A. Ozcan gratefully acknowledges the support of the Presidential Early Career Award for Scientists and Engineers (PECASE), Army Research Office (ARO) Young Investigator Award, National Science Foundation (NSF) CAREER Award, Office of Naval Research (ONR) Young Investigator Award and National Institutes of Health (NIH) Director's New Innovator Award DP2OD006427 from the Office of the Director, National Institutes of Health.

In this section, we introduce the experimental protocols for our cell-phone based wide-field fluorescence microscopy13 and opto-fluidic imaging cytometry platform14. We will use fluorescent beads and fluorescently labeled white blood cells to test these imaging platforms.
A. Preparation of Cell-phone Based Wide-field Fluorescent Microscope and Opto-fluidic Imaging Flow Cytometer
The cell-phone based wide-field fluorescent microscope or flow cytometer consists of two major parts: a camera phone and a compact opto-fluidic add-on attachment.
1. The Camera Phone
While the presented techniques are applicable to almost any camera phone, we have chosen Sony Erickson Aino as the base for these devices. This cell-phone has an ~ 8 MegaPixel RGB CMOS sensor installed on it and a built-in lens that has a focal length (f1) of ~ 4.65 mm.
2. Opto-fluidic Attachment for Wide-field Fluorescent Microscopy
The optical attachment is designed by Autodesk and is printed by a Dimension Elite 3-D printer using ABSplus thermoplastic material. In this printing process, model and support materials are heated in an extrusion head within the printer and are deposited layer by layer on a modeling base. When this step is completed, the supporting material can be dissolved, leaving a robust 3D model of the desired prototype Our optical attachment design consists of LEDs (center wavelength at ~ 470 nm, Digikey), a plastic filter (#NT54-46, Edmund Optics), a sample tray, and a plano-convex lens f2=15 mm (# NT45-302, Edmund Optics). All the LEDs and plastic filters can be easily changed based on the fluorophores' spectra. The steps for assembling this opto-fluidic attachment include:
<ol>
	<li>Place the lens into the attachment within its specific lens holder position.</li>
	<li>Place the plastic filter onto the filter tray and slide it into the attachment; or tape the plastic filter in front of the cell-phone camera lens.</li>
	<li>Insert the LED tray into the attachment.</li>
	<li>Place the sample glass slides into the sample tray. Slide the sample tray into the attachment. Face the LEDs toward the sample.</li>
	<li>Clip the attachment onto the cell-phone, such that the extra lens is directly in touch with the cell-phone camera lens.</li>
	<li>Use the switch on the attachment to turn on the LEDs.</li>
	<li>Image the sample of interest with the cell-phone camera unit using its "night mode".</li>
</ol>
3. Opto-fluidic Attachment for Fluorescent Imaging Cytometry
When there is a need to screen large volumes of liquid samples for the detection of rare events, optofluidic flow cytometry device could be preferred. We can modify our wide-field fluorescent microscope design and convert it into a flow cytometer, where a PDMS based microfluidic channel is used to continually deliver the liquid sample through the imaging volume. The optical attachment is also designed by Autodesk and printed by Dimension Elite 3-D printer. It also consists of LEDs (center wavelength at ~ 470 nm, Digikey), a plastic filter (#NT54-46, Edmund Optics), a sample tray, and an aspherical lens (f = 4.5 mm) (product # C230TME-A; Thorlab). The steps for assembling this opto-fluidic attachment include:
<ol>
	<li>Place the aspherical lens into the attachment.</li>
	<li>Place the plastic filter onto the filter tray and slide it into the attachment; or tape the plastic filter in front of the cellphone camera lens.</li>
	<li>Slide the microfluidic channel into the same opto-fluidic attachment.</li>
	<li>Clip the attachment onto the cell-phone such that the extra lens is directly in touch with the cell-phone camera lens.</li>
	<li>Use the switch on the attachment to turn on the LEDs.</li>
	<li>Connect the microfluidic channel to the syringe pump and deliver the liquid sample into the microfluidic device at a constant flow rate.</li>
	<li>Capture a movie of the fluorescent cells/particles flowing through the microfluidic channel using the video mode of the cell-phone camera.</li>
</ol>
B. Sample Preparation
4. Preparation of Fluorescent Micro-particle Samples
<ol>
	<li>Fluorescent beads with 10 &mu;m diameter (red beads: product #F8834 excitation/emission 580nm/605nm; green beads: product #F8836: excitation/emission 505nm/515nm) are purchased from Invitrogen (Carlsbad, CA).</li>
	<li>Mix 10 &mu;l of green fluorescent beads, 10 &mu;l of red fluorescent beads with 40 &mu;l of DI water.</li>
	<li>Place10 &mu;l of this bead mixture on a glass slide using a micropipette and put another glass slide on the top of it to make a sandwich structure.</li>
	<li>Insert this sandwich structure into the sample tray and slide it into the cell-phone attachment.</li>
</ol>
5. Preparation of Fluorescently Labeled White Blood Cells
<ol>
	<li>Take SYTO16 nucleic acid fluorescent labeling kit (#S7578, Life Technology) and phosphate buffered saline (PBS) out from the refrigerator and bring them to room temperature.</li>
	<li>Transfer 200 &mu;l whole blood sample from EDTA blood collection tube to 1.5 ml polystyrene tube (# 05-408-129, Fisher Scientific).</li>
	<li>Add 1 ml red blood cell lysing buffer (# R7757, Sigma-Aldrich) to the 200 &mu;l whole blood sample and mix thoroughly.</li>
	<li>After 5 min, centrifuge the lysed blood sample and remove the supernatant solution.</li>
	<li>Resuspend the white blood cell pellet into 200 &mu;l PBS buffer and gently mix them.</li>
	<li>Add 5 &mu;l 1 mM SYTO16 solution to the white blood cell sample. Wrap the sample with aluminum foil and incubate in dark environment for ~ 30 min.</li>
	<li>Centrifuge the sample again. Supernatant is removed and the labeled white blood cell pellet is re-suspended into PBS buffer.</li>
	<li>Place 5-10 &mu;l labeled white blood cell liquid sample to a cover slip, and place a second cover slip on the top of the sample.</li>
	<li>Insert the sandwiched sample slide into the sample tray and image it using the cell-phone fluorescent microscope.</li>
</ol>
Alternatively
<ol start="10">
	<li>Continually deliver the fluorescently labeled white blood cells through a microfluidic channel using an automated syringe pump, while also capturing a fluorescent microscopic movie of the flowing cells using the cell-phone camera in video mode. We should also emphasize that a portable battery powered syringe pump or even gravity force can be utilized to drive the flow through the microfluidic channel.</li>
</ol>

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Dr. Ozcan is the founder of a start-up company that aims to commercialize computational imaging and microscopy tools.

We have presented our recent results on cell-phone based wide-field fluorescent microscopy and opto-fluidic imaging flow cytometry using light-weight and compact opto-fluidic attachments to cell-phone cameras. Using this platform technology we imaged fluorescent objects including micro-particles and labeled white blood cells in whole blood samples. Therefore, this compact and cost-effective cell-phone based fluorescent imaging toolset might be useful for point-of-care diagnosis, especially in resource limited regions for...

Microscopy and flow-cytometry are widely used techniques1-12 in biomedical and scientific research as well as clinical diagnosis for counting and characterization of various cell types. However, conventional microscopes and flow-cytometry instruments are relatively complex and expensive, which limits their use to mainly well-established central laboratories. Recently we have developed a compact and lightweight fluorescent imaging cytometry and microscopy device integrated on a cell-phone,13,14 which shows promise to cost-effectively translate fluorescent microscopy, flow-cytometry and related micro-analysis techniques to resource-limited environments for various telemedicine applications impacting global health.
In the optofluidic flow-cytometry configuration (see e.g. Figure 1C and 1D), a custom-designed polydimethysiloxane (PDMS) based microfluidic channel is positioned in front of the cell-phone camera-unit, where light-emitting-diodes (LEDs) are butt-coupled to the edges of the channel. This microfluidic chip, together with the liquid sample inside, forms an opto-fluidic planar waveguide (composed of for example PDMS-liquid-PDMS) such that the excitation light is guided to uniformly pump the fluorescent labeled specimens inside the micro-channel. The fluorescence emission from these labeled objects, e.g. cells, is further imaged through an additional lens placed right after the cell-phone camera-unit and is mapped onto the cell-phone Complementary Metal-Oxide-Semiconductor (CMOS) image sensor. Since the fluorescent emission is collected perpendicular to the excitation light path, an inexpensive plastic absorption filter is sufficient to remove the scattered excitation light and can provide a decent dark-field background required for fluorescent imaging. Using a similar opto-fluidic design, we can also image the fluorescent objects in static mode (see Figure 1A and 1B), in which the fluorescent particles are sandwiched between two glass slides instead of flowing through a microfluidic channel and the fluorescent emission from these fluorescent particles are captured by the cell-phone CMOS image sensor for particle counting and characterization. Based on different application requirements, flow cytometry or wide-field fluorescent microscopy can be chosen. For example, cell-phone flow cytometry device could be especially useful for screening large volumes of liquid samples (e.g. a few ml) for the detection of rare cells or pathogens.
In this manuscript we review some of our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone-based micro-analysis, imaging cytometry and sensing platforms could provide various opportunities for telemedicine and point-of-care diagnostics, especially impacting our fight against global health challenges in resource limited regions of the world.

<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td>Company</td>
			<td>Catalog Number </td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell-phone</td>
			<td>Sony</td>
			<td>Sony Ericsson Aino</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plano-convex lens</td>
			<td>Edmund Optics</td>
			<td># NT45-302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspherical lens</td>
			<td>Thorlab</td>
			<td># C230TME-A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Edmund Optics</td>
			<td>#NT54-46</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue LED</td>
			<td>Digikey</td>
			<td>#365-1201-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Battery</td>
			<td>Digikey</td>
			<td>#P032-ND</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polystyrene tube</td>
			<td>Fisher Scientific</td>
			<td>#05-408-129</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYLGARD 184 SILICONE ELASTOMER KIT</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Red fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8834</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Green fluorescent beads (10 &mu;m)</td>
			<td>Life Technologies</td>
			<td>#F8836</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SYTO16 nucleic acid fluorescent labeling</td>
			<td>Life Technologies</td>
			<td># S7578</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

With our opto-fluidic pumping/excitation scheme (Figures 1C and 1D), fluorescently labeled cells can be continuously delivered into the microfluidic channel using a syringe pump while the cell-phone camera records a time-lapse fluorescent microscopic movie of the flowing cells. These fluorescent movies can then be rapidly analyzed using contour-detection and tracking algorithms14,15 to automatically determine the absolute number and the density of the cells flowing through the...

Watch this Scientific Journal Video about Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone at JoVE.com

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

wide-field-fluorescent-microscopy-fluorescent-imaging-flow-cytometry

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Bioengineering

23.5K Views.  University of California, Los Angeles. We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.