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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.


Related in: MedlinePlus

(a) Simulated reflector shapes with contact angles Θs = 30°, Θp = 60°. The size of the liquid meniscus increases with time as the vapor is deposited.The meniscus corresponding to the maximum enhancement of fluorescence collection is drawn in magenta. (b) The predicted enhancement factors resulting from ray tracing simulations for various substrate (Θs) and particle (Θp) contact angles. The set of contact angles best matching the experimental case is shown in magenta. (c) Calculated emitted intensity improvement of the magenta-colored reflector shape vs. the numerical aperture of the optical system (contact angles: Θs = 30° ; Θp = 60°) using a spherical 5 μm fluorescent particle. The light is mainly redirected towards the optical axis of the imaging system. During the non-sequential ray tracing simulations, 2000 emitters are placed randomly into the fluorescent sphere with uniform distribution. Every emitter releases 2000 rays into the full 4π solid angle with uniform distribution for a total starting ray count of 10 million. The simulation assumes a perfect spherical particle before the micro-reflector deposition, thus ~50% of the emission is initially confined inside the particle due to the total internal reflection on the particle surface. Inset shows the detectors located ~10 cm above and below the bead. The two hemispheres detect the emitted intensity into angles between −80° to 80° with an angular bin size of 1°.
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f3: (a) Simulated reflector shapes with contact angles Θs = 30°, Θp = 60°. The size of the liquid meniscus increases with time as the vapor is deposited.The meniscus corresponding to the maximum enhancement of fluorescence collection is drawn in magenta. (b) The predicted enhancement factors resulting from ray tracing simulations for various substrate (Θs) and particle (Θp) contact angles. The set of contact angles best matching the experimental case is shown in magenta. (c) Calculated emitted intensity improvement of the magenta-colored reflector shape vs. the numerical aperture of the optical system (contact angles: Θs = 30° ; Θp = 60°) using a spherical 5 μm fluorescent particle. The light is mainly redirected towards the optical axis of the imaging system. During the non-sequential ray tracing simulations, 2000 emitters are placed randomly into the fluorescent sphere with uniform distribution. Every emitter releases 2000 rays into the full 4π solid angle with uniform distribution for a total starting ray count of 10 million. The simulation assumes a perfect spherical particle before the micro-reflector deposition, thus ~50% of the emission is initially confined inside the particle due to the total internal reflection on the particle surface. Inset shows the detectors located ~10 cm above and below the bead. The two hemispheres detect the emitted intensity into angles between −80° to 80° with an angular bin size of 1°.

Mentions: where Δp is the pressure difference across the air-liquid interface, ρPEG is the liquid PEG density, g is the acceleration due to gravity, h(r) is the height of the liquid-air interface, γ is the PEG surface tension, and Km is the local mean curvature of the interface. Equation (6) becomes a nonlinear second-order ordinary differential equation when the explicit expression26 for Km is substituted. We solve this equation numerically using an initial-value approach. We compute the interface shape for a set of different contact points at the bead, all having a given contact angle with the bead of θp, lying within the range 55° to 65°, based on the side-view optical micrograph shown in Fig. 1B. Out of the set of interface shapes corresponding to different contact points on the bead, we select the interface shape that provides a close match (within 0.3°) to the desired contact angle at the substrate θs, which is known from the side-view optical micrograph in Fig. 1b to lie within the range 30° to 35°. We perform this computation for a large range of Δp values, which correspond to different micro-reflector volumes V. These computations give us the shape of the micro-reflector at each stage of growth in volume. The rate at which the micro-reflector grows and adopts these shapes is then computed via Equation (1), the results of which are summarized in Fig. 3a.


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

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

(a) Simulated reflector shapes with contact angles Θs = 30°, Θp = 60°. The size of the liquid meniscus increases with time as the vapor is deposited.The meniscus corresponding to the maximum enhancement of fluorescence collection is drawn in magenta. (b) The predicted enhancement factors resulting from ray tracing simulations for various substrate (Θs) and particle (Θp) contact angles. The set of contact angles best matching the experimental case is shown in magenta. (c) Calculated emitted intensity improvement of the magenta-colored reflector shape vs. the numerical aperture of the optical system (contact angles: Θs = 30° ; Θp = 60°) using a spherical 5 μm fluorescent particle. The light is mainly redirected towards the optical axis of the imaging system. During the non-sequential ray tracing simulations, 2000 emitters are placed randomly into the fluorescent sphere with uniform distribution. Every emitter releases 2000 rays into the full 4π solid angle with uniform distribution for a total starting ray count of 10 million. The simulation assumes a perfect spherical particle before the micro-reflector deposition, thus ~50% of the emission is initially confined inside the particle due to the total internal reflection on the particle surface. Inset shows the detectors located ~10 cm above and below the bead. The two hemispheres detect the emitted intensity into angles between −80° to 80° with an angular bin size of 1°.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: (a) Simulated reflector shapes with contact angles Θs = 30°, Θp = 60°. The size of the liquid meniscus increases with time as the vapor is deposited.The meniscus corresponding to the maximum enhancement of fluorescence collection is drawn in magenta. (b) The predicted enhancement factors resulting from ray tracing simulations for various substrate (Θs) and particle (Θp) contact angles. The set of contact angles best matching the experimental case is shown in magenta. (c) Calculated emitted intensity improvement of the magenta-colored reflector shape vs. the numerical aperture of the optical system (contact angles: Θs = 30° ; Θp = 60°) using a spherical 5 μm fluorescent particle. The light is mainly redirected towards the optical axis of the imaging system. During the non-sequential ray tracing simulations, 2000 emitters are placed randomly into the fluorescent sphere with uniform distribution. Every emitter releases 2000 rays into the full 4π solid angle with uniform distribution for a total starting ray count of 10 million. The simulation assumes a perfect spherical particle before the micro-reflector deposition, thus ~50% of the emission is initially confined inside the particle due to the total internal reflection on the particle surface. Inset shows the detectors located ~10 cm above and below the bead. The two hemispheres detect the emitted intensity into angles between −80° to 80° with an angular bin size of 1°.
Mentions: where Δp is the pressure difference across the air-liquid interface, ρPEG is the liquid PEG density, g is the acceleration due to gravity, h(r) is the height of the liquid-air interface, γ is the PEG surface tension, and Km is the local mean curvature of the interface. Equation (6) becomes a nonlinear second-order ordinary differential equation when the explicit expression26 for Km is substituted. We solve this equation numerically using an initial-value approach. We compute the interface shape for a set of different contact points at the bead, all having a given contact angle with the bead of θp, lying within the range 55° to 65°, based on the side-view optical micrograph shown in Fig. 1B. Out of the set of interface shapes corresponding to different contact points on the bead, we select the interface shape that provides a close match (within 0.3°) to the desired contact angle at the substrate θs, which is known from the side-view optical micrograph in Fig. 1b to lie within the range 30° to 35°. We perform this computation for a large range of Δp values, which correspond to different micro-reflector volumes V. These computations give us the shape of the micro-reflector at each stage of growth in volume. The rate at which the micro-reflector grows and adopts these shapes is then computed via Equation (1), the results of which are summarized in Fig. 3a.

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.


Related in: MedlinePlus