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.
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.
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. 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. 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. 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.
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
2. Whole-organ Ex Vivo Fluorescence Imaging
3. Standards and Image Processing
4. Quantitative Tissue Histology
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.
Figure 1A displays representative ex vivo whole-organ images of the organs from the treated animals. The heat map overlay shows the fluorescence levels in radiant efficiency within the tissues. By drawing ROIs around the individual tissues and by plotting those values on their corresponding standard curve, standardized fluorescence values illustrated in Figure 1B are generated. The standardized fluorescence values can then be used for accurate comparisons of the relative concentrations of the different compounds. These data accurately describe both the effect of ELP modification on the penetration into specific tissues, as well as the effect of administration route on each compound. With these results, it is immediately clear that ELP delivery via IN administration is the most effective combination to reach the CNS.
While these results help determine the best penetrating compound and delivery method, they do not describe the actual concentration or regional localization of compounds within the CNS. Quantitative histology is therefore ideal to complete the description of the bio-distribution of these compounds, as it allows for quantitative measurements of the selected brain regions. Figure 2A displays a representative slice from the ELP IN-treated animals, and Figure 2B describes the quantification of the ROIs separating the olfactory bulbs, hemispheres, and cerebellum. From these data, it is clear that while ELP administration by the IN route may result in high CNS concentrations, it does this primarily in the olfactory bulbs and cerebellum. Conversely, ELP delivered IV has lower overall concentrations, but it is equally distributed throughout the brain. Depending on the target of these administered compounds and the potential for effects in other organs, either IN or IV administration may be the better choice. This could not be concluded without the addition of the quantitative histology. Technically, quantitative histology could have been used in every tissue of every group, but by utilizing both methods together, a great deal of time and labor was saved while still resulting in the same conclusion.
Figure 1: Ex Vivo Whole-organ Fluorescence Measurements. A) Representative images from the ELP group. The heat map scale was set as radiant efficiency. B) Average radiant efficiency values obtained from the creation of ROIs around the individual tissues that were analyzed on a standard curve in order to generate the standardized fluorescence values used for the comparison of bio-distributions. Error bars represent SEM. Scale bar = 5 mm. (Modified from McGowan, et al.; Drug Design, Development, and Therapy; 2016) Please click here to view a larger version of this figure.
Figure 2: Quantitative Tissue Histology Measurements. A) Representative image of ELP IN- or IV-treated animals created by slicing brains 20 μm thick, mounting the slices on slides, and then scanning those slides using a fluorescence slide scanner. B) Average values obtained from the creation of ROIs around the olfactory bulb, hemispheres, and cerebellum, measured as the mean grey value and fit to a standard curve to generate μg/mL concentration measurements. Error bars represent SEM. Scale bar = 5 mm. (Modified from McGowan, et al.; Drug Design, Development, and Therapy; 2016) Please click here to view a larger version of this figure.
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. Conversely, long wavelengths of light undergo significantly less scattering and absorbance, resulting in greater penetration and more accurate results with thicker tissues10. Fluorophores which fall in the far-red and near infrared (NIR) range (excitation ∼ 600 nm) of the spectrum are commonly chosen for whole animals or larger tissues. For experiments using these long-wavelength fluorophores, the auto-fluorescence of particular tissues or materials is likely a key consideration. For example, the bile inside the gallbladder of rodents is highly auto-fluorescent at wavelengths in the 500 – 600 nm range. Because this can greatly skew liver values, it is often best to remove the gallbladder prior to imaging. Alternatively, if the intact liver and gall bladder are required, choosing a fluorophore with an excitation wavelength at the very far end of the spectrum can limit the influence of the bile auto-fluorescence. Additionally, many of the common rodent chows contain unpurified alfalfa, which fluoresces highly throughout the mid- and far-ranges of the spectrum. If the chosen labels fall within those wavelengths and the digestive tract is to be imaged, a change in diet is likely the best option.
While live imaging systems are designed to accurately measure thick tissues, with non-homogenous and particularly thick tissues (e.g., whole rat livers), more accurate measurements can often be obtained by spreading the tissue out, such that the lobes of the liver overlap as little as possible. Also, in cases where tissues have a highly skewed distribution of labeled molecules, imaging from multiple sides of the tissue and averaging those measurements may increase accuracy. Ultimately, the types of tissue to be imaged, the auto-fluorescence of the materials associated with those tissues, and the potential distribution of the administered labeled molecules should be carefully considered in order to choose a fluorophore with the appropriate excitation and emission wavelengths that limit interference as much as possible.
When performing quantitative tissue histology, there are several important details to keep in mind. In order for the results to be accurate, slices must be taken throughout the whole tissue or region to be analyzed. This means that these tissues will not be available for other processes. It may be possible to fix and stain after scanning, but it is typically best to allow the slices to dry slightly before putting them into the scanner, which could affect future staining. Likewise, the scanning will cause some level of photo-bleaching, reducing the potential signal in subsequent processes. For downstream microscopy applications, superior sections are typically achieved by fixing the tissues and equilibrating them in a sucrose solution prior to sectioning. However, this process is best avoided in the above quantitative histology protocol due to the effects of fixation and/or long sucrose soaks on fluorescence intensity. Also, depending on the amount of signal present, slices may experience photo-bleaching if exposed to even low levels of ambient light. Keeping the slides cold and away from light will significantly improve the accuracy of the resulting measurements. Overall, careful handling and preparation can drastically improve the final outcome.
There are a variety of other methods employed for studying the bio-distribution and pharmacokinetics of administered macromolecules that vary widely in technical difficulty, throughput, accuracy, and the equipment required11. Quantitative immunoassays and bioactivity assays can both be technically complicated and require antibodies or reagents specific to the administered molecules, adding significantly to the cost and labor. Multiple methods based on radioisotope labeling have been used in the past, with varying accuracy, but the integration of radioisotopes to macromolecules, as well as the collection of data itself, can require significant preparation and can often render tissues completely unusable for further analyses12. Finally, other methods shown to be effective, including positron emission tomography and mass spectrometry, are typically precluded simply due to a lack of the required equipment. The protocol described above uses two methods in conjunction for efficiently determining the bio-distribution of labeled molecules in a quick, cost-effective, accurate, and easily accessible manner due to the regular availably of the required equipment.
This method was used to generate the data above, identifying the best vector and administration route for the delivery of labeled molecules to the CNS. From the ex vivo data, ELP administered IN and ELP administered IV were identified for further characterization via quantitative tissue histology. Quantitative tissue histology then provided the concentrations of labeled molecules in specific regions of the CNS, allowing the final conclusions to be drawn. The two methods together represent a fast and effective way to perform a detailed examination of the bio-distribution of multiple specific compounds.
The authors have nothing to disclose.
Partial salary support for GLB is provided by NIH grant R01HL121527. JWDM is supported by the Currier Fellowship in Neurology.
Reagents | |||
Maleimide derivitized fluorophors (e.g. tetramethylrhodamine-5-maleimide, AlexaFluor 633-C5-maleimide) | Thermo Fisher | T6027, A20342 | Thiol reactive fluorescent dyes for protein labeling |
Phosphate Buffered Saline | Sigma | 1002243569 | PBS Buffer for rinsing |
Optimal Cutting Temperature Compound | Tissue-Tek | 4585 | Used for freezing and mounting |
Equipment | |||
IVIS Spectrum | Perkin Elmer | For ex vivo whole organ imaging | |
Cryomicrotome | Thermo | For cryosectioning | |
Fluorescence slide scanner | Perkin Elmer | For slide scanning |