This work was supported by DARPA grant number 84389.01.44908, an NSF CAREER award (DBI-1253293), an NIH exploratory/developmental research grant (CA195709), and NIH New Innovator Awards (HD080351, DP2-AR068129-01), and a New Directions grant from the UCSF resource allocation program.

1. SU8 Master Fabrication
<ol>
	<li>Design the microfluidic structures for three layer fabrication using design software and have the designs printed by a vendor on circuit board film with 10 &#181;m resolution. The details of device design are given in an attached reference 6 and the channel geometries are shown in Figure 1. The layers should include alignment marks to help collocate features from each fabrication layer 8.</li>
	<li>Place a pre-cleaned 3 inch diameter silicon wafer on a spin coater and turn on the vacuum to affix it to the chuck. Apply 1 ml of SU8-3050 in the center of the wafer and spin for 20 sec at 500 rpm, then 30 sec at 1,750 rpm, providing a layer thickness of 80 &#181;m.</li>
	<li>Remove the wafer and bake on a 135 &#176;C hotplate for 30 min. Allow the wafer to cool to RT before moving on to the next step.</li>
	<li>Expose the coated wafer to the 1st layer mask under a collimated 190 mW, 365 nm LED for 3 min. After exposure, place the wafer on a 135 &#176;C hotplate for 1 min, then cool to RT before proceeding to the next step.</li>
	<li>Place the wafer on the spin coater and turn on the vacuum to affix it to the chuck. Apply 1 ml of SU8-3050 in the center of the wafer and spin for 20 sec at 500 rpm, then 30 sec at 5,000 rpm, resulting in a layer that provides an additional thickness of 40 &#181;m.</li>
	<li>Remove the wafer and bake on a 135 &#176;C hotplate for 30 min, then cool to RT before moving to the next step.</li>
	<li>Align the 2nd layer mask onto the geometry patterned in 1.3 and expose the coated wafer to a collimated 190 mW, 365 nm LED for 3 min. After exposure, place on a 135 &#176;C hotplate for 4 min 30 sec, then cool to RT before proceeding to the next step.</li>
	<li>Place the wafer on the spin coater and turn on the vacuum to affix it to the chuck. Apply 1 ml of SU8-3050 in the center of the wafer and spin for 20 sec at 500 rpm, then 30 sec at 1,000 rpm, resulting in a layer that provides an additional thickness of 100 &#181;m.</li>
	<li>Remove the wafer and bake on a 135 &#176;C hotplate for 30 min, then cool to RT before moving to the next step.</li>
	<li>Align the 3rd layer mask onto the geometry patterned in 1.3 and expose the coated wafer to a collimated 190 mW, 365 nm LED for 3 min. After exposure, place on a 135 &#176;C hotplate for 9 min, then cool to RT before proceeding to the next step.</li>
	<li>Develop the masks by immersing in a stirred bath of propylene glycol monomethyl ether acetate for 30 min. Wash the wafer in isopropanol and bake on a 135 &#176;C hotplate for 1 min. Place the developed master in a 100 mm Petri dish for polydimethylsiloxane (PDMS) molding.</li>
</ol>
2. PDMS Device Fabrication
<ol>
	<li>Prepare 10:1 PDMS by combining 50 g of silicone base with 5 g of curing agent in a plastic cup. Mix the contents with a rotary tool fitted with a stir stick. Degas the mixture inside a desiccator for 30 min, or until all air bubbles are removed.</li>
	<li>Pour the PDMS to give a thickness of 3 mm over the master and place back into the desiccator for further degassing. Once all bubbles are removed, bake the device at 80 &#176;C for 80 min.</li>
	<li>Cut the device from the mold using a scalpel and place on a clean surface with the patterned side up and punch the fluidic inlets and outlets with a 0.75 mm biopsy punch. The device must be cut so that the 120 &#181;m and 220 &#181;m tall geometries are accessible from the side of the device.</li>
	<li>Plasma treat the device, with feature side up, along with a pre-cleaned 2 inches by 3 inches glass slide at 1 mbar O2 plasma for 20 sec in a 300 W plasma cleaner. Bond the PDMS device by placing the patterned side of the PDMS device onto the plasma-treated side of the glass slide. Place the device in a 80 &#176;C oven and bake the assembled device for 40 min.</li>
	<li>Render the channels hydrophobic by using a syringe to flush the device with a fluorinated surface treatment fluid. Immediately bake the device at 80 &#176;C for 10 min to evaporate the solvent.</li>
</ol>
3. Preparation of Optical Components
<ol>
	<li>Prepare the laser excitation fiber by removing the insulation from the last 5 mm of a 105 &#181;m core, 125 &#181;m cladding, NA = 0.22 optical fiber.</li>
	<li>Prepare the 2nd color laser excitation fiber by repeating 3.1 for a 2nd identical fiber.</li>
	<li>Prepare the fiber for collecting the fluorescence signal by repeating 3.1 for a 200 &#181;m core, 225 &#181;m cladding, NA = 0.39 optical fiber.</li>
	<li>Inspect the tips of all the fibers &#8212; if the tips do not end in a flat surface, re-cleave the ends with a fiber scribe.</li>
	<li>Attach a laser fiber coupler to a 50 mW, 405 nm laser and attach one of the 105 &#181;m core fibers to the laser. Direct the stripped end at the sensor of the laser power meter, and use the fine adjustments of the laser coupler to maximize the laser power.</li>
	<li>Repeat 3.5 for a 50 mW, 473 nm laser.</li>
	<li>Mount a 446/510/581/703 nm quad bandpass filters on the PMT using lens tubes to block laser light and transmit emitted fluorescence. Attach fiber coupler so that light travels through the filters before hitting the PMT.</li>
	<li>Attach the fiber from 3.3 to the unit assembled in 3.7.</li>
</ol>
4. Offline Mixed Emulsion Generation
<ol>
	<li>Obtain a flow focus microfluidics dropmaker device with a 60 &#181;m x 60 &#181;m orifice.</li>
	<li>Fill a 1 ml syringe with HFE 7500 oils with 2% ionic fluorosurfactant 9.</li>
	<li>Fill a series of 1 ml syringes with the following combinations of fluorescein (FITC) and dextran-conjugated blue dyes (CB) in PBS: 1 nM FITC/10 nM CB, 10 nM FITC/10 nM CB, 1 nM FITC/100 nM CB, and 10 nM FITC/100 nM CB.</li>
	<li>Mount the dropmaker device on the stage of an inverted microscope and the aqueous and oil syringes on syringe pumps. Couple the syringes to the devices using PE-2 tubing.</li>
	<li>Running syringe pumps at 500 &#181;l/hr for the oil and 250 &#181;l/hr for the aqueous solutions, create a mixture of 80 &#181;m droplets containing the solutions from 4.3. For each type of aqueous sample, use a fresh dropmaker device to eliminate cross-contamination. Collect ~200 &#181;l of emulsion from each dye combination into an empty syringe.</li>
	<li>After the 4 droplets types have been collected in a single collection syringe, mix the emulsion by repeatedly rotating the syringe.</li>
	<li>Repeat steps 4.2-4.6 with aqueous solutions containing 0.1, 1, 10, and 100 nM FITC in PBS.</li>
</ol>
5. Optical Fiber Insertion
<ol>
	<li>Place the microfluidic chip fabricated in step 2 on the stage of an inverted microscope coupled with a digital camera capable of &#60; 100 &#181;sec shutter speeds.</li>
	<li>Working carefully from the side, insert the fiber coupled to the 473 nm laser into the farthest upstream 120 &#181;m channel, taking care not to puncture through to the main flow channel.</li>
	<li>Insert the fiber coupled to the 405 nm laser into the farthest downstream 120 &#181;m high side channel, providing fiber spacing of 300 &#181;m.</li>
	<li>Insert the PMT-coupled fiber into the 220 &#181;m tall channel normal to the two laser excitation fibers.</li>
</ol>
6. Fluorescence Detection of Mixed Emulsions&#160;
<ol>
	<li>Mount a 5 ml syringe filled with HFE 7500 with 2% ionic fluorosurfactant to the spacer oil inlet of the detection device with PE-2 tubing.</li>
	<li>Mount the syringe containing the mixed FITC/CB emulsion on a vertically oriented syringe pump and couple to the device&#39;s droplet reinjection inlet using PE-2 tubing. Connect a length of PE-2 tubing from the device exit to a waste container.</li>
	<li>Prime the device by running each of the pumps at 1,000 &#181;l/hr until both oil and droplets are seen to be regularly combining in the device and flowing downstream.</li>
	<li>Adjust the flow rates such that the spacer oil runs at 6,000 &#181;l/hr and the droplets at 100 &#181;l/hr, providing significant spacing between droplets traveling through the detection region.</li>
	<li>Turn on the lasers, start the data acquisition program, and adjust the PMT gain to provide signals that are more than 100x the baseline noise floor. Adjust the laser power so that all of the doublet peaks are clearly visible on a single, linearly scaled timetrace.</li>
	<li>Acquire 60 sec of the PMT voltage timetrace at at least 20 kHz. Import the data into computing program such as Matlab and measure the heights of peaks of the recorded doublets using the custom computing program script. 
	NOTE: This is provided as supplementary information.</li>
	<li>Repeat steps 6.2-6.7 using the FITC-only mixed emulsion created in 4.7, with the 405 nm laser turned off.</li>
</ol>

<ol>
	<li>Teh, S. Y., Lin, R., Hung, L. H., Lee, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+microfluidics.">Droplet microfluidics.</a> Lab Chip. 8, 198-220 (2008).</li><li>Mazutis, L., Gilbert, J., Ung, W. L., Weitz, D. A., Griffiths, A. D., Heyman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+and+sorting+using+droplet-based+microfluidics.">Single-cell analysis and sorting using droplet-based microfluidics.</a> Nat Protocol. 8 (5), 870-891 (2013).</li><li>Hindson, B. J., Ness, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+droplet+digital+PCR+system+for+absolute+quantitation+of+DNA+copy+number.">High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.</a> Anal Chem. 83, 8604-8610 (2011).</li><li>Agresti, J. J., Antipov, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh-throughput+screening+in+drop-based+microfluidics+for+directed+evolution.">Ultrahigh-throughput screening in drop-based microfluidics for directed evolution.</a> Proc Nat Acad Sci USA. 107 (14), 4004 (2010).</li><li>Eastburn, D. J., Sciambi, A., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picoinjection+Enables+Digital+Detection+of+RNA+with+Droplet+RT-PCR.">Picoinjection Enables Digital Detection of RNA with Droplet RT-PCR.</a> PLOS ONE. 8 (4), (2013).</li><li>Cole, R. H., de Lange, N., Gartner, Z. J., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compact+and+modular+multicolour+fluorescence+detector+for+droplet+microfluidics.">Compact and modular multicolour fluorescence detector for droplet microfluidics.</a> Lab Chip. 15 (13), 2754-2758 (2015).</li><li>Guo, F., Lapsley, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+droplet-based,+optofluidic+device+for+high-throughput,+quantitative+bioanalysis.">A droplet-based, optofluidic device for high-throughput, quantitative bioanalysis.</a> Anal Chem. 84, 10745-10749 (2012).</li><li>. Lithography Available from: <a target="_blank" href="https://www.memsnet.org/mems/processes/lithography.html">https://www.memsnet.org/mems/processes/lithography.html</a> (2015)</li><li>DeJournette, C. J., Kim, J., Medlen, H., Li, X., Vincent, L. J., Easley, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Biocompatible+Oil-Water+Interfaces+without+Synthesis:+Direct+Interactions+between+Primary+Amines+and+Carboxylated+Perfluorocarbon+Surfactants.">Creating Biocompatible Oil-Water Interfaces without Synthesis: Direct Interactions between Primary Amines and Carboxylated Perfluorocarbon Surfactants.</a> Anal Chem. 85 (21), (2013).</li><li>Fallah-Araghi, A., Baret, J. C., Ryckelynck, M., Griffiths, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+completely+in+vitro+ultrahigh-throughput+droplet-based+microfluidic+screening+system+for+protein+engineering+and+directed+evolution.">A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution.</a> Lab Chip. 12, 882 (2012).</li><li>Eastburn, D. J., Sciambi, A., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh-Throughput+Mammalian+Single-Cell+Reverse-Transcriptase+Polymerase+Chain+Reaction+in+Microfluidic+Drops.">Ultrahigh-Throughput Mammalian Single-Cell Reverse-Transcriptase Polymerase Chain Reaction in Microfluidic Drops.</a> Anal Chem. 85 (16), 8016-8021 (2013).</li><li>Martini, J., Recht, M. I., Huck, M., Bern, M. W., Johnson, N. M., Kiesel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+encoded+multicolor+fluorescence+detection+in+a+microfluidic+flow+cytometer.">Time encoded multicolor fluorescence detection in a microfluidic flow cytometer.</a> Lab Chip. 12 (23), 5057-5062 (2012).</li><li>Bliss, C. L., McMullin, J. N., Backhouse, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+fabrication+of+a+microfluidic+device+with+integrated+optical+waveguides+for+DNA+fragment+analysis.">Rapid fabrication of a microfluidic device with integrated optical waveguides for DNA fragment analysis.</a> Lab Chip. 7 (10), 1280-1287 (2007).</li><li>Martinez Vazquez, R., Osellame, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sensing+in+microfluidic+lab-on-a-chip+by+femtosecond-laser-written+waveguides.">Optical sensing in microfluidic lab-on-a-chip by femtosecond-laser-written waveguides.</a> Anal Bioanal Chem. 393, 1209-1216 (2009).</li><li>Vishnubhatla, K. C., Bellini, N., Ramponi, R., Cerullo, G., Osellame, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+control+of+microchannels+fabricated+in+fused+silica+by+femtosecond+laser+irradiation+and+chemical+etching.">Shape control of microchannels fabricated in fused silica by femtosecond laser irradiation and chemical etching.</a> Opt Express. 17 (10), 8685-8695 (2009).</li></ol>

The authors have nothing to disclose.

Fiber optic detection requires the alignment of optical fibers with respect to fluid channels. Because our device utilizes guide channels fabricated with multilayer photolithography, placement of masks with respect to each other is of great importance. If the fiber guide channels are too close to the fluid channel, there is a potential for fluid leakage; if the guide channels are located too far away or misaligned, the fluorescence signal gathered by the detection fiber may be significantly diminished. Proper alignment can be aided by designing alignment marks, such as concentric circles into masks to be co-located during photo patterning. Additionally, manual insertion of fiber optics into the device is a delicate process with the potential to break the tips of fibers within the device, rendering them unusable. Restraining fiber against the glass slide bonded to the PDMS device and inserting the fibers slowly while observing with the microscope are keys to successful fiber insertion. Last, reinjection of pre-formed emulsions should occur with a minimum of handling. Ideally, once the emulsion is made and deposited directly into an empty syringe, it should not be transferred until it is used for droplet reinjection.
Multicolor detection requires that the peaks in a signal doublet be distinct from one another and that signals from consecutive droplets do not overlap. The detection setup that we use here uses a pair of 125 &#181;m optical fibers separated at a center-to-center distance of 300 &#181;m, providing a non-illuminated region of ~175 &#181;m between excitation regions. This allows an 80 &#181;m droplet to be excited by only one light source at a time and ensures that each of the peaks in a doublet is solely a result of one type of laser excitation. Designs where the separation between the excitation regions is similar to the droplet diameter give problematic signals with overlapping emission peak doublets. The width of a signal doublet correlates to the time that it takes for the leading edge of a droplet to enter the first excitation regions to the time the trailing edge of the droplet to leave the final excitation region, a distance of ~500 &#181;m in the device we describe. In order to maintain distinctness of adjacent detected droplets, signal doublets need to be spaced by a low signal region at least as wide as the signal doublet. For the configuration described here, 80 &#181;m droplets are spaced at least 1 mm apart.
Although multi-color detection with a single photodetector allows for significant savings in terms of cost and complexity compared to standard techniques, there is some loss of robustness because emitted light is not filtered by wavelength. This can be problematic when detecting droplets that contain multiple fluorophores that excite at the same wavelengths, because the fluorescence source is indistinguishable. These limitations can be overcome by performing experiments with nonoverlapping excitation profiles, or by designing experiments where spectral bleed over does not occur into a &#34;channel&#34; where a sensitive assay is being performed. This is compatible with many droplet microfluidic detection applications, where a bright background dye is used to indicate the presence of a droplet, and a second fluorophore is used to report the result of a molecular biology assay 10,11.
Compared to standard techniques, our approach has two main advantages: 1) spatially registered excitation regions eliminate the need for complicated light filtering, and 2) use of fiber optics removes the needs for a microscope and optical hardware to direct light. Because we address common technical concerns with microfluidic detection systems, previous investigators have developed methods to address similar concerns. For instance, Martini et al. frequency-encode multicolor fluorescence recorded by a single detector by projecting an excitation source through uniquely patterned &#34;bar code&#34; filters 12. Our use of an optical fiber array allows the utilization of a similar temporal encoding scheme using standard, off the shelf components. The literature shows optical numerous optical coupling techniques that are more controlled that the simple fiber insertion that we describe. Bliss et al. use liquid filled optical wave guides for the carefully controlled delivery and collection of light from specific microchannel locations 13, Martinez Vasquez et al. use waveguides laser etched into fused silica substrates to deliver excitation light 14, and Vishnubhatla et al. are able to precisely fabricate fiber insertion channels using a combination of laser irradiation and chemical etching 15. Each of these techniques require additional steps beyond those used to fabricate the base fluidic architecture of a microfluidic chip. While these fabrication procedures may be necessary for highly sensitive detection applications, we have found that fiber insertion into a molded PDMS channel is adequate for most of the everyday applications in our lab. The detection system was able to detect FITC concentrations down to 100 pM in 80 &#181;m droplets, roughly equivalent to the detection of ~17,000 fluorophore molecules. This sensitivity is adequate for most detection applications in our laboratory currently using an epifluorescence-based system, the most common of which being PCR-activated cell sorting of assay positive droplets containing the equivalent of 106 fluorophore molecules 11. The detection sensitivity also compares favorably to recently reported sensitivities of other optical fiber based systems &#8212; for instance, it is 200x more sensitive than the detection limit of 20 nM of Alexa Fluor 488 reported in &#62; 100 &#181;m droplets7.
We have presented a compact and modular droplet microfluidic multi-color detection scheme as an alternative to more costly, complex, and bulky epifluorescence microscope based systems. The necessary detection components used here (lasers, fibers, fiber adapters, filter, and PMT) can be purchased for &#60;$10,000, making the multi-color droplet detection accessible to a great number of investigators, and allowing individual investigators to afford multiple detection stations in a laboratory. The system also remove some expertise-based barriers to entry, as a degree of familiarity is required to align free space optical systems in conjunction with an epifluorescence microscope. Additionally the small footprint of the detections setup makes it ideally suited for portable and diagnostic applications.

Droplet microfluidics provide a platform for high throughput biology by compartmentalizing experiments in a large number of aqueous droplets suspended in a carrier oil 1. Droplets have been used for applications as varied as single cell analysis 2, digital polymerase chain reaction (PCR) 3, and enzyme evolution 4. Fluorescent assays are the standard mode of detection for droplet microfluidics, as their bright signals and fast time response are compatible with detecting sub-nanoliter droplet volumes at kilohertz rates. Many applications require fluorescence detection for at least two colors simultaneously. For instance, our lab commonly performs PCR-activated droplet sorting experiments that use one detection channel for the result of an assay, and uses a secondary background dye to make assay-negative droplet countable 5.
Typical detection stations for droplet microfluidics are based on epifluorescence microscopes, and require complicated light manipulations schemes to introduce excitation light from free space lasers into microscope to be focused on the sample. After fluorescence is emitted from a droplet, the emitted fluoresced light is filtered so that each detection channel utilizes one photomultiplier tube (PMT) centered on a wavelength band. Epifluorescence microscope-based optical detection systems provide a barrier to entry due to their expense, complexity, and required maintenance. Optical fibers provide the means to construct a simplified and robust detection scheme, since fibers can be manually inserted into microfluidic devices, removing the need for mirror-based light routing, and allowing light paths to be interfaced using optical fiber connectors.
In this paper, we describe the assembly and validation of a compact and modular scheme to perform multicolor fluorescence detection by utilizing an array of optical fibers and a single photodetector 6. Optical fibers are coupled to individual lasers and are inserted normal to an L shaped flow channel at regular spatial offsets. A fluorescence collection fiber is oriented parallel to the excitation regions and is connected to a single PMT. Because a droplet passes through the laser beams at different times, data recorded by the PMT shows a temporal offset that allows the user to distinguish between the fluorescence emitted after the droplet is excited by each distinct laser beam. This temporal shift eliminates the need to separate emitted light to separate PMTs using a series of dichroic mirrors and bandpass filters. To validate the efficacy of the detector, we quantitate fluorescence in droplet populations encapsulating dyes of different color and concentration. The sensitivity of the system is investigated for single color fluorescein detection, and shows the ability to detect droplets with concentrations down to 0.1 nM, a 200x sensitivity improvement as compared to recent fiber based approaches reported in the literature 7.

<table>
	<tbody>
		<tr>
			<td>Photomasks</td>
			<td>CadArt Servcies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3&#34; silicon wafers, P type, virgin test grade</td>
			<td>University Wafers</td>
			<td>447</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3035</td>
			<td>Microchem</td>
			<td>Y311074</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050</td>
			<td>Microchem</td>
			<td>Y111072</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>Krayden</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringes</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>27 gaugue needles</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>PE 2 polyethylene tubing</td>
			<td>Scientific Commodities, Inc.</td>
			<td>B31695-PE/2</td>
			<td></td>
		</tr>
		<tr>
			<td>Novec 7500</td>
			<td>Fisher Scientific</td>
			<td>98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr>
			<td>Ionic Krytox Surfactant</td>
			<td></td>
			<td></td>
			<td>Synthesis instructions in ref #10</td>
		</tr>
		<tr>
			<td>Dextran- conjugated cascade blue dye</td>
			<td>Life Technologies</td>
			<td>D-1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein sodium salt</td>
			<td>Sigma</td>
			<td>28803</td>
			<td></td>
		</tr>
		<tr>
			<td>Quad bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-446/510/581/703-25</td>
			<td></td>
		</tr>
		<tr>
			<td>PMT</td>
			<td>Thorlabs</td>
			<td>PMM02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber port</td>
			<td>Thorlabs</td>
			<td>PAFA-X-4-A</td>
			<td></td>
		</tr>
		<tr>
			<td>lens tube</td>
			<td>Thorlabs</td>
			<td>SM1L05</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch cable with 200 um core / 225 um cladding optical fiber with one stripped end and one FC/PC connector</td>
			<td>Thorlabs</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch cable with 105 um core / 125 um cladding optical fiber with one stripped end and one FC/PC connector</td>
			<td>Thorlabs</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr>
			<td>125 um fiber stripping tool</td>
			<td>Thorlabs</td>
			<td>T08S13</td>
			<td></td>
		</tr>
		<tr>
			<td>225 um fiber stripping tool</td>
			<td>Thorlabs</td>
			<td>T10S13</td>
			<td></td>
		</tr>
		<tr>
			<td>laser fiber adapter</td>
			<td>OptoEngine</td>
			<td>FC/PC Adapter</td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm CW laser at 50 mW</td>
			<td>OptoEngine</td>
			<td>MDL-III-405</td>
			<td>Distributor for CNI lasers</td>
		</tr>
		<tr>
			<td>473 nm CW laser at 50 mW</td>
			<td>OptoEngine</td>
			<td>MLL-FN-473-50</td>
			<td></td>
		</tr>
	</tbody>
</table>

multicolor fluorescence detection for droplet microfluidics using optical fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Fabrication of a PDMS device that allows for the insertion of optical fibers requires a multistep photolithography procedure to create channels of varying height (Figure 1). First, an 80 &#181;m tall layer of SU-8 is spun onto a silicon wafer and patterned using a mask to create the fluid handling geometry. Next, an additional 40 &#181;m layer of SU-8 is spun onto the wafer, and patterned using a second mask to create features that will form 120 &#181;m tall laser fiber insertion channels. Last, 100 &#181;m more SU-8 is spun onto the wafer, and patterned to give 220 &#181;m tall detection fiber insertion channels. The master is developed and used to mold a PDMS device, which is in turn bonded to a glass slide. The final fabrication contains 80 &#181;m tall flow geometry, accessed via holes punched in the top of the device and 120 &#181;m and 220 &#181;m tall optical fiber channels accessed through the side of the device.
The geometry of the device used for detection is shown in Figure 2. Externally generated 80 &#181;m emulsions are re-injected into a drop reinjection port and spaced by spacer oil before they flow into a L shape detection channel. Two laser-coupled fibers are inserted into 120 &#181;m tall channels provide excitation at discrete spatial locations in the flow channel. Because the multimode fibers inserted into these channels have a 125 &#181;m external diameter and a 105 &#181;m core diameter, the entire cross section of the flow channel is illuminated by laser light. In our device the detection flow channel makes an abrupt 90 degree turn after the last laser excitation region to create close optical access for a 225 &#181;m OD fiber used to detect emitted fluorescence. The larger fiber is used because of its high numerical aperture (NA = 0.39), which is similar to a standard microscope objective.
A droplet flowing through the detection channel encounters discrete excitation regions in front of each of the laser-coupled optical fibers (Figure 3A). The single PMT coupled to the detection fiber records emitted fluorescence with a temporal shift the maps to the excitation source. For a droplet that contains dyes that excite at 473 nm (region &#34;1&#34; in Figure 3A) and at 405 nm (region &#34;2&#34; in Figure 3B), the resulting PMT signal contains a signal doublet with a peak separation that correlates with the time it takes a droplet to move from one excitation region to the next. By temporally encoding spectral data in this manner, the need for further light filtering and separate PMTs for each fluorophore color is eliminated. Figure 3B shows signal doublets for a train of 4 droplets with different dye combinations. The first peak in the double corresponds to the fluorescence emitted after excitation by the 473 nm laser in region &#34;1&#34; and the second peak corresponds to fluorescence emitted after excitation by the 405 nm laser in region &#34;2&#34;.
The efficacy of the detection scheme was tested by detecting a mixed emulsion of droplets containing dyes that excite at 405 nm (dextran-conjugated blue, CB) and at 473 nm (fluorescein, FITC). Droplets were flowed through the detection regions at roughly 50 Hz and data was recorded for 60 sec, then post-processed with a computing program script. The data is plotted in Figure 4 and shows clear separation between the four re-injected droplet types. Because the detection system does not differentiate between different colors of emitted light, each droplet is described by its maximum total fluorescence after being excited in region &#34;1&#34; (peak 1) and in region 2 (peak 2).
The dynamic range and absolute sensitivity of the detector is investigated by turning off the 405 nm laser and measuring the fluorescence of an emulsion containing fluorescein-only droplets (Figure 5). The data shows the ability to detect dye concentration that range from 0.1 to 100 nM FITC on a single detector with the same detection settings.
<img alt="Figure 1" src="/files/ftp_upload/54010/54010fig1.jpg" /> 
Figure 1. Multi-layer photolithography. The microfluidic device is fabricated by creating a master with three different feature heights. First, a 80 &#181;m tall layer of SU-8 is spun on and patterned using a mask for the fluid channels. Next, 40 &#181;m more SU-8 is spun on and 120 &#181;m tall features are patterned with a 2nd mask through both of the SU-8 layers to yield geometry to accommodate 125 &#181;m tall optical fibers. After this, 100 &#181;m more SU-8 is spun on and patterned with a 3rd mask to yield 225 &#181;m tall features. After developing, PDMS molding, and plasma bonding to glass, the resultant device contains large channels accessible from the side for fiber insertion, in addition to standard enclosed fluid channels. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54010/54010fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54010/54010fig2.jpg" /> 
Figure 2. Device design and optical layout. The fluidic portions of the device are comprised of a droplet reinjector and an oil spacer, coupled to an L-shaped channel. Fibers coupled to individual lasers are inserted normal to the direction of flow. A detection fiber is inserted perpendicular to the laser fibers in order to collect fluorescent light. This light is filtered through a bandpass filter, before being detected by a single PMT. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54010/54010fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54010/54010fig3.jpg" /> 
Figure 3. Temporal encoding of multicolor information. (A) Droplets flow through excitation regions &#34;1&#34; and &#34;2&#34; at different times, providing time shift that allows the identification of the emitted light. (B) Data from a train of 4 different droplets flowing through the detection channel. The first peak of each doublet corresponds to the fluorescence emitted after excitation in region &#34;1&#34; and the second peak in each double corresponds to the fluorescence emitted after excitation in region &#34;2&#34;. The height of droplet peaks are proportional the amount of dye excited at a given wavelength, except when spectral bleed occurs due to the use of dyes with overlapping excitation spectra. The width of the peaks are determined by the amount of time that it takes a droplet to traverse an excitation region. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54010/54010fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54010/54010fig4.jpg" /> 
Figure 4. Multicolor detection of a mixed droplet population. Scatter plot showing maximum droplet intensities for a mixed droplet population after excitation and emission by a 473 nm laser (peak 1) and a 405 nm laser (peak 2). The droplets contain combinations of fluorescein (FITC) and cascade blue (CB) at nM concentrations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54010/54010fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54010/54010fig5.jpg" /> 
Figure 5. Single color detection on highly varied droplet population. Histogram of emissions by a mixed droplet population containing 0.1-100 nM of fluorescein after excitation by a 473 nm laser. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54010/54010fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers at JoVE.com

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

multicolor-fluorescence-detection-for-droplet-microfluidics-using

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.4K Views.  University of California, San Francisco. Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Video: Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...