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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

Self assembled micro-reflector formation.(a) As the liquid condenses around the fluorescent particle, a meniscus forms over time. (b) Side view images of the same 10 μm fluorescent particle before and after the meniscus formation using the same illumination and camera settings. Both reflected bright field and fluorescent 20× microscope images are shown. Note the absence of the reflection from the coverslip holding the sample, as this light gets redirected downward into the coverslip as indicated in (d). Also note that the condensation process forms a thin liquid layer on the fluorescent particle, reducing its surface roughness, thus lowering the scattered light intensity from the bead and confining some of the fluorescent emission within the bead body. PEG is highly transparent, thus the visibility of the meniscus is low. (c) Bottom view of the same particle before and after the meniscus formation using a 4× microscope objective, which qualitatively shows the increase of the detected fluorescent light. The measurements were taken using the same illumination and camera settings. (d) Fluorescent light emitted by the bead outside the acceptance angle of the optical system will be redirected towards the detector due to reflections on the surface of the meniscus.
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f1: Self assembled micro-reflector formation.(a) As the liquid condenses around the fluorescent particle, a meniscus forms over time. (b) Side view images of the same 10 μm fluorescent particle before and after the meniscus formation using the same illumination and camera settings. Both reflected bright field and fluorescent 20× microscope images are shown. Note the absence of the reflection from the coverslip holding the sample, as this light gets redirected downward into the coverslip as indicated in (d). Also note that the condensation process forms a thin liquid layer on the fluorescent particle, reducing its surface roughness, thus lowering the scattered light intensity from the bead and confining some of the fluorescent emission within the bead body. PEG is highly transparent, thus the visibility of the meniscus is low. (c) Bottom view of the same particle before and after the meniscus formation using a 4× microscope objective, which qualitatively shows the increase of the detected fluorescent light. The measurements were taken using the same illumination and camera settings. (d) Fluorescent light emitted by the bead outside the acceptance angle of the optical system will be redirected towards the detector due to reflections on the surface of the meniscus.

Mentions: Here we present a simple alternative method to increase the fluorescent sensitivity of optical imaging systems by depositing a liquid micro-reflector around the target sample through vapor condensation (Fig. 1a). Vapor deposition and droplet growth on transparent surfaces have intrigued physicists for more than a century24. Recently, self-assembly of liquid nano-lenses around nano-particles has been used for enhancing the signal-to-noise ratio in holographic on-chip microscopy, enabling label-free detection of sub-40 nm particles2526. For this purpose, polyethylene glycol (PEG) is an ideal material for creating evaporation-based self-assembled optical components as it is non-toxic, and has a sufficiently high vapor pressure to be able to evaporate at moderate temperatures (e.g. ~100 °C), but low enough to be stable for several hours at room temperature. Furthermore, evaporation based self-assembled PEG nanolenses have been successfully integrated with cost-effective and field-portable holographic imaging devices27. The shape of the PEG meniscus depends on the contact angles of the polymer on the sample substrate and on the particle. Unlike its prior uses in digital holography for label-free bright-field imaging, where the enhancement of the scattered signal is achieved by depositing liquid nanolenses with a low surface contact angle, here we engineer these self-assembled vapor-condensed lenses to improve the sensitivity of fluorescence microscopy by using larger substrate contact angles to shape the meniscus as a reflector that increases the excitation efficiency and redirects laterally-emitted fluorescent light away from the sides and towards a detector located below the micro-reflector, thereby strongly enhancing the measured fluorescence signal for each micro-particle (see Fig. 1).


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

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

Self assembled micro-reflector formation.(a) As the liquid condenses around the fluorescent particle, a meniscus forms over time. (b) Side view images of the same 10 μm fluorescent particle before and after the meniscus formation using the same illumination and camera settings. Both reflected bright field and fluorescent 20× microscope images are shown. Note the absence of the reflection from the coverslip holding the sample, as this light gets redirected downward into the coverslip as indicated in (d). Also note that the condensation process forms a thin liquid layer on the fluorescent particle, reducing its surface roughness, thus lowering the scattered light intensity from the bead and confining some of the fluorescent emission within the bead body. PEG is highly transparent, thus the visibility of the meniscus is low. (c) Bottom view of the same particle before and after the meniscus formation using a 4× microscope objective, which qualitatively shows the increase of the detected fluorescent light. The measurements were taken using the same illumination and camera settings. (d) Fluorescent light emitted by the bead outside the acceptance angle of the optical system will be redirected towards the detector due to reflections on the surface of the meniscus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Self assembled micro-reflector formation.(a) As the liquid condenses around the fluorescent particle, a meniscus forms over time. (b) Side view images of the same 10 μm fluorescent particle before and after the meniscus formation using the same illumination and camera settings. Both reflected bright field and fluorescent 20× microscope images are shown. Note the absence of the reflection from the coverslip holding the sample, as this light gets redirected downward into the coverslip as indicated in (d). Also note that the condensation process forms a thin liquid layer on the fluorescent particle, reducing its surface roughness, thus lowering the scattered light intensity from the bead and confining some of the fluorescent emission within the bead body. PEG is highly transparent, thus the visibility of the meniscus is low. (c) Bottom view of the same particle before and after the meniscus formation using a 4× microscope objective, which qualitatively shows the increase of the detected fluorescent light. The measurements were taken using the same illumination and camera settings. (d) Fluorescent light emitted by the bead outside the acceptance angle of the optical system will be redirected towards the detector due to reflections on the surface of the meniscus.
Mentions: Here we present a simple alternative method to increase the fluorescent sensitivity of optical imaging systems by depositing a liquid micro-reflector around the target sample through vapor condensation (Fig. 1a). Vapor deposition and droplet growth on transparent surfaces have intrigued physicists for more than a century24. Recently, self-assembly of liquid nano-lenses around nano-particles has been used for enhancing the signal-to-noise ratio in holographic on-chip microscopy, enabling label-free detection of sub-40 nm particles2526. For this purpose, polyethylene glycol (PEG) is an ideal material for creating evaporation-based self-assembled optical components as it is non-toxic, and has a sufficiently high vapor pressure to be able to evaporate at moderate temperatures (e.g. ~100 °C), but low enough to be stable for several hours at room temperature. Furthermore, evaporation based self-assembled PEG nanolenses have been successfully integrated with cost-effective and field-portable holographic imaging devices27. The shape of the PEG meniscus depends on the contact angles of the polymer on the sample substrate and on the particle. Unlike its prior uses in digital holography for label-free bright-field imaging, where the enhancement of the scattered signal is achieved by depositing liquid nanolenses with a low surface contact angle, here we engineer these self-assembled vapor-condensed lenses to improve the sensitivity of fluorescence microscopy by using larger substrate contact angles to shape the meniscus as a reflector that increases the excitation efficiency and redirects laterally-emitted fluorescent light away from the sides and towards a detector located below the micro-reflector, thereby strongly enhancing the measured fluorescence signal for each micro-particle (see Fig. 1).

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