A custom made confocal detection setup was used for the droplet based experiments described in this thesis. Figure 2.11 shows the schematic diagram of the confocal fluorescence spectrometer used for the confocal fluorescence detection and the picture of the setup is presented in Figure 2.13. All the optical components such as the beam steering optics, an olympus IX71 microscope (Olympus UK Ltd, UK) and detectors were mounted on an optical table (Thorlabs, Ltd, UK). A 488nm diode laser (10mW, Coherent UK Ltd, UK) was used as the excitation source for all droplet based microfluidic experiments performed in this
44
thesis. Lasers are used for the confocal fluorescence detection as they are bright, highly monochromatic, and have Gaussian beam profiles. Also, 488nm wavelength is highly compatible with many of the fluorescent dyes used in the experiments in this thesis. A dichroic mirror (DC1: z488rdc, Chroma Technology Corp., USA) was used to selectively reflect the blue laser beam by 90° whilst letting red laser through without deflection. This allows one to make use of other laser sources with different wavelengths such as 632.8 nm HeNe laser if necessary although this option wasn’t utilized in this thesis. The neutral density filter set housed in a filter wheel (FW2A, Thorlabs, Ltd, UK) was placed in the laser pathway to attenuate the laser intensity. By rotating the filter wheel, appropriate optical density values can be selected for each experiment. An iris (I1: ID20, Thorlabs, Ltd, UK) was placed to provide a reference point for the height and direction of the laser pathway. Then the laser beam passed through a 5X beam expander (BE: BE05M-A, Thorlabs, Ltd, UK) with a maximum input 1/e2
beam diameter of 2.25 mm. In other words, it expands the 488 nm laser beam from 0.7 mm to 3.5 mm after beam expansion. Through the use of the beam expander, the laser beam fills the entire back aperture of an objective lens which reduces the laser spot at the focal point as the size of the focused beam after the objective lens is inversely proportional to the incident beam diameter. Thus it allows one to minimize the detection probe volume and perform highly sensitive fluorescence detection. Two Mirrors (M1, M2: BB1-E02, Thorlabs, Ltd, UK) reflect the expanded laser beam into the microscope body and allow for the fine adjustments and alignment of the laser beam with respect to the microscope for sensitive fluorescence detection. Two Irises, I2 and I3, ensure the perfect alignment of the laser beam with respect to the microscope body. The incident laser beam is then reflected towards a high NA objective lens and focused onto the sample. A high NA objective was used for the experiments as it maximizes the fluorescence signals collected from the focal plane by minimizing the probe volume. Fluorescence signals from the sample then pass through the dichroic mirror, filtered by emission filter, and focused onto a tight spot by a tube lens prior to passing through a 75 µm precision pinhole (PH: P75S, Thorlabs, Ltd, UK)
45
46
Figure 2.12. (a) Confocal Fluorescence detection setup for microfluidic experiments. (a) Picture of the confocal fluorescence detection setup (laser pathway). (b) 488 nm excitation laser source. (c) Attenuator for attenuating laser intensity. (d) Optical mirror pathway into the microscope for fluorescence detection with the beam expander for sensitive fluorescence detection.
47
mounted on a XY translation stage (ST1XY-A/M, Thorlabs, Ltd, UK). After passing through the pinhole, a dichroic mirror (DC3: z630rdc, Chroma Technology Corp., USA) transmits light with wavelengths above 630nm and reflects those below 630nm to split fluorescence signals into two detection pathways. Then a band-pass emission filter (em2: ET525/50m 25mmR, Chroma Technology Corp., USA) further filters light directed towards the green channel by transmitting wavelengths from 500nm to 550nm and blocking other wavelengths. In the red channel, a long pass emission filter (em3: hq640lp, Chroma Technology Corp., USA) transmits light with wavelengths greater than 640nm whilst blocking those below 640nm. In both of the detection channels, plano-convex lenses (L1, L2: LA1951-A, f = 25.4 mm, 25.4 mm diameter, Thorlabs, Ltd, UK) focus light onto avalanche photodiode detectors (APD1, APD2: SPCM- AQR-14, Perkin Elmer, Canada) connected to the data acquisition unit.
48
2.6. References
1. McDonald JC, et al. (2000) Fabrication of microfluidic systems in poly(dimethylsiloxane).
Electrophoresis 21:27 - 40.
2. Sia SK & Whitesides GM (2003) Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis 24:3563-3576.
3. Sollier E, Murray C, Maoddi P, & Di Carlo D (2011) Rapid prototyping polymers for microfluidic devices and high pressure injections. Lab Chip 11:3752 - 3765.
4. Tiggelaar RM, et al. (2007) Fabrication, mechanical testing and application of high-pressure glass microreactor chips. Chem. Eng. J. 131:163 - 170.
5. Kim JY, deMello AJ, Chang SI, Hong J, & O'Hare D (2011) Thermoset polyester droplet-based microfluidic devices for high frequency generation. Lab on a Chip 11:4108 - 4112.
6. Mendes LAV, Pinho RR, Avila LF, Lima CRA, & Rocco MLM (2007) AZ-1518 Photoresist analysis with synchrotron radiation using high-resolution time-of-flight mass spectrometry. Polymer
Degradation and Stability 92:933-938.
7. del Campo A & Greiner C (2007) SU-8: a photoresist for high aspect-ratio and 3D submicron lithography. J. Micromech. Microeng. 17:R81-R95.
8. Kim JY, et al. (2012) Lab-chip HPLC with integrated droplet-based microfluidics for separation and high frequency compartmentalisation. Chem Commun. 73:9144-9146.
9. Huebner A, et al. (2007) Quantitative detection of protein expression in single cells using droplet microfluidics. Chem Comm:1218-1220.
10. Baret JC, et al. (2009) Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. Lab on a Chip 9:1850-1858.
11. Srisa-Art M, et al. (2009) Analysis of Protein-Protein Interactions by using droplet based microfluidics. Chembiochem 10:1605-1611.
12. Monpichar SA, deMello AJ, & Edel JB (2007) High-Throughput DNA Droplet Assays Using Picoliter Reactor Volumes. Anal. Chem. 79:6682-6689.
13. Solvas XC, Srisa-Art M, deMello AJ, & Edel JB (2010) Mapping of Fluidic mixing in microdroplets with 1us time resolution using fluorescence lifetime imaging. Analytical Chemistry 82:3950- 3956.
14. Choi J-W, Kang D-K, Park H, deMello AJ, & Chang S-I (2012) High-Throughput Analysis of Protein-Protein Interactions in Picoliter-Volume Droplets Using Fluorescence Polarization.
49