Name: the use of ex vivo whole-organ imaging and quantitative tissue histology to determine the bio-distribution of fluorescently labeled molecules
Uploaded: 2016-12-24T15:00:00
Description: Watch this Scientific Journal Video about The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules at JoVE.com

Partial salary support for GLB is provided by NIH grant R01HL121527. JWDM is supported by the Currier Fellowship in Neurology.

NOTE: All animal use in this protocol was approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.
1. Treatment of Animals and Tissues
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	<li>Administration and Sacrifice
	<ol>
		<li>Anesthetize the animals using 3% isoflurane for 5 min and verify the depth of anesthesia by observing the absence of the toe-pinch reflex. Apply veterinary ointment to the eyes to prevent dryness while under anesthesia.</li>
		<li>Weigh the animal and pipett...

<ol>
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While ex vivo whole-organ imaging is generally straightforward, the adherence to some basic concepts and techniques can improve the accuracy of bio-distribution measurements. Short wavelengths of light experience a high degree of scattering and absorbance in most tissues, which significantly impacts the utility of the short-wavelength fluorophores. These fluorophores have limited application in deep-tissue studies, but they have been used effectively in experiments looking at surface tissues or into the eye. Con...

Fluorescent labeling is an easily utilized and effective method for determining the bio-distribution of compounds, using a range of equipment that is common to many laboratories. Fluorophores are widely available, relatively inexpensive, and come in a variety of wavelengths, such that multiple labeled molecules can be used simultaneously without interference. Most fluorophores have a range of chemistries for conjugation to different reactive groups on target compounds, and the process of conjugation is generally straightforward for most types of reactive sites. Additionally, the equipment required for the measurements of fluorescently labeled compounds are common in many labs. Fluorescent microscopes, imagers, and slide scanners can all be used in different circumstances, making the use of fluorescent labeling highly accessible. Fluorescent labels are frequently used to determine the bio-distribution and kinetics of compounds both in vivo and ex vivo with live imaging devices, such as the IVIS Spectrum Imager, and by quantitative tissue histology2,3.
The use of ex vivo whole-organ imaging using live-imaging devices has increased over time due to their ease of use and ability to quickly create an accurate comparison of the relative concentrations of labeled compounds without requiring the further processing of tissues4. However, while ex vivo whole-organ imaging can allow for easy analysis and comparison, it does not generate a quantitative measure of absolute compound concentrations within a tissue. This is due to light scattering effects within intact organs.&#160; Since light scattering (and, to a lesser extent, absorbance) varies by tissue size and density, whole-organ imaging can underestimate tissue levels in large or dense organs.&#160; Formulating appropriate standards for absolute concentration measurements is also difficult because one must mimic the thickness and density of each individual organ. On the other hand, whole-organ imaging is a rapid method of obtaining the relative tissue levels of an agent, and it is ideal for comparing the relative bio-distribution of multiple related molecules (such as in drug targeting studies). An alternative strategy is to utilize quantitative fluorescence histology, a technique derived from the method of quantitative autoradiography, to obtain absolute tissue levels of a test agent5,6. Rather than placing an entire animal or organ into an imagining device, quantitative tissue histology requires that each tissue be sliced, mounted on slides, scanned, and analyzed individually.&#160; Standards of the test agent are prepared and sliced at the same thickness as the organ samples. By cutting all organs and standards to the same thickness, variability due to light scattering or absorbance is eliminated, and tissue fluorescence intensity can be fit to the standard curve to determine absolute concentration. While this method, when done properly, is quantitative, it is also labor-intensive and easily mishandled. Given the more complex nature of quantitative histology and the significantly higher cost of labor when compared to whole-organ imaging, it becomes worthwhile to examine where each process is most practical to use when examining the bio-distribution of fluorescently labeled compounds. This protocol provides a detailed description of how these methods can be used together to efficiently compare the bio-distribution of rhodamine-labeled Elastin-like polypeptide (ELP), with or without the addition of the SynB1 or Tat cell-penetrating peptides, via the intranasal (IN) and intravenous (IV) administration routes.

<table border="0" cellpadding="0" cellspacing="0" width="819">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Reagents</td>
			<td style="width:99px;"></td>
			<td style="width:159px;"></td>
			<td style="width:332px;"></td>
		</tr>
		<tr height="77">
			<td height="77" style="height:77px;width:231px;">Maleimide derivitized fluorophors (e.g. tetramethylrhodamine-5-maleimide, AlexaFluor 633-C5-maleimide)</td>
			<td style="width:99px;">Thermo Fisher</td>
			<td style="width:159px;">T6027, A20342</td>
			<td>Thiol reactive fluorescent dyes for protein labeling</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:231px;">Phosphate Buffered Saline</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:159px;">1002243569</td>
			<td>PBS Buffer for rinsing</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:231px;">Optimal Cutting Temperature Compound</td>
			<td style="width:99px;">Tissue-Tek</td>
			<td style="width:159px;">4585</td>
			<td>Used for freezing and mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Equipment</td>
			<td style="width:99px;"></td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">IVIS Spectrum</td>
			<td style="width:99px;">Perkin Elmer</td>
			<td style="width:159px;"></td>
			<td>For ex vivo whole organ imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Cryomicrotome</td>
			<td style="width:99px;">Thermo</td>
			<td style="width:159px;"></td>
			<td>For cryosectioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fluorescence slide scanner</td>
			<td style="width:99px;">Perkin Elmer</td>
			<td style="width:159px;"></td>
			<td>For slide scanning</td>
		</tr>
	</tbody>
</table>

the use of ex vivo whole-organ imaging and quantitative tissue histology to determine the bio-distribution of fluorescently labeled molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The data below describe the delivery of three compounds: a drug-delivery vector known as ELP and two versions of ELP modified with cell-penetrating peptides (SynB1-ELP and Tat-ELP)9. All three compounds were labeled with tetramethylrhodamine-5-maleimide and delivered via two administration routes (IN and IV). The goal of these experiments was to determine which compound and administration route would result in the best penetration into the central nervous system (CNS)3.
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Watch this Scientific Journal Video about The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules at JoVE.com

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

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