The authors would like to extend their gratitude for financial support from the Pasteur Foundation of New York (to J.D., C.S., C.S.), the EU-FP7 Program "Automation" (to S.L.S.), the Institut Carnot Program 11 (to J.D., A.H., A.R., R.T., S.L.S.) and Project IMNOS (to R.T., S.L.S.), the Conny-Maeve Charitable Foundation (S.L.S.), the European Masters in Molecular Imaging (to I.T.), the Region Ile de France programs MODEXA (S.L.S.), SESAME (S.L.S.) and DimMalInf (S.L.S., R.T.), the ANR Program Grandes Investissement de l'avenir Infrastructures Nationales en Biologie-Sant&#233;: France LifebioImaging (FLI) France Life Imaging (R.T., S.L.S.), France Bioimaging (J.D., S.L.S) and the Institut Pasteur, Paris. Further, the authors would like to thank, Jos&#233; Bengoechea and Herbert Schweizer for reagents. Further, The authors would like to thank Cindy Fevre who generated the antibodies.

1. Reagents
<ol>
	<li>Purchase or develop luminescent bacteria and appropriate culture media such as modified Escherichia coli expressing the luxABCDE21, Vibrio fischeri, Photobacterium sp. Klebsiella pneumoniae22.</li>
	<li>Prepare physiological saline (0.9% NaCl) and culture media solutions according to standard recipes. E. coli and K. pneumoniae were grown in Luria Bertani (LB) at 37 &#176;C, and V. fischeri and Photobacterium sp in LB supplemented with 0.5 M NaCl at 22 &#176;C.</li>
	<li>Purchase or acquire fluorescent probes such as Q-tracker 705 (40 nm diameter, referred to as QD705), Q-tracker 800 (40 nm diameter, referred to as QD800), fluorescent (40 nm diameter) and non-fluorescent polystyrene microspheres (48 nm diameter), and conventional fluorescent dyes.</li>
	<li>Prepare the necessary reagents for bacterial labeling.</li>
	<li>Ensure access to a whole animal bioluminescence imager, such as an IVIS Spectrum, that is capable of detecting signal under a wide range of emission wavelengths and exposure times. A plate reader capable of bioluminescence measurements will suffice for most of the experiments described here.</li>
</ol>
2. Basic Recapitulation of FUEL
<ol>
	<li>Starting from individual colonies on standard culture plates, start overnight cultures of luminescent bacteria. Here, V. fischeri were used.</li>
	<li>The day of experimentation, initiate fresh subcultures and allow them to progress until an OD600 of 1-1.5 is achieved. In order to produce comparable results it is best to use bacteria in similar growth states in which they produce intense signals. This can be assured by keeping the OD600 constant.</li>
	<li>Combine aliquots of 100 &#956;l from each with either 5&#160;&#956;l of QD705 or physiological saline (PS), and then add to 895 &#956;l of PS into standard spectroscopic cuvettes. Place the filled cuvettes into the IVIS Spectrum and take the measurements under the appropriate filter sets. Here, the 710-730 nm emission filter was used.</li>
</ol>
3. FUEL Over Varying Distances
<ol>
	<li>Fill two reduced volume (1 ml) plastic photometric cuvettes with either 50 &#956;l of QD705 or 97.2 &#956;l of non-fluorescent 48 nm polystyrene microspheres in a total volume of 1 ml PS. This ensures similar solid surface area per total volume between the two entities.</li>
	<li>Prepare a light source cuvette by encasing a third cuvette with standard black tape or some other opaque material capable of blocking light. Carefully prepare two identical optical windows on opposite sides of the cuvette.</li>
	<li>Place the previously filled cuvettes directly onto either side of the light source cuvette. Add a 1 ml aliquot of V. fischeri or other culture (i.e. luminescent E. coli or Photobacterium sp) from a fresh subculture into the light source cuvette and cover it with an opaque material, such as black paper to reduce any light contamination.</li>
	<li>Visualize the three cuvettes under the appropriate emission filters such as the Total Light and 710-730 nm emission filters, with exposure times of 10, 30, and 30 sec, respectively.</li>
	<li>Reposition both external cuvettes at equivalent further distances from the central cuvette, and then visualize again. Repeat until a final face-to-face (central to reduced volume cuvette) distance of 3cm is achieved. At each step, acquire a fluorescence image (450-480 nm excitation, 710-730 nm emission) to validate the QD705 location.</li>
</ol>
4. Investigating Potential FUEL Pairs
<ol>
	<li>Fill two reduced volume cuvettes with 1 ml solutions containing either a fluorophore or a fluorescent nanoparticle of interest in one, and PS or PS with non-fluorescent nanoparticles as the negative control in the other. In the work demonstrated here, a variety of potential commercially available FUEL fluorophores and fluorescent nanoparticles were investigated as outlined in Table 1.</li>
	<li>Add a 1 ml aliquot of fresh V. fischeri or other luminescent bacteria to a blacked-out third cuvette containing two physically equal optical windows situated on opposite sides. Cover the cuvette with an opaque substance such as a piece of black paper.</li>
	<li>At a short but equal distance place the two reduced volume cuvettes on either side of the blacked-out cuvette and visualize under the appropriate filters. Here, a distance of 0.7 cm was used. Repeat with the other fluorophores.</li>
</ol>
5. Targeted FUEL
<ol>
	<li>Prepare biotinylated antibodies specific to the luminescent bacteria. For example, here primary antibodies specific to K. pneumoniae (&#945;-Kp) were biotinylated using a solution containing EZ-Link Sulfo-NHS-LC-Biotin.</li>
	<li>Remove excess reagent from the antibodies using desalting columns under centrifugation (1,500 x g) for 2 min.</li>
	<li>Use a standard Bradford assay to determine the protein concentration and determine the degree of antibody biotinylation by HABA assay.</li>
	<li>Obtain the &#945;-Kp-biot biotinylated antibody (Ab) batches by mixing 100 &#956;l (10 mM, dissolved in 1x PBS) with 40 &#956;l &#945;-Kp antibody, resulting in a final degree of substitution of 16 biotin residues per Ab and a final protein concentration of 10 mg ml-1.</li>
	<li>Using multiple overnight replicate cultures, target the washed bacteria with the aforementioned antibodies following the appropriate protocol.
	<ol>
		<li>Specifically, wash the K. pneumoniae in 1x PBS, and resuspend in 1x PBS to an OD of 4 (ca. 4 x 108 CFU ml-1 OD-1).</li>
		<li>For each replicate, incubate 100 &#956;l PBS, &#945;-Kp antibodies (10 &#956;l &#945;-Kp-biotB or 10 &#956;l PBS for the control) and 100 &#956;l cells for 90 min at 30 &#176;C. Wash the cells three times in 1x PBS, and resuspend in 196 &#956;l PBS.</li>
	</ol>
	</li>
	<li>Label the cells with the QD705 streptavidin conjugate and divide them into two equivalent volumes.</li>
	<li>Wash one solution three times and then resuspend it into the appropriate volume.</li>
	<li>Distribute 100 &#956;l of the washed and unwashed solutions into individual wells of a black 96-well plate. Measure the resulting luminescence under the Total Light, 490-510 nm, and 710-730 nm filters. Fluorophore concentration can be determined using 450-480 nm excitation and 710-730 nm emission.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging:+progress+and+applications.">Bioluminescence imaging: progress and applications.</a> Trends in Biotechnology. 29, 624-633 (2011).</li><li>Maywood, E. 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The authors declare competing financial interests. J.D., S.B., K.L.R. and S.L.S. contributed to European Patent EP10290158.4 and World Intellectual Property Organization Patent Application WO/2011/117847.

The fundamental demonstration of FUEL can be achieved simply by mixing luminescent bacteria with fluorescent nanoparticles or QDs. The two entities will be physically separated and remain beyond any efficient RET distance. More difficult is the FUEL signal optimization both in vitro and in vivo. Under in vitro conditions, both with and without an optical absorber present, usually the addition of excess fluorophore will be sufficient to maximize the FUEL response. However, at high concentrations phenomena such as static or collisional quenching can lead to a loss of fluorescent signal. Performing a dilution series by independently varying the concentration of the luminescent source and the fluorophore will help to optimize the desired concentrations. The establishment and optimization of FUEL under in vivo concentrations is much more difficult and needs to be addressed on a case-by-case basis. It can be difficult to create a condition where the fluorescent entity can be accessed optically by the luminescent source. As such, beginning with direct co-injections of the two moieties can provide information regarding the success of FUEL under optimal conditions.
Standard protocols exist for labeling bacteria and eukaryotic cells with fluorescent entities such as the Alexa series and QDs. Often this requires surface functionalization or activation with antibodies, which can lead to unwanted effects like reduced cell viability or altered metabolic activity. To overcome this, it is important to determine the optimal amount of antibody or activation agent needed that minimizes cellular perturbations while maximizing the fluorescent labeling. The use of QDs is advantageous because of their characteristically broad excitation spectra, narrow and tunable emission spectra, and the possibility of a large Stokes shift. However, QDs can be cytotoxic and may not be desirable in some cases.
FUEL is a phenomenon that is present in many BRET experiments13 and is applicable to a variety of luminescent and fluorescent sources. Until now, the photons resulting from FUEL were considered the product of non-specific interactions or an unfortunate background signal resulting from poorly designed BRET experiments. It is only with the type of experiments demonstrated here that we were able to identify the utility of this unwanted signal. In the shown examples, the luminescent bacteria act as a diffuse excitation source capable of eliciting a standard fluorescent response from a wide variety of fluorescent entities. Furthermore, due to the substantial working distance, it is safe to conclude that while FUEL can be constructed without the occurrence of BRET, in general BRET cannot be observed without a contribution from FUEL. Importantly, due to the lack of a targeting requirement, FUEL can be used to cover the spatial gap that exists between BRET and conventional whole-animal imaging techniques.

The genetic modification of organisms, such as viruses1,2, bacteria3, or small mammals4 to either induce or constitutively express bioluminescence, has been highly successful and widely demonstrated5-7. Bioluminescence, an in vivo chemiluminescent reaction involving naturally occurring reagents, has the advantage of producing light without the need for an external light source. As such, bioluminescent imaging does not suffer from the common drawbacks of auto- and non-specific signal found from fluorescence imaging8. Consequently, bioluminescence has a significant signal-to-noise ratio since any detected signal originates solely from the intended source. While many models have exploited the lux operon from Photorhabdus luminescens (emission maximum centered between 480 and 490 nm) for in vitro and in vivo applications9, its use in small mammals has been problematic due to the very nature of the imaging conditions; the pervading existence of optical absorbers, such as hemoglobin, and scattering agents, such as tissue and bone, strongly affect blue to yellow wavelengths3. The expression of an engineered firefly luciferase (emission maximum at 617nm) has been recently developed and incorporated, providing a tool that greatly overcomes optical absorption10, but is still subject to scattering effects.
In response, there have been multiple attempts to red-shift the emitted signal into the desired optical window of 650-900 nm, a region of minimized absorption and scatter, using bioluminescence resonance energy transfer (BRET)11-13. As a tool to enhance signal detection, BRET, which uses a bioluminescent source as the donor and an added fluorophore as the acceptor, has found limited success. As a seminal example of this phenomenon, &#34;self-illuminating quantum dots&#34; (SIQDs)14 consist of modified Renilla reniformis luciferases bound to the external polymer-lysine layer of commercially available quantum dots (QDs). Upon substrate addition, the resulting bioluminescent reaction induces fluorescence emission from the QDs, generating a significant production of red photons. However, these SIQDs have limited applicability to in vivo visualization of physiologically relevant events. This limited applicability is likely due to the difficulty of linking the dual probe to the organ, cell or gene of interest, since the SIQDs cannot be genetically encoded and therefore would require a secondary modification of the polymer shell. To improve their applicability, alternative SIQDs, where the luciferases are bound directly to the luminescent core, have recently been employed15. Building off of the SIQD concept, a more applicable BRET system was achieved by attaching Cypridina luciferase to an indocyanine dye16, which was capable of specifically targeting tumors in mice while producing a substantial red shift from 460 nm to 675 nm. To undergo non-radiative energy transfer, BRET follows the same primary constraints as its fluorescent counterpart: there must be a strong spectral overlap between the donor emission and the acceptor excitation spectra and the working distance between the two moieties must be on the order of the F&#246;rster radius (5-14 nm depending on the donor-acceptor pair, with an effective maximum distance of twice the F&#246;rster radius17). This distance dependence greatly limits the types of events that can be observed using BRET as a means to enhance detection.
Recently a new approach was identified and demonstrated under both in vitro and in vivo conditions. Building off the foundation of BRET, Fluorescence by Unbound Excitation from Luminescence (FUEL)18,19 also requires a strong spectral overlap between the luminescent and fluorescent components. However, unlike BRET, FUEL is a completely radiative process whereby the emitted photon from the luminescent source is absorbed by an optically accessible fluorophore, which subsequently emits a red-shifted photon according to the fluorophore quantum yield. Akin to BRET, this approach can also be used to overcome the constraints of imaging in the presence of optical absorbers. The resulting red shift provides an overall increase and specificity in the detected signal due to a decrease in attenuation and a reduction of optical scattering effects. FUEL has been reported to occur between bioluminescent Escherichia coli expressing the lux operon and QDs18,19. While experimentally similar to the SIQDs, a fundamental difference exists: in FUEL, it is not necessary for the luminescent source to be physically bound to the fluorophore, which allows for genetic encoding of the luminescent probe. Due to the successful detection of FUEL between luminescent bacteria and QDs, it is possible that this technique could be applied to both superficial (skin) and deep tissue (lung, liver) infections such as Staphylococcus aureus and Klebsiellia pneumoniae.
Since the report of its experimental significance, FUEL has evolved to include a robust mathematical model20 that can be used to predict acceptable luminescent and fluorescent pairs, and its applications have expanded to include use in identification of photophysical characteristics such as quantum yield. We describe below some of the basic techniques of FUEL. First, we show evidence for this phenomenon over both short (&#181;m) and long (cm) working distances, which fundamentally distinguishes FUEL from BRET. Second, we expand upon the possible FUEL pairs by examining a wide variety of fluorophores and fluorescent nanoparticles. Third, FUEL applications are investigated by comparing targeted and non-targeted FUEL pairs.

<table border="0" cellpadding="0" cellspacing="0" style="width:764px;" width="764">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Name of Material/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Escherichia coli expressing the luxABCDE operon&#160;</td>
			<td style="width:119px;"></td>
			<td>kindly provided by Jos&#233; A. Bengoechea with permission from Herbert P. Schweizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Klebsiella pneumoniae 52145&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td>52145 is a serotype K2 reference strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Bertani (LB)&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:119px;"></td>
			<td>standard growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q-Tracker 705&#160;</td>
			<td style="width:175px;">Life Sciences</td>
			<td>Q21061MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Q-Tracker 800</td>
			<td style="width:175px;">Life Sciences</td>
			<td>Q21071MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa 555</td>
			<td style="width:175px;">Life Sciences</td>
			<td style="width:119px;">S21381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa 568</td>
			<td style="width:175px;">Life Sciences</td>
			<td style="width:119px;">S11226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa 633</td>
			<td style="width:175px;">Life Sciences</td>
			<td style="width:119px;">S21375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa 700</td>
			<td style="width:175px;">Life Sciences</td>
			<td style="width:119px;">S21383</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Non-fluorescent microspheres</td>
			<td style="width:175px;">Polysciences, Inc</td>
			<td align="right" style="width:119px;">15913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Pink microspheres</td>
			<td style="width:175px;">Life Sciences</td>
			<td>F8887</td>
			<td>40nm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">yellow microspheres</td>
			<td style="width:175px;">Life Sciences</td>
			<td style="width:119px;">F8888</td>
			<td>40nm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Ivis Spectrum</td>
			<td style="width:175px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td style="width:175px;">Thermo Scientific Pierce</td>
			<td align="right">21425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeba Spin Desalting Columns 7K MWCO</td>
			<td style="width:175px;">Thermo Scientific Pierce</td>
			<td align="right">21425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HABA assay kit&#160;</td>
			<td style="width:175px;">Thermo Scientific Pierce</td>
			<td align="right">28005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Bradford assay</td>
			<td style="width:175px;">Bio-Rad</td>
			<td>500-0201</td>
			<td></td>
		</tr>
	</tbody>
</table>

a step beyond bret: fluorescence by unbound excitation from luminescence (fuel)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

Watch this Scientific Journal Video about A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL at JoVE.com

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

a-step-beyond-bret-fluorescence-unbound-excitation-from-luminescence

Research

JoVE Journal

Bioengineering

11.5K Views.  Institut Pasteur. Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.