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
<ol>
	<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 pipette a 50 mg/kg dose of either rhodamine-labeled ELP, SynB1-ELP, or Tat-ELP into a 1.5-mL tube. Save 100 &#181;L of the administered labeled compounds for formulation of standard curves in later steps. 
		Note: It is important that standards be made from the same batch of labeled protein administered to the animals in order to avoid differences due to variability in fluorescent labeling efficiency. In order to properly remove background fluorescence from later fluorescence measurements, an untreated animal must be sacrificed using the same methods/reagents as the treated animals.</li>
		<li>Administer an IV injection as previously described1 using either the tail vein or the femoral vein (which is accessed via a 1-cm incision in the groin). For analgesia, if making a groin incision, administer carprofen subcutaneously at a dose of 5 mg/kg, and apply sensorcaine to the incision site before closure with a wound clip. To administer via the intranasal (IN) route, place the animal in the supine position.</li>
		<li>Using a 2-&#181;L pipette, draw up a 1-&#181;L drop of labeled compound. Briefly slide the animal&#8217;s head from the anesthesia collar to allow access to the nares.</li>
		<li>Place the tip of the pipette to the opening of a single nare and slowly depress the plunger, allowing the droplet to run down the nare into the nasal cavity. Repeat every 1 min, alternating nares every drop until the full dose is administered. 
		Note: The maximum volume administered via IN should not exceed 10 - 15 &#181;L for mice.</li>
		<li>Leave the animals in the supine position under anesthesia for 10 min after the final administration before moving them each into an individual cage. 
		Note: Do not leave animals unattended until they have regained sufficient consciousness to maintain sternal recumbency.</li>
		<li>1 h after administration, re-anesthetize the animals as before and sacrifice them via transcardial perfusion using PBS (210.0 mg/L KH2PO4, 9,000.0 mg/L NaCl, 726 mg/L Na2HPO4.7H2O) at 10 mL/min for a total of 20 mL. 
		Note: the perfusion of the animals at sacrifice to remove all blood from the tissues is required to determine the extravascular concentrations of the labeled compounds. Perfusion reagents can result in changes to background fluorescence and should be kept consistent. The method of sacrifice should be consistent within groups. Different methods of sacrifice can result in varying levels of blood within the vasculature, changing the background fluorescence of the tissues.</li>
	</ol>
	</li>
	<li>Collection of Tissues
	<ol>
		<li>Prepare PBS as described above for rinsing the removed tissues. 
		Note: If another buffer was used as the perfusion liquid, rinse tissues in the same buffer.</li>
		<li>Remove the heart, lungs, stomach, liver, spleen, and kidneys by simple dissection. Briefly rinse in PBS before using lab wipes to pat dry or wick away excess moisture and placing the tissues onto the imaging mat.</li>
		<li>Decapitate the mouse and remove the top of the skull using bone cutters or rongeurs. Using forceps, carefully remove the brain, keeping the olfactory bulbs intact. Rinse, dry, and place it on the provided imaging mat.</li>
		<li>To remove the spinal cord via rapid ejection7, fill a 10-mL syringe with PBS and attach a dulled 18-gauge needle. 
		Note: 18 gauge fits the adult spinal canal of many mouse lines. The required diameter will vary with animal size.</li>
		<li>Place the animal in the prone position, ensuring that the opening of the spinal canal at the decapitation site is cleanly cut and free of blood.</li>
		<li>Remove the skin from around the spinal column. Using sharp bone cutters or scissors, cut through the spinal column in the lumbar section directly rostral to the hips.</li>
		<li>Briefly rinse it with PBS to visualize the spinal canal. Gently insert the needle into the spinal canal. 
		Note: The needle should feel snug and may require some slow rolling back and forth during insertion. Should the needle feel loose, a more rostral cut can be made or a larger diameter needle might be needed.</li>
		<li>Using the thumb and index finger, apply pressure to the spinal column around the inserted needle. Depress the plunger with moderate and constant pressure. The spinal cord will eject intact out of the rostral opening in the spinal column. 
		Note: The complete removal, rinsing, and drying of all major organs can be reasonably completed in 5 - 7 min. This short time prevents the need for extended storage in PBS, which could result in excessive exposure to light and the leaching of labeled proteins from the tissue.</li>
	</ol>
	</li>
</ol>
2. Whole-organ Ex Vivo Fluorescence Imaging
<ol>
	<li>Imaging Parameters
	<ol>
		<li>Start the image acquisition software. In the bottom right of the acquisition control panel, click &#8220;initialize.&#8221;</li>
		<li>Under &#8220;imaging mode,&#8221; select the &#8220;fluorescent&#8221; checkbox. Under &#8220;exposure time,&#8221; select &#8220;AUTO.&#8221;</li>
		<li>Under &#8220;binning,&#8221; select &#8220;small&#8221; and set the F/Stop to 2.</li>
		<li>Select the 535-nm excitation and 580-nm emission filters. 
		Note: This will vary depending on the label used.</li>
		<li>Select &#8220;Field of View B&#8221; and set the target height to 0.3 cm. 
		Note: This will vary depending on the tissues to be imaged.</li>
		<li>Once the temperature indicator turns green (meaning the machine is fully initialized), place the tissues onto the provided black background mat, ensuring that the tissues are completely separated from one another. Place the mat in the imager, ensuring that all tissues are within the selected area grid. Larger and non-homogenous tissues, such as the whole liver, should be laid out so as to reduce overlap of the lobes. 
		Note: Tissues can be arranged together in a single group, allowing one image to accommodate a single animal or all tissues of a similar type. This makes analysis and organization easier later on. If large differences in fluorescence intensity are present among various tissues, it is best to image them individually or as groups of the same tissue type, as a single exposure time may not be ideal for all tissue intensities.</li>
		<li>Click &#8220;Acquire.&#8221;</li>
		<li>After imaging, remove the tissues immediately and store or freeze them for quantitative histology, as outlined in step 4.</li>
	</ol>
	</li>
</ol>
3. Standards and Image Processing
<ol>
	<li>Standards
	<ol>
		<li>In PBS, create two-fold serial dilutions of each protein used in the treatment of the animals, starting at the original concentration, with a total of 15 dilutions. 
		Note: The concentration range should aim to include the expected tissue concentrations and may require additional dilutions of the original sample, depending on the starting concentration.</li>
		<li>Pipette 100 &#181;L of each standard into each well of a black 96-well plate. Include one well of pure PBS to obtain a measure of background fluorescence.</li>
		<li>Image the standards with the same parameters used for the tissue imaging.</li>
		<li>In the image acquisition software &#8220;Tool Palette&#8221; under &#8220;ROI Tools,&#8221; select the &#8220;Region of Interest (ROI)&#8221; circle and draw the ROI around one well. Copy that ROI to all of the other filled wells and click &#8220;measure the ROIs.&#8221;</li>
		<li>Subtract the value in average radiant efficiency of the pure PBS from the values of the other standards; this removes the background fluorescence. Plot the values against the known concentration on a scatter plot. 
		Note: The resulting equation of the linear trend line can then be used to calculate the relative fluorescence value.</li>
	</ol>
	</li>
	<li>Image Processing
	<ol>
		<li>In the image acquisition software, select the freeform ROI tool and draw ROIs around each of the individual tissues. Click &#8220;measure.&#8221;</li>
		<li>With the resulting average radiant efficiency measurements, subtract the values measured from the untreated animal for each tissue type and plot those values on the previously created standard curve. 
		Note: The resulting relative fluorescence values provide a quick and accurate snapshot of the biodistribution of the labeled compound.</li>
	</ol>
	</li>
</ol>
4. Quantitative Tissue Histology
<ol>
	<li>Preparation and Freezing of Tissues for Slicing
	<ol>
		<li>Create or purchase containers of sufficient size to freeze the chosen tissues and cryostat to be used. Wrapping aluminum foil around an appropriately sized and shaped object works well.</li>
		<li>Prepare a mixture of isopropanol and dry ice by filling half of a 500-mL beaker with dry ice and adding ~ 350 mL of isopropanol so that there is sufficient liquid above the dry ice to partially submerge the freezing container. 
		Note: Liquid nitrogen often results in the fracture of the optimal cutting temperature compound (OCT), and embedded tissues and should be avoided.</li>
		<li>Fill the freezing container partially with OCT, ensuring that there are no bubbles present. If there are bubbles, work the bubbles out of solution using forceps or a similar implement.</li>
		<li>Remove the tissue from PBS and dry it thoroughly, either by gently patting it with a lab wipe or by touching the edges of delicate tissues to a lab wipe and allowing the liquid to wick away.</li>
		<li>Place the tissue gently into the OCT inside the freezing container, ensuring not to introduce bubbles underneath the tissue. Once the tissue is in place, add OCT until the tissue is completely covered.</li>
		<li>Submerge the freezing container as much as is possible without allowing the isopropanol to come in contact with the exposed OCT inside the container. Allow 30 - 90 s (or potentially more for larger tissues) for the OCT and tissue to freeze completely before storing at -80 &#176;C for as long as needed.</li>
	</ol>
	</li>
	<li>Preparation of Standards for Slicing.
	<ol>
		<li>Prepare and appropriately label 1-mL single-use syringes by removing the end of the barrel, such that the entire length of the syringe is one diameter. This will allow the frozen cylinder to be pushed out later.</li>
		<li>Using OCT, create a 15-step two-fold serial dilution of administered compounds, as was done with PBS previously, but with a final resulting volume of 1 mL. Include one sample of pure OCT for removal of background fluorescence values.</li>
		<li>The viscosity of OCT makes adequate mixing difficult. Invert the tubes and allow the OCT to completely settle before inverting again. Repeat this 10 - 20 times to ensure a uniform mixture. Keep the standard solutions at 4 &#176;C and away from light during mixing.</li>
		<li>After mixing, again prepare a mixture of isopropanol and dry ice, as before. 
		Note: Liquid nitrogen can also be used in place of the isopropanol/dry ice mixture, provided the syringes are quickly removed after they have frozen solid.</li>
		<li>Draw the OCT standard into the syringe, taking care to introduce as few bubbles as possible. After drawing up the OCT solution, immediately dip the syringe directly into the isopropanol and allow the solution to freeze completely inside the syringe for 10 - 20 s. Store the syringes at -80 &#176;C for as long as necessary.</li>
	</ol>
	</li>
	<li>Cutting of Tissues and Standards
	<ol>
		<li>Place both tissues and standards in -20 &#176;C and allow enough time for all samples to come to temperature. 
		Note: The ideal cutting temperature generally falls around the -10 &#176;C range for most tissues, but it ultimately depends on tissue size and fat content and must be refined empirically.</li>
		<li>Using a cryostat, cut tissues into 20-&#181;m thick slices and mount them onto slides, as previously described8. Store the slides at -20 &#176;C and keep them away from light as much as possible during cutting and until scanning. 
		Note: It is important here that the sections to be measured are appropriately distributed throughout the entire thickness of the tissue.</li>
		<li>To mount the standards onto the cryostat chuck, take the syringe from the freezer and warm the outside for 3 - 5 s by holding it in hand. Push the plunger until a short section (~ 1 cm) of the OCT cylinder is outside of the syringe.
		<ol>
			<li>Using a razorblade, cut through the cylinder and place the resulting cylinder section perpendicularly onto the chuck, using OCT to hold it in place. This configuration should result in well-defined circles being cut.</li>
		</ol>
		</li>
		<li>Slice the cylinders at 20 &#181;m, placing one slice from each dilution onto a single slide, so that one slide represents the entire standard curve. Store the slides at -20 &#176;C until ready for scanning.</li>
	</ol>
	</li>
	<li>Scanning and Quantitation
	<ol>
		<li>Start the scan array software and click &#8220;543 laser&#8221; to begin the laser startup procedure. Wait for 15 min for the laser to get ready.</li>
		<li>Once the laser is ready, remove the slides from -20 &#176;C and move them into a large, light-protected container to come to temperature and to dry for 3 min. This will prevent moisture buildup in the slide scanner.</li>
		<li>Load the slide into the slide receptacle on the slide scanner. Click &#8220;scan&#8221; and select &#8220;run easy scan.&#8221;</li>
		<li>Set the scan resolution to 50 &#181;m.</li>
		<li>Select the first &#8220;Use&#8221; checkbox under &#8220;Fluorophore&#8221; and select &#8220;TRITC.&#8221; Set the PMT Gain to 80%. 
		Note: These settings will vary depending on the fluorophore and the concentration of the labeled molecules.</li>
		<li>On the left-hand side, ensure that the scan area includes the whole slide. Click &#8220;Start.&#8221;</li>
		<li>After scanning, select &#8220;file&#8221; and &#8220;save as,&#8221; and then save the image as a .tif. 
		Note: Slides of tissues and standards should be scanned on the same settings.</li>
		<li>Load the resulting images into ImageJ and select ROIs around the tissues and standards. Under the &#8220;analyze&#8221; tab, select &#8220;set measurements&#8221; and select &#8220;mean grey value.&#8221; Click &#8220;measure.&#8221; 
		Note: As with the previous technique, background fluorescence must be subtracted from all measured values.</li>
		<li>Plot the mean grey value measurements from the standards against the known concentration to create a standard curve. 
		Note: The resulting measurements from the tissues should be averaged within each tissue and plotted on that standard curve to determine the concentration within the tissue.</li>
	</ol>
	</li>
</ol>

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GLB is owner of Leflore Technologies, LLC, a private company working to develop ELP-based drug delivery technology. Leflore Technologies provided no support for the publication of this paper or for the generation of data within it.

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><tbody><tr><td>&lt;u&gt;Reagents&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Maleimide derivitized fluorophors (&lt;em&gt;e.g.&lt;/em&gt;, tetramethylrhodamine-5-maleimide, AlexaFluor 633-C5-maleimide)</td><td>Thermo Fisher</td><td>T6027, A20342</td><td>Thiol reactive fluorescent dyes for protein labeling</td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma</td><td>1002243569</td><td>PBS Buffer for rinsing</td></tr><tr><td>Optimal Cutting Temperature Compound</td><td>Tissue-Tek</td><td>4585</td><td>Used for freezing and mounting</td></tr><tr><td>&lt;u&gt;Equipment&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>IVIS Spectrum</td><td>Perkin Elmer</td><td></td><td>For &lt;em&gt;ex vivo&lt;/em&gt; whole organ imaging</td></tr><tr><td>Cryomicrotome</td><td>Thermo</td><td></td><td>For cryosectioning</td></tr><tr><td>Fluorescence slide scanner</td><td>Perkin Elmer</td><td></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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9.5K Views.  The University of Mississippi Medical Center. 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.

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

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Medicine

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Bioengineering

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Cancer Research

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Whole&#45;Organ Pathogen Tracking via Fluorescent Reporter Quantification

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

Immunology and Infection

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

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

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In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

Hybrid µCT-FMT imaging and image analysis

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

High-Resolution Three-Dimensional Whole-Organ Tomography of Microbial Infections

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids