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Three dimensional microfluidics with embedded microball lenses for parallel and high throughput multicolor fluorescence detection
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10.1063/1.4818944
/content/aip/journal/bmf/7/4/10.1063/1.4818944
http://aip.metastore.ingenta.com/content/aip/journal/bmf/7/4/10.1063/1.4818944
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Figures

Image of FIG. 1.
FIG. 1.

(a) Schemetic of 3D parallel microfluidic device with an embedded microball lens array. (b) and (c) Pictures of the device consisting of 32 parallel sample channels, 64 sheath flow channels, and 32 micro ball lenses deployed in a linear array.

Image of FIG. 2.
FIG. 2.

(a) Process flow for fabricating microball lens arrays embedded in PDMS. (b) An optical image showing part of a 100 × 100 microball lens array. (c) A SEM image showing the cross-section of microball lenses embedded right underneath the PDMS surface.

Image of FIG. 3.
FIG. 3.

(a) Schematic of multiple PDMS thin film layers stacked in our 3D microfluidic device. (b) and (c) Pictures of a device consisting of 32 parallel sample channels, 64 sheath-flow channels, and 32 microball lenses.

Image of FIG. 4.
FIG. 4.

Ray tracing simulation of (a) parallel light rays and (b) light rays from a point source propagating through a microball lens embedded in PDMS and focused in water. Light rays propagating through the edge of the ball lens undergoes spherical aberration and results in a photonic jet effect. Light rays illuminate at the middle of the ball lens get tightly focused to the focal point. It provides a 52∘ collection angle with small spherical aberration.

Image of FIG. 5.
FIG. 5.

A light beam propagates through a PDMS embedded 75 m ball lens and gets focused to excite fluorescence dye in a 30 m high channel. The fluorescence images are taken by a 10× objective lens on a fluorescence microscope to calibrate the excitation range after the lens. (a) Topview of the fluorescence image captured at the focal point. (b) The bright field and the (c) fluorescent sideview images of a ball lens. A column-shape of fluorescence excitation is observed after the ball lens. (d) The intensity profiles extracted from the topview fluorescence image in (a). (e) The intensity profile extracted from a sideview fluorescence image in (c). It shows the smallest spot size is 5 m. The intensity distribution along the center propagation axis after the ball lens is plotted in (f). It shows the column-shape excitation beam can penetrate 27 m deep into the channel.

Image of FIG. 6.
FIG. 6.

(a) Schematic of the custom constructed optical system for parallel fluorescence detection in our device. A high power laser diode provides fluorescence excitation on cells through a micro ball lens array. Multicolor fluorescence emission from cells are collected by the same microlens array and imaged on to a high speed CMOS camera through telescope optics. Optical images of different colors are split on the detection image plane by a custom built prism to achieve continuous, simultaneous, multicolor detection of cells passing through all 32 channels. (b) Schematic of the ray tracing shows how multicolor fluorescence signals are collected by a microball lens, color split by a small angle prism, and imaged on to the image plane.

Image of FIG. 7.
FIG. 7.

Example of parallel, multicolor fluorescence detection in 32 channels. (a) Image without an emission filter and a prism added in the optical pathway. The blue, green, and red signals from the ball lens array overlap on the image plane. (b) An emission filter is added to remove the blue excitation light from the laser. An array of yellow dots is observed since the green and red fluorescence signals from the same ball lens overlap. (c) A prism is added to split images of different colors on the detection plane. (d) An emission filter and a prism are added. Green and red fluorescence signals from 32 channels are simultaneously picked up. (e) Comparison of the signal to noise ratio improvement without and with a microball lens array. An 18 times improvement has been achieved with microball lenses.

Image of FIG. 8.
FIG. 8.

(a) A representative fluorescence image of red and green beads passing through the detection zone. Green beads with long fluorescence emission tail in spectrum showed both green and red signals in the corresponding channels, while red beads show only red signals. (b) A fluorescence image showing two green beads flowing through two adjacent channels with little fluorescence signal crosstalk between channels.

Image of FIG. 9.
FIG. 9.

(a) The results of bead counting using sample 1: green beads with concentration of 2 × 10 beads/ml (b) Three different bead locations can cause different peak intensity profiles in (a). The high camera frame rate and the small excitation spot (∼5 m) allow our flow cytometer to distinguish closely positioned beads. (c) The results of bead counting using sample 2: green and red beads mixed at a 1:1 ratio with a total concentration of 2 × 10 bead/ml.

Image of FIG. 10.
FIG. 10.

(a) A high throughput detection of Ramos cells at 12 800 cells/sec per channel was achieved. (b) Multicolor detection of a mixture with red HeLa cells and green Ramos cells mixed at a 1:10 ratio. The results show good agreement with the original ratio of cells we prepared. Microscopic images of cell mixtures collected at the flow cytometer outlet and taken at different modes, from (c) to (e), the bright field, the green fluorescence with a FITC filter, and the red fluorescence with a TRITC filter.

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/content/aip/journal/bmf/7/4/10.1063/1.4818944
2013-08-21
2014-04-23
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Three dimensional microfluidics with embedded microball lenses for parallel and high throughput multicolor fluorescence detection
http://aip.metastore.ingenta.com/content/aip/journal/bmf/7/4/10.1063/1.4818944
10.1063/1.4818944
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