Imaging and analysis guidance was provided by the Research Resources Center &#8211; Research Histology and Tissue Imaging Core at the University of Illinois at Chicago established with the support from the office of the Vice Chancellor for Research. The work was supported by NIH/NCI RO1CA191317 to CLP, by NIH/NIAMS (SBDRC grant 1P30AR075049-01) to Dr. A. Paller, and by support of the Robert H. Lurie Comprehensive Cancer Center to the Immunotherapy Assessment Core at Northwestern University.

Mouse spleen and HLF16 mouse tumor tissues23 were obtained from our laboratory. Human tonsil tissue was purchased from a commercial vendor. Details are provided in the Table of Materials.
1. Tissue Embedding
<ol>
	<li>Embed fresh tissue in OCT (optimal cutting temperature) solution and snap freeze using either dry ice or liquid nitrogen.</li>
	<li>Store tissues at -80 &#176;C.</li>
</ol>
2. Cryosectioning
<ol>
	<li>Cut 8 &#956;m sections in a cryostat with temperatures set at -25 &#176;C. 
	NOTE: The preferred section thickness can be adjusted to generate crisper images.</li>
	<li>Place sections on charged glass slides.</li>
	<li>Air-dry the sections for 1 h at room temperature (RT) prior to fixing in histology grade ice-cold acetone for 10 min. 
	NOTE: Acetone causes coagulation of water-soluble proteins and extracts lipids but does not impact carbohydrate-containing components. In contrast, formalin preserves most lipids and has little impact on carbohydrates24. The choice of fixative is important depending on the choice of marker being detected.</li>
	<li>Store the slides at -20 &#176;C.</li>
</ol>
3. Selection of Antibodies and Fluorophores
NOTE: Before tissue staining, antibody clones that will robustly and specifically stain their antigens of interest within sequential sections from acetone fixed tissue must be validated. Some antibodies may require a different fixative, and their compatibility with other antibodies in the panel will also have to be empirically determined. The goal is also to identify fluorophores with minimal overlap that can be detected with the epifluorescence filters for DAPI, FITC, Cy3, Texas Red, and Cy5.
<ol>
	<li>Confirm staining by conventional IHC or immunofluorescence (IF) detection in tissue sections with known expression target antigen.</li>
	<li>Using the excitation and emission filter sets available on the semiautomated imaging system and after testing various combinations of fluorophore-conjugated primary antibodies, prepare fluorophores to be used that have minimal spectral overlap (e.g., see Table 1).</li>
</ol>
4. Staining
NOTE: The tissue rehydration and slide washes were performed in a Coplin jar. The blocking and antibody incubation steps were performed in a humidified slide box.
<ol>
	<li>Allow the slides to warm to RT for 5&#8211;10 min.</li>
	<li>Rehydrate in phosphate buffered saline (PBS) for 5 min.</li>
	<li>Perform a blocking step prior to staining tissues with antibodies. For mouse sections, use specialized blocking solution (see Table of Materials) for 10 min at RT. For human sections, use 10% normal pooled human serum (NHS) diluted in PBS for 15 min at RT. 
	NOTE: Different blocking buffers may be tested as needed to preserve the specific properties of the sections depending on the follow-up procedures to be used.</li>
	<li>Wash the slides for 5 min in PBS after blocking.</li>
	<li>For multiplex staining, prepare a cocktail of antibodies with compatible fluorophores at predetermined optimal dilutions.</li>
	<li>Add the cocktail of fluorophore-conjugated antibodies to the slides. For single-marker staining, add only the primary-conjugated antibody to the slide.</li>
	<li>Include a control unstained slide that undergoes the same staining procedure without the addition of any primary-conjugated antibody.</li>
	<li>Incubate the slides for 1 h at RT in the dark and then wash the slides 2x with PBS for 5 min each. From here on, the resulting slide is referred to as multiplex-stained.</li>
	<li>To counterstain, add DAPI to the multiplex-stained slide, incubate for 7 min in the dark at RT, and wash the slides 2x with PBS for 5 min each. Do not counterstain single-stained and unstained slides.</li>
	<li>To coverslip, add a drop of the mounting medium, and gently place the glass coverslip over the tissue.</li>
</ol>
5. Preparing a Spectral Library
<ol>
	<li>Image acquisition
	<ol>
		<li>Set the lamp power to 100%. Usually the power is set to 10% because fluorescence detection on FFPE tissues includes a signal amplification step.</li>
		<li>Begin by opening the microscope operating software (see Table of Materials).</li>
		<li>Select Edit Protocol and then New Protocol.</li>
		<li>Provide a &#34;Protocol Name&#34;, and select Fluorescence&#160;under Imaging Mode, and provide a &#34;Study Name&#34;.</li>
		<li>Place the single-stained slides on the stage and examine each marker in its corresponding fluorescence channel to ensure staining. Choose a region on the tissue expressing the strongest signal for the marker.</li>
		<li>Adjust the exposure times using the Autofocus and Autoexpose options.</li>
		<li>Acquire snapshots for the single-stained and unstained slides and save the protocol. 
		NOTE:&#160;The following steps are performed in machine learning software (see&#160;Table of Materials), using the single stained and unstained slides to verify specific staining as well as to determine antibody cross talk.</li>
		<li>Under the Build Libraries tab in the software, load each single-stained slide image, choose the appropriate Fluor, and click Extract. The software will automatically extract the fluorescence signal chosen in the Fluor.</li>
		<li>To save the extracted color, click on Save to Store. A &#34;New Group&#34; may be created or the extracted color can be stored to an existing group.</li>
	</ol>
	</li>
	<li>Verifying the spectral library
	<ol>
		<li>Check the emission spectral curve window, located to the right of the extracted image, for each filter set. 
		NOTE: The extracted signal is correct if the spectral curve is observed only in the filter sets where the fluorophore is detected. If a spectral curve is observed in the wrong filter set, it can mean that either the primary signal expected in the filter set is not strong enough, or the software is detecting another signal that is too high, possibly due to spectral overlap. In this case, first try using the Draw Processing Regions tool to draw regions around areas expressing the fluorescent marker and autofluorescence in the image. This trains the software to detect the true signal and remove any interfering signals. If this does not work, repeat the staining process for the single-stained slide to test different antibody titrations.</li>
	</ol>
	</li>
</ol>
6. Multispectral Imaging
NOTE: Once the spectral library is created and verified, perform the following steps for the multiplex-stained slide.
<ol>
	<li>Whole slide scan
	<ol>
		<li>Adjust focus and exposure times on the multiplex-stained slide as mentioned under in section 5.1.</li>
		<li>Under Scan Slides, create a New Task and choose the protocol saved above.</li>
		<li>Perform a whole slide scan on the multiplex-stained slide.</li>
		<li>Using the whole slide scan software, open the whole slide scan image. This image has not been spectrally unmixed.</li>
		<li>Select regions of interest (ROI) across the whole slide scan image using the Stamp or ROI tool. These ROIs will be scanned using the exposure times set in 6.1.1 to be used for spectral unmixing and analysis.</li>
		<li>Click Process Slide to acquire ROIs at 20x magnification</li>
	</ol>
	</li>
	<li>Spectral unmixing
	<ol>
		<li>Once the ROIs have been acquired, in the machine learning software, under the Manual Analysis tab, load the multiplex stained images by clicking Open under File.</li>
		<li>In the Spectral Library Source dropdown menu click Select Fluors.</li>
		<li>A new window will open. Here, choose the spectral library or group created above.</li>
		<li>Load the unstained slide image. Click the AF ink marker icon located above the selected spectral library and draw a line or region on the unstained slide to identify autofluorescence in the tissue.</li>
		<li>Under the Edit Markers and Colors tab, assign names for each marker. Pseudo colors can be assigned at this step. 
		NOTE: The color for the Autofluorescence image defaults to DarkSlateGray. Change this to Black.</li>
		<li>Click Prepare All.</li>
	</ol>
	</li>
	<li>Verifying spectrally unmixed images 
	NOTE: When the spectral unmixing step is completed, a composite image consisting of all the colors is created.
	<ol>
		<li>Click the Edit the View eye icon. Here, each color in the &#34;Component Display&#34; can be turned off or on to view the staining of each individual marker.</li>
		<li>Visually inspect the staining and the morphology of the cells to ensure that there is no overlapping of marker, unless it is biologically relevant. A pathologist can help verify the staining as well. 
		NOTE: The staining on the multiplexed slide should be validated by leaving out one fluorophore at a time and reviewing the staining pattern. In addition, the validation will also help to identify strong fluorophores that appear in adjacent spectra due to antibody cross talk or bleed-through.</li>
		<li>For Pathology Views, which simulate brightfield images for each fluorescent marker, click the Select a Component Image button. Here, choose a marker to view a simulated brightfield image.</li>
	</ol>
	</li>
</ol>
7. Analyzing Multispectral Images via Cell Segmentation and Phenotyping
NOTE: After verifying the spectrally unmixed image, cell segmentation can be performed using the machine learning software, which will provide step-by-step instructions. Tissue segmentation was not performed here. If the panel includes one or more tissue specific marker and especially if the tissue is messy, tissue segmentation should be performed.
<ol>
	<li>Select &#34;Cytoplasm&#34; and &#34;Membrane&#34; under the &#39;Segment&#39; option. 
	NOTE: &#34;Nuclei&#34; is chosen by default.</li>
	<li>Select a marker from the panel. Configure the marker to detect either nuclei, cytoplasm, or membrane. For example, DAPI can be selected to detect nuclei and CD3 for membrane.</li>
	<li>Click on the ellipsis button (&#39;...&#39;) to select an option under &#34;use this signal to find&#34;. For example, &#39;nuclei&#39; for DAPI. Multiple markers from the panel can be selected for segmentation. 
	NOTE: For cytoplasmic or membrane markers, select the &#34;Use this signal to assist in nuclear segmentation&#34; option.</li>
	<li>The software automatically detects and creates a mask each for the nucleus, cytoplasm, and membrane in the image.</li>
	<li>Ensure all the cells are &#39;masked&#39; for segmentation. To adjust, switch to the Pathology View for the specific marker chosen in 7.3. and use the configuration options in the software.</li>
	<li>Click &#34;Segment All&#34; to segment cells.</li>
	<li>After cell segmentation, proceed with phenotyping cells. In this step, choose the markers needed for phenotyping and manually select at least five cells that are brightly stained with the chosen marker. This trains the software to then automatically detect all cells stained with the chosen marker in the image.</li>
	<li>A phenotype map is created. Analyze to ensure that the cell stained with the marker is correctly phenotyped. 
	NOTE: Cell phenotyping can be an iterative process. If the software is unable to phenotype the cells correctly, it means that the training is inadequate or incorrect. In this case, the user has to manually select more cells and retrain the software and repeat this step until the user is satisfied with the training.</li>
	<li>Create a group named &#34;Others&#34; and include cells that are not stained for any of the markers. 
	NOTE: This step is important to train the software to exclude all the unstained cells from phenotyping.</li>
</ol>
8. Exporting Images and Analysis Tables
<ol>
	<li>Click the Export button to view the Export Settings panel.</li>
	<li>In the &#34;Export Directory&#34;, click Browse to select a location to export the images.</li>
	<li>In the &#34;Image Export Options&#34;, choose the Image Output Format.</li>
	<li>In the &#34;Images to Export&#34; list, select the images to be exported. &#34;Composite image&#34; is the final pseudocolored unmixed image. &#34;Pathology Views&#34; are the individual simulated brightfield images and the &#34;Component Images (multi-image TIFF)&#34; is a multi-image TIFF file of component data that can be used by third party analysis software.</li>
	<li>Click &#34;Export for All&#34; button to export the images. 
	NOTE: Tables from analysis can also be selected and exported at this step.</li>
</ol>

The authors have no conflicts of interest to disclose.

Frozen tissues have extensively been used for mIF imaging to traditionally detect three to four markers31 on a tissue using the direct and indirect method32. In the direct method, antibodies are conjugated to fluorescing dyes or quantum dots33 to label the tissue, whereas in the indirect method, an unconjugated primary antibody is used to label the tissue followed by a fluorophore-conjugated secondary antibody that specifically recognizes the primary...

Recent advances in microscopic imaging techniques have significantly improved our knowledge and understanding of biological processes and disease states. In situ detection of proteins in tissues via chromogenic immunohistochemistry (IHC) is routinely performed in pathology. However, detection of multiple markers using chromogenic IHC staining is challenging1 and newer methods to use multiplex immunofluorescence (mIF) staining approaches, wherein multiple biological markers are labeled on a single tissue sample, are being developed. The detection of multiple biological markers is useful, because information related to tissue architecture, spatial distribution of cells, and antigen co-expression are all captured in a single tissue sample2. The use of multispectral fluorescence imaging technology has made detection of multiple biological markers possible. In this technology, using specific optics the fluorescence spectra of each individual fluorophore can be separated or &#34;unmixed&#34;, enabling the detection of multiple markers without any spectral crosstalk3. Multispectral fluorescence imaging is becoming a critical approach in cell biology, preclinical drug development, clinical pathology, and tumor immune-profiling4,5,6. Importantly, the spacial distribution of immune cells (specifically CD8 T cells) can serve as a prognostic factor for patients with existing tumors7.
Various approaches to multiplex fluorescence staining have been developed and can be performed either simultaneously or sequentially. In the simultaneous staining method, all the antibodies are added together as a cocktail in a single step to label the tissue. UltraPlex technology uses a cocktail of hapten-conjugated primary antibodies followed by a cocktail of fluorophore-conjugated anti-hapten secondary antibodies. InSituPlex technology8 uses a cocktail of unique DNA-conjugated primary antibodies that are simultaneously added to the tissue followed by an amplification step and finally fluorophore-conjugated probes that are complementary to each unique DNA sequence on the primary antibody. Both of these technologies enable the detection of four markers plus 4&#8217;,6-diamino-2-phenylindole (DAPI) for nuclear staining. Two other approaches for simultaneous multiplex staining are based on secondary ion mass spectrometry9. The Hyperion Imaging System uses imaging mass cytometry10 to detect up to 37 markers. This technology uses a cocktail of metal-conjugated antibodies to stain the tissues, and specific areas of the tissues are ablated by a laser and transferred to a mass cytometer where the metal ions are detected. Another similar technology is the IONPath, which uses multiplexed ion beam imaging technology11. This technology uses a modified mass spectrometry instrument and an oxygen ion source instead of laser to ablate the metal-conjugated antibodies. While all these simultaneous multiplex staining approaches enable the detection of multiple markers, the costs involved for conjugating DNA, haptens, or metals to antibodies, the loss of tissue due to ablation, and the extensive image processing for unmixing cannot be underestimated. Moreover, kits and staining protocols are currently available only for FFPE tissues and developing custom panels entails additional time and expenditure.
The sequential multiplex staining method, in contrast, includes labeling the tissue with an antibody to one marker, stripping to remove the antibody, followed by sequential repeats of this process to label multiple markers12. The tyramide signal amplification (TSA) is the most frequently used sequential multiplexing method. Two other multiplexing technologies use a combination of simultaneous and sequential staining methods. The CODEX platform13 employs a cocktail of antibodies conjugated to unique DNA oligonucleotide sequences that are eventually labeled with a fluorophore using an indexed polymerization step followed by imaging, stripping, and repeating the process to detect up to 50 markers. The MultiOmyx multiplex staining approach14 is an iteration of staining with a cocktail of three to four fluorophore-conjugated antibodies, imaging, quenching the fluorophores, and repeating this cycle to detect up to 60 markers on a single section. Similar to the simultaneous multiplex staining method, while a broad range of markers can be detected, the time involved in staining, image acquisition, processing, and analysis is extensive. The stripping/quenching step involves heating and/or bleaching the tissue sample and thus, the sequential multiplex staining approach is commonly performed on FFPE tissues that maintain tissue integrity upon heating or bleaching.
Formalin fixation and subsequent paraffin embedding is readily performed in a clinical setting, tissue blocks are easy to store, and several multiplex staining protocols are available. However, the processing, embedding, and deparaffinization of FFPE tissues, as well as antigen retrieval15, a process by which antibodies can better access epitopes, is time-consuming. Furthermore, the processing involved in FFPE tissues contributes to autofluorescence16 and masks target epitopes, resulting in the variability and lack of antibody clone available to detect antigens in FFPE tissues17,18,19. An example is the human leukocyte antigen (HLA) class I alleles20. In contrast, snap freezing of tissues does not involve extensive processing steps prior to or after fixing, circumventing the need for antigen retrieval21,22, and making it beneficial for detecting a wider range of targets. Therefore, using frozen tissues for multispectral fluorescence imaging can be valuable to detect targets for preclinical and clinical studies.
Given the above mentioned limitations when using FFPE tissues, we asked whether multispectral fluorescence imaging can be performed on frozen tissues. To address this question, we tested a simultaneous multiplex staining method using a panel of fluorophore-conjugated antibodies to detect multiple antigens and analyzed the staining using a semiautomated multispectral imaging system. We were able to simultaneously stain up to six markers in a single tissue section within 90 min.

<table>
	<tbody>
		<tr>
			<td>Acetone (histological grade)</td>
			<td>Fisher Scientific</td>
			<td>A16F-1GAL</td>
			<td>Fixing tissues</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-mouse CD3</td>
			<td>BioLegend</td>
			<td>100212</td>
			<td>Clone - 17A2; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488, eBioscience anti-human CD20</td>
			<td>ThermoFisher Scientific</td>
			<td>53-0202-82</td>
			<td>Clone - L26; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 Mouse anti-Ki-67</td>
			<td>BD Biosciences</td>
			<td>558617</td>
			<td>Primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 anti-human CD3</td>
			<td>BioLegend</td>
			<td>300446</td>
			<td>Clone - UCHT1; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 anti-mouse CD8a</td>
			<td>BioLegend</td>
			<td>100758</td>
			<td>Clone - 53-6.7; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-human CD8a</td>
			<td>BioLegend</td>
			<td>372906</td>
			<td>Clone - C8/144B; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-mouse CD206 (MMR)</td>
			<td>BioLegend</td>
			<td>141711</td>
			<td>Clone - C068C2; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-mouse CD4 Antibody</td>
			<td>BioLegend</td>
			<td>100426</td>
			<td>Clone - GK1.5; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>C57BL/6 Mouse</td>
			<td>Charles River Laboratories</td>
			<td>27</td>
			<td>Mouse frozen tissues used for multispectral training</td>
		</tr>
		<tr>
			<td>Coplin Jar</td>
			<td>Sigma Aldrich</td>
			<td>S6016-6EA</td>
			<td>Rehydrating and washing slides</td>
		</tr>
		<tr>
			<td>DAPI Solution</td>
			<td>BD Biosciences</td>
			<td>564907</td>
			<td>Nucleic Acid stain</td>
		</tr>
		<tr>
			<td>Diamond White Glass Charged Slides</td>
			<td>DOT Scientific</td>
			<td>DW7590W</td>
			<td>Adhering tissue sections</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline 1x (without Ca and Mg)</td>
			<td>Fisher Scientific</td>
			<td>MT21031CV</td>
			<td>Washing and diluent</td>
		</tr>
		<tr>
			<td>Gold Seal Cover Slips</td>
			<td>ThermoFisher Scientific</td>
			<td>3306</td>
			<td>Protecting stained tissues</td>
		</tr>
		<tr>
			<td>Human Normal Tonsil OCT frozen tissue block</td>
			<td>AMSBio</td>
			<td>AMS6023</td>
			<td>Human frozen tissue used for multispectral staining</td>
		</tr>
		<tr>
			<td>Human Serum 1X</td>
			<td>Gemini Bio-Products</td>
			<td>100-512</td>
			<td>Blocking and diluent for human tissues</td>
		</tr>
		<tr>
			<td>inForm</td>
			<td>Akoya Biosciences</td>
			<td>Version 2.4.1</td>
			<td>Machine learning software</td>
		</tr>
		<tr>
			<td>PerCP/Cyanine5.5 anti-human CD4</td>
			<td>BioLegend</td>
			<td>300529</td>
			<td>Clone - RPA-T4; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>PerCP-Cy 5.5 Rat Anti-CD11b</td>
			<td>BD Biosciences</td>
			<td>550993</td>
			<td>Clone - M1/70; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Phenochart</td>
			<td>Akoya Biosciences</td>
			<td>Version 1.0.8</td>
			<td>Whole slide scan software</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P36965</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Research Cryostat</td>
			<td>Leica Biosystems</td>
			<td>CM3050 S</td>
			<td>Sectioning tissues</td>
		</tr>
		<tr>
			<td>Superblock 1X</td>
			<td>ThermoFisher Scientific</td>
			<td>37515</td>
			<td>Blocking mouse tissues</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T Solution</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td>Embedding tissues</td>
		</tr>
		<tr>
			<td>Vectra 3.0 Automated Quantitative Pathology Imaging System, 6 Slide</td>
			<td>Akoya Biosciences</td>
			<td>CLS142568</td>
			<td>Semi-automated multispectral imaging system</td>
		</tr>
		<tr>
			<td>Vectra Software</td>
			<td>Akoya Biosciences</td>
			<td>Version 3.0.5</td>
			<td>Software to operate microscope</td>
		</tr>
	</tbody>
</table>

a rapid method for multispectral fluorescence imaging of frozen tissue sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

Detection of single-stained markers on frozen spleen sections 
As the semiautomated imaging system uses a liquid crystal tunable filter (LCTF) system that allows for a wider range of wavelength detection25, and because no signal amplification steps were performed here, we first optimized the detection of our primary-conjugated antibodies for each marker on the microscope. An example is shown in Figure 1, where each single-stained marker is pseudo...

Watch this Scientific Journal Video about A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections at JoVE.com

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

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7.7K Views.  Northwestern University. We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Video: A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. 
Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device.

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...