μm fluorescent particles, several local maxima in time (i.e., blinking) were observed.
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f4: Enhancement of fluorescence collection during the PEG micro-reflector formation process for 10 μm, 5 μm, and 2 μm fluorescent beads, respectively.In order to match our simulations, the emission data was normalized to the first minimum on the curve, corresponding to a smoothened fluorescent bead (through PEG deposition), and the square root of the intensity was taken to account for the increased excitation caused by the micro-reflector. Regardless of the bead size, the peak values for the enhancement of fluorescence collection are ~2.3-2.4, agreeing very well with our model results shown in Fig. 3b. For 2 μm fluorescent particles, several local maxima in time (i.e., blinking) were observed.

Mentions: A second conceptual difference between our model and experiments is that the optical model only calculates the fluorescent light emitted from the particle and does not consider possible enhancement of the excitation light due to the micro-reflector. When we compensate for these two conceptual differences between our model and experiments, we can find even better agreement between the experimental results and numerical simulations. To compensate for the particle smoothening effect, we rescale the measurement results to take the minimum point of the intensity curve as the new normalization factor, instead of the initial intensity point, which we used in Fig. 2. This change effectively makes our reference point that of a perfect sphere (after PEG deposition) instead of a rough native sphere. In addition to this, we also aimed to isolate the effects of increased excitation due to the micro-reflector shape. In our ray tracing calculations, a substantial amount of the enhancement of the fluorescent collection originates from the light rays that get reflected from the meniscus surface and leave the sample plane adjacent to the bead (see Fig. 1d). Since the same lens is used for both excitation and collection of the fluorescent emission, by using a geometrical optics approximation we can treat the light rays as reversible, and for a given reflector shape we can consider the excitation enhancement factor due to the micro-reflector to be approximately equal to the enhancement factor of the fluorescence collection. In other words, the physical source of the excitation enhancement can be considered to be the internal reflections of the excitation rays at the micro-reflector surface, re-directing them toward the embedded body of fluorophores within a given particle. By using this assumption, we can take the square root of the experimentally-measured intensity curves to separate the component of the enhancement that is due to increases in the collection efficiency alone, apart from the component that is due to excitation enhancement. Figure 4 shows our experimental data after making these two adjustments: in both the numerical model results for fluorescence collection (Fig. 3b) and the normalized/adjusted experimental results (Fig. 4), the peak values for the enhancement of fluorescence collection are ~2.3-2.4×, providing a decent agreement to each other. Here, we should also emphasize that the overall fluorescence enhancement peak factor due to these self-assembled micro-reflectors is larger as shown in Fig. 2 since it also includes an additional source of improvement due to enhanced excitation.


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

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

Enhancement of fluorescence collection during the PEG micro-reflector formation process for 10 μm, 5 μm, and 2 μm fluorescent beads, respectively.In order to match our simulations, the emission data was normalized to the first minimum on the curve, corresponding to a smoothened fluorescent bead (through PEG deposition), and the square root of the intensity was taken to account for the increased excitation caused by the micro-reflector. Regardless of the bead size, the peak values for the enhancement of fluorescence collection are ~2.3-2.4, agreeing very well with our model results shown in Fig. 3b. For 2 μm fluorescent particles, several local maxima in time (i.e., blinking) were observed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Enhancement of fluorescence collection during the PEG micro-reflector formation process for 10 μm, 5 μm, and 2 μm fluorescent beads, respectively.In order to match our simulations, the emission data was normalized to the first minimum on the curve, corresponding to a smoothened fluorescent bead (through PEG deposition), and the square root of the intensity was taken to account for the increased excitation caused by the micro-reflector. Regardless of the bead size, the peak values for the enhancement of fluorescence collection are ~2.3-2.4, agreeing very well with our model results shown in Fig. 3b. For 2 μm fluorescent particles, several local maxima in time (i.e., blinking) were observed.
Mentions: A second conceptual difference between our model and experiments is that the optical model only calculates the fluorescent light emitted from the particle and does not consider possible enhancement of the excitation light due to the micro-reflector. When we compensate for these two conceptual differences between our model and experiments, we can find even better agreement between the experimental results and numerical simulations. To compensate for the particle smoothening effect, we rescale the measurement results to take the minimum point of the intensity curve as the new normalization factor, instead of the initial intensity point, which we used in Fig. 2. This change effectively makes our reference point that of a perfect sphere (after PEG deposition) instead of a rough native sphere. In addition to this, we also aimed to isolate the effects of increased excitation due to the micro-reflector shape. In our ray tracing calculations, a substantial amount of the enhancement of the fluorescent collection originates from the light rays that get reflected from the meniscus surface and leave the sample plane adjacent to the bead (see Fig. 1d). Since the same lens is used for both excitation and collection of the fluorescent emission, by using a geometrical optics approximation we can treat the light rays as reversible, and for a given reflector shape we can consider the excitation enhancement factor due to the micro-reflector to be approximately equal to the enhancement factor of the fluorescence collection. In other words, the physical source of the excitation enhancement can be considered to be the internal reflections of the excitation rays at the micro-reflector surface, re-directing them toward the embedded body of fluorophores within a given particle. By using this assumption, we can take the square root of the experimentally-measured intensity curves to separate the component of the enhancement that is due to increases in the collection efficiency alone, apart from the component that is due to excitation enhancement. Figure 4 shows our experimental data after making these two adjustments: in both the numerical model results for fluorescence collection (Fig. 3b) and the normalized/adjusted experimental results (Fig. 4), the peak values for the enhancement of fluorescence collection are ~2.3-2.4×, providing a decent agreement to each other. Here, we should also emphasize that the overall fluorescence enhancement peak factor due to these self-assembled micro-reflectors is larger as shown in Fig. 2 since it also includes an additional source of improvement due to enhanced excitation.

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.