Studies presented here were supported by funding from the National Institutes of Health (NIH U01 GM111243 and NIH NIDDK P30 DK079312). Intravital microscopy studies were conducted at the Indiana Center for Biological Micros&#173;copy. We thank Dr. Malgorzata Kamocka for technical assistance with microscopy.

All animal experiments were approved and conducted according to the Institutional Animal Care and Use Committee guidelines of Indiana University, and adhered to the NRC guide for the care and use of animals.
1. Surgical Preparation for Intravital Microscopy
NOTE: This is not a survival surgery. Once section 1 &#34;Surgical preparation for intravital microscopy&#8221; is begun, work cannot be paused until the completion of section 2 &#34;Intravital mic...

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There are multiple critical steps in this protocol. First, minimization of motion during intravital imaging of the liver is essential for generating movies that are usable for capillary flow analysis using STAFF. Due to the proximity of the diaphragm, short periods of respiration-induced motion occur, with the secured liver returning to its initial position after each breath. Securing the surgically exposed liver against the coverslip-bottomed dish using gauze, then imaging from below using an inverted microscope serves ...

The microvasculature plays a critical role in physiology, ensuring effective perfusion of tissues under changing conditions. Microvascular dysfunction is associated with myriad conditions including long-term cardiovascular morbidity and mortality, development of dementia, and disease of both liver and kidney and thus is a key factor of interest in a broad range of biomedical investigations1,2,3,4,5. While multiple techniques have been used to evaluate tissue perfusion, only intravital microscopy enables data collection at the temporal and spatial resolution necessary to characterize blood flow at the level of individual capillaries.
Microvascular flow can be visualized in fluorescence microscopy either by the movement of fluorescent microspheres or by the movement of red blood cells against the background of membrane-impermeant fluorescent markers (e.g., fluorescently-labeled dextran or albumin)6,7. Microvascular flow can be imaged in superficial cell layers using widefield microscopy, or at depth using either confocal or multiphoton microscopy. However, capillary flow rates are such that the passage of red blood cells cannot generally be captured at speeds less than 60 frames/s. Since most laser scanning confocal and multiphoton microscopes require 1&#8211;5 s to scan a full image field, this speed can generally be accomplished only by limiting the field of view, sometimes to a single scan line8. The process of limiting measurements to selected capillary segments (1) has the potential to introduce selection bias and (2) makes it impossible to capture spatial and temporal heterogeneity in the rates of capillary blood flow. In contrast, images of capillary networks can be collected at speeds exceeding 100 fps using widefield digital microscopes equipped with scientific complementary metal oxide semiconductor (sCMOS) cameras9,10. These inexpensive systems, common in typical biomedical laboratories make it possible to image microvascular flow across entire two-dimensional networks, essentially continuously. The problem then becomes one of finding an analysis approach that is capable of extracting meaningful quantitative data from the massive and complex image datasets generated by high-speed video microscopy.
To enable analysis of full-field flow data we have developed STAFF, novel image analysis software that can continuously measure microvascular flow throughout entire microscope fields of image series collected at high speed11. The approach is compatible with a variety of different experimental systems and imaging modalities and the STAFF image analysis software is implemented as a macro toolset for the FIJI implementation of ImageJ12. The underlying principle used here to visualize microvascular flow is that first, some contrast must be provided to be able to image the red blood cells within capillaries. In our studies, contrast is provided by a bulk fluorescent probe that is excluded by the red blood cells. The velocity of flow can then be quantified from the displacement of the red blood cells that appear as a negative stain within the fluorescently labeled plasma in images collected at high speed from a living animal8. We then use STAFF to make plots of distance along each capillary segment over multiple intervals of time called kymographs, then detect the slopes present in the kymographs13, and from those slopes calculate the rates of microvascular flow. The approach can be applied to images collected from any capillary bed that can be accessed for imaging. Here we describe the application of IVM and STAFF to studies of blood flow in the liver.

<table border="0" cellpadding="0" cellspacing="0" style="width:1020px;" width="1021">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">#5 forceps</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:120px;">11251-20</td>
			<td style="width:431px;">Dumont #5 Inox Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">C57BL/6 mice</td>
			<td style="width:211px;">Jackson Labs</td>
			<td style="width:120px;"></td>
			<td>male 9-12 weeks old</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Cannula</td>
			<td style="width:211px;">Instech</td>
			<td style="width:120px;">BTPE-10</td>
			<td>Polyethylene Tubing .011x.024in</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">CMOS camera</td>
			<td style="width:211px;">Hamamatsu</td>
			<td style="width:120px;">C11440-42U30</td>
			<td>4.0LT Scientific CMOS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Coverslip-bottomed dish</td>
			<td style="width:211px;">Electron Microscopy Sciences</td>
			<td style="width:120px;"></td>
			<td>WillCo Dish glass bottom GWST5040</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Dissecting scissors</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:120px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Fiji ImageJ Image analysis software</td>
			<td style="width:211px;"></td>
			<td style="width:120px;"></td>
			<td>https://fiji.sc/ ; https://downloads.imagej.net/fiji/Life-Line/fiji-win64-20170530.zip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Fluorescein dextran</td>
			<td style="width:211px;">Thermo Fisher, Invitrogen</td>
			<td style="width:120px;">D1822</td>
			<td>Dextran, Fluorescein, 70,000 MW, Anionic, Lysine Fixable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Gauze sponge</td>
			<td style="width:211px;">Fisher</td>
			<td>22-415-504</td>
			<td>2x2 inch Dukal sterile gauze sponges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Heating pad</td>
			<td style="width:211px;">Reptitherm</td>
			<td style="width:120px;">RH-4</td>
			<td>between mouse and stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Heating pad</td>
			<td style="width:211px;">Sunbeam</td>
			<td>000732-500-000U</td>
			<td>over mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Inverted epifluorescence microscope</td>
			<td style="width:211px;">Nikon</td>
			<td style="width:120px;"></td>
			<td>Nikon TiE inverted microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Isis Rodent electric shaver</td>
			<td style="width:211px;">Braun Aesculap</td>
			<td style="width:120px;">GT420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Isofluorane</td>
			<td style="width:211px;">Abbott GmbH</td>
			<td style="width:120px;">PZN4831850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Luer stub adapter</td>
			<td style="width:211px;">Fisher</td>
			<td>14-826-19E</td>
			<td>Catheter adapter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Micro scissors</td>
			<td>Castro Viejo</td>
			<td style="width:120px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Microscope objective</td>
			<td style="width:211px;">Nikon</td>
			<td style="width:120px;"></td>
			<td>Plan Fluor 20x, NA 0.75 water immersion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Needle</td>
			<td style="width:211px;">Fisher</td>
			<td></td>
			<td>30 Ga.x1/2&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Needle holder</td>
			<td style="width:211px;">Olsen-Hegar</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Objective heater</td>
			<td style="width:211px;">BioScience Tools</td>
			<td style="width:120px;">MTC-HLS-025</td>
			<td>Temperature controller with objective heater</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Rectal thermometer</td>
			<td style="width:211px;">Braintree Scientific, INC</td>
			<td style="width:120px;">TH-5A</td>
			<td>Mouse Body Temperature monitoring</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">STAFF macros</td>
			<td style="width:211px;"></td>
			<td style="width:120px;"></td>
			<td>https://github.com/icbm-iupui/STAFF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Suture string</td>
			<td>Harvard Bioscience</td>
			<td style="width:120px;">723288</td>
			<td>silk black suture, 6-0, spool</td>
		</tr>
	</tbody>
</table>

spatial temporal analysis of fieldwise flow in microvasculature

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, appearance of defects in microcirculation can be a primary indicator in the development of multiple pathologies. Advances in optical imaging have made intravital microscopy (IVM) a practical approach, permitting imaging at the cellular and subcellular level in live animals at high-speed over time. Yet, despite the importance of maintaining adequate tissue perfusion, spatial and temporal variability in capillary flow is seldom documented. In the standard approach, a small number of capillary segments are chosen for imaging over a limited time. To comprehensively quantify capillary flow in an unbiased way we developed Spatial Temporal Analysis of Fieldwise Flow (STAFF), a macro for FIJI open-source&#160;image analysis software. Using high-speed image sequences of full fields of blood flow within capillaries, STAFF produces&#160;images that represent motion over time called kymographs for every time interval for every vascular segment. From the kymographs STAFF calculates velocities from the distance that red blood cells move over time, and outputs the velocity data as a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. In normal mouse livers, STAFF analyses quantified profound differences in flow velocity between pericentral and periportal regions within lobules. Even more unexpected are the differences in flow velocity seen between sinusoids&#160;that are side by side and fluctuations seen within individual vascular segments over seconds. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of capillary flow.

STAFF analysis generates a complete census of microvascular velocities across entire microscope fields over periods of time extending from seconds to minutes. Representative results are presented in Figure 1, Figure 2, Figure 3, and Figure 4. Figure 1 shows an example of a time series of the microvascular network in the liver of a mouse, the generation of the skeletonized i...

Watch this Scientific Journal Video about Spatial Temporal Analysis of Fieldwise Flow in Microvasculature at JoVE.com

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses.

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, ...

Since microvascular flow varies between capillaries and changes over a time course of seconds, a method for the continuous quantification of flow across entire fields is needed. Staff enables a nearly continuous measurement of flow velocities across entire microscope fields, within hours, that would otherwise take months to obtain manually. Although these procedures are straightforward, visual demonstration will significantly help new users navigate staff for the first time.To use TrakEM2 to define the vascular network, first select File, New, and TrakEM2 Blank to setup a new blank TrakEM2 project, and select the new folder containing the single image from the movie as the project folder. The TrakEM2 windows will open. Right-click in the main work area and select Import and Import Image.Navigate to the single saved image and select the image. Right-click in the main work area again and select Display and Auto Resize Canvas/LayerSet. Then, left-click in the main work window.The image will fill the work area. To select the areas in the image that contain the vascular network, in the smaller TrakEM2 window, right-click on template Anything, and select Add New Child and Area List. In the smaller TrakEM2 window, drag template Anything onto Project Objects, untitled project.Then drag template Anything, area list, onto Project Objects, untitled projects, Anything. Under Project Objects, untitled project, the Anything area list will now be present. In the main TrakEM2 window under the Z Space tab, a bar labeled Area List will have been created.Click to select the list. In the main TrakEM2 window, select the paintbrush tool and click Shift while rolling the mouse wheel to select a paintbrush size that is smaller than the diameter of the vasculature of interest. Press Control S to save the project.Using the paintbrush tool, paint the vascular network, excluding any out of focus regions. When the labeling is complete, right-click in the main TrakEM2 window and select Export and Area Lists as Labels. In the pop-up window, select Scale, 100%and export all Area List.Close the TrakEM2 windows and select Yes to save the project. The image of Area List will open and may appear as a blank, black image. In the the main Fiji menu, select Image, Adjust, and Brightness/Contrast.In the Brightness and Contrast window, click Auto and the Area List will become visible. In the main Fiji menu, select Image, Lookup Tables, and Invert Lookup Table. Save this image of black labels on white background as a t-i-f file and close the image.Open the Labels file in Fiji and select Plugins, Skeleton, and Skeleton Eyes. Then, save the Skeleton Eyes image as a t-i-f file. To create a new staff project, select Plugins, Macros, and Open/Create Project and follow the prompts to create or update a configuration file.From the Open/Create project menu, navigate to and select the project directory Input File folder and the Input Movie and Skeleton files. Input the values for the Maximum Measured Speed, the Maximum Speed Mapped, the Minimum Segment Length, the Pixel Size, and the Frame Rate. Check the Flicker correction box if the images have periodic background intensity flicker and click OK.Adjust the parameters for the best visualization of the data.Set the Maximum Speed Mapped to include about 95 percent of the data, so that the data is mapped across the full range of the color scale. To analyze the skeleton, select Plugins, Macros, and Analyze Skeleton. The skeleton file will open and the Region of Interest manager will open and run.In the Region of Interest manager, click Show All and Label and click OK.To select the time intervals, select Plugins, Macros, and Edit Time Intervals and wait for the macro to generate a kymograph from a single segment over the total time for the movie. Use the Control plus and minus keys to increase the size of the kymograph as needed and use the Rectangle selection tool to draw a rectangle around each time interval. Press the T key or click the Add button to record a Time Interval selection and repeat the selection for as many time intervals as desired.To evaluate the parameter choices, select three to four time intervals and complete the analysis for just those intervals. Click OK when the time interval selection has been completed. A pop-up window will appear when the selected intervals have been saved in the project folder.Click OK.To analyze the flow, select Plugins, Macros, and Analyze Flow. The Analyze Flow Parameters dialogue box will open and display the values entered in the Open/Create Project step. Edit these values if necessary and click OK.The Output File Names dialogue box will open and display the names entered in the Open/Create Project step.Edit these names as necessary and click OK to begin the Flow Analysis. When a dialogue box appears indicating that the flow has been analyzed, click OK to store the analysis results in CSV Spreadsheet files. To produce spatial maps, select Plugins, Macros, and Produce Spatial Map.The Spatial Map Parameters window will open displaying the values entered in the Open/Create Project step. Edit these values as necessary and click OK.A temporal sequence of Spatial Maps of Flow Velocities will be generated with each color indicating a flow speed. Scroll through the generated stack of one image per interval to visualize the spatial and temporal variations in flow and save the stack as an a-v-i file to share the file as a movie.Staff Analysis generates the complete census of the microvascular velocities across entire microscope fields, over periods of time extending from seconds to minutes. For example, using a single image in this time series of the microvascular network in the liver of a mouse, the generated skeletonized image was used to define the axis of the microvascular flow, allowing the Staff generated map of individual vascular segments to be identified for subsequent flow quantification. From the individual vascular segments generated from the skeletonized image, kymographs can be generated representing the intensity along each line segment over time.The Skeleton and User Supply time intervals can be used to break the kymograph of each segment into individual segment time intervals. Staff then identifies the predominant angle in the kymograph from each segment time interval and provides the calculated velocity measurements as CSV data files. Staff CSV output can be used to analyze the overall velocity distribution to enable plotting of the velocity in the individual vascular segments over time.And can be visualized as stacks of color-coded velocity map images. Note that the results that came from Staff depend upon the quality of the image series, with respect to the sample stability, frame rate, resolution, and signal-to-noise ratio. The Staff Velocity Map Outputs allow an intuitive exploration of spatial flow patterning while Staff CSV outputs enable the statistical analysis of flow velocities, both within and between treatment groups.

spatial-temporal-analysis-of-fieldwise-flow-in-microvasculature

Research

JoVE Journal

Bioengineering

5.7K visualizações.  Indiana University. Uma vez que o fluxo microvascular varia entre capilares e alterações ao longo de um curso de tempo de segundos, um método para a quantificação contínua do fluxo através de campos inteiros é necessário. A equipe permite uma medição quase contínua das velocidades de fluxo em campos inteiros de microscópio, em poucas horas, que levaria meses para obter manualmente. Embora esses procedimentos sejam simples, a demonstração visual ajudará significativamente novos usuários a navegar...