Keywords
Intravital Microscopy, 2-Photon Microscopy, Kidney, Single Nephron GFR, ImageJ, R
This article is included in the NEUBIAS - the Bioimage Analysts Network gateway.
Intravital Microscopy, 2-Photon Microscopy, Kidney, Single Nephron GFR, ImageJ, R
Figure 1 was insufficiently described in the figure caption and hard to understand without the context of the entire article. In the new version we added more information to the figure caption to address this issue.
See the authors' detailed response to the review by Christopher Schmied
See the authors' detailed response to the review by Bruce A. Molitoris
See the authors' detailed response to the review by Georgina Gyarmati
See the authors' detailed response to the review by Anna Schueth
Glomerular filtration rate (GFR) is a key parameter of kidney function and deviations from normal GFR are a hallmark of renal diseases1,2. GFR describes the filtration of substances from blood in the glomerular capillaries, to the primary urine in the tubular system of the kidney. Therefore, changes in GFR serve to monitor disease progression1,2. GFR is also measured in animal models to study effects of pharmacological intervention on kidney function3. Advances in intravital imaging and multiphoton microscopy allow repetitive assessment of GFR and morphological changes in the smallest functional unit of the kidney – the nephron3–5. Longitudinal imaging of single nephrons (sn) enable direct correlation of structural and functional data3–5.
After intravenous injection of the freely filtered, non-resorbable, fluorescent dye LuciferYellow (LY), a time series was captured by multiphoton microsopy. Filtration was observed from the glomerular capillaries to the proximal tubule (PT) and the tubular signal intensity shift is analyzed to calculate the filtration rate. Translated to an image processing task, this can be generalized as the flow rate in a tube. Previous methods for this analysis3,4 relied on two manually annotated measurement points in the PT and stereotypic estimation of PT volume in 2D images. Since results we obtained with this approach were highly variable, we expanded the analysis of image data via 3D modelling with open source software, to increase overall reproducibility and reliability of the analysis when comparing renal function of different experimental groups.
Animal experiments were performed in accordance with the Federation of European Laboratory Animal Science Associations (FELASA) Guidelines for the Care and Use of Laboratory Animals and the Federal Law on the Use of Experimental Animals in Germany and approved by the ethical review committee at the Landesdirektion Sachsen (license DD-24.1-5131/338/37). For microscopy, male, 10–12 week old C57BL/6 mice were prepared as previously described5,6. In brief, a titanium abdominal imaging window (AIW) covered with a coverslip is surgically implanted above the kidney. The kidney is glued to the coverslip with cyanoacrylate glue before securing the AIW by tightening the skin in the AIW groove. Microscopy was performed one day after AIW implantation.
A custom-built temporary intravenous catheter (polyethylene tubing #587360 by Science Products GmbH with 0.3×12mm needle) was placed in the lateral tail vein. Fluorescent dyes were administered into the tail vein prior (Hoechst, AngioSpark) or during (LuciferYellow) microscopy (detailed information in Table 1).
All efforts were made to ameliorate harm to animals. Imaging (including injections of the fluorescent dyes) and the implantation is done under isoflurane anaesthesia. The image data of the five animals presented for the comparison of the extended workflow with the previous workflow in this manuscript were generated previously as part of an independent experiment (license DD-24.1-5131/338/37).
Imaging was performed on an upright Leica SP8 multiphoton laser scanning microscope at the Core Facility Cellular Imaging. Settings for signal acquisition are summarized in Table 2.
Image processing and analysis was done in ImageJ7–9 (1.53c) with 3D ImageJ Suite10 and Bio-Formats11 for the use of 3D image processing plugins and the Bio-Formats Importer. Data analysis was performed in R12 (4.0.2), with RStudio13 (1.2.5033) including ggplot214 (including dependencies) installed as additional library. The script executed the ImageJ macro from command line and subsequently analyzed and visualized the results. A detailed description of the algorithm is associated with the scripts on GitHub15.
The line region of interest (ROI) set for the extended workflow to manually define direction and position of the proximal tubule (PT) was also used to determine the two measuring points (beginning and end of line) for analysis of image material based on the previously described approach3,4. Tubular diameter was calculated as the mean of five manually measured diameters.
In the time series acquired after application of LY, a line ROI was set to manually define the position and direction of the measurement. Along this ROI, x-y plots measured the dye intensity in the PT in every frame (Figure 1) and numerical results were saved.
For the automatic 3D modelling of PT volume the z-stack of the same field of view was acquired. Additional channels (Ch3: AngioSpark - vessels, Ch4: Hoechst - nuclei, Figure 2A) were subtracted plane by plane from Ch2 (target channel, LY intensity) to remove spectral bleed-through artifacts (Figure 2B). With the 3D watershed, the PT was segmented (Figure 2C, 3D-model) and saved for visual verification. The cumulative PT volume was measured over the distance along the ROI and plotted in subsequent data analysis (Figure 3A). The position is now recalculated to the cumulative PT volume at each point along the ROI. From intensity measurements a threshold intensity was set to the turning point of fluorescence intensity over time at every volume (maximum slope, Figure 3B). The volume with this intensity was approximated in each frame and used for linear regression (Figure 3C, intersect of horizontal threshold at every frame with intensity curves). The slope of the regression line equals the snGFR after conversion of µm³ per frame to nl per minute. Together with information about PT length, PT volume and R-squared the results were summarized and saved in a data table.
Repeated analysis (five times) of 15 individual glomeruli by the same researcher showed that results obtained with the presented workflow had higher consistency (lower intrasample variance, CV=10.35%) compared to the previous approach (CV=38.75%, Figure 4). Due to the high variance with the previous approach a direct correlation of the workflows was not possible; however, the final result - the mean snGFR - was comparable (previous workflow: 1.71±0.91, extended workflow: 1.70±0.78) and a two-sample Kolmogorov-Smirnof test of both result vectors showed that the distributions were not statistically different (p=0.4662). Numerical results of the repeated analysis with both workflows are listed in Table 3.
The progressive development of microscopy techniques like measurement of snGFR in experimental animals needs to be accompanied by improvements in analysis algorithms to use their full potential. In this manuscript we present a workflow by extending an existing analysis method via 3D modelling, for increased reproducibility, accuracy, but also transparency in the measurement of snGFR. By reducing user interaction, intrasample variance was markedly improved.
Additionally, the automatically saved user input and intermediate results (z-stack of watershed of PT as shown in Figure 2C and graphs in Figure 4) for every analyzed dataset provide full possibility to verify every analysis step. These results can be used to objectively evaluate the measurement. Although the snGFR in this manuscript was very low for healthy animals compared to previously published values3, the range was comparable in both methods and not an artifact produced by the workflow but more likely caused by the general experimental setup.
Taken together, this workflow extension contributes to an overall improvement of the interpretation of snGFR measurements. Applied to experimental data this can cumulate in a higher power to detect statistically significant differences between experimental groups and even decrease the necessary sample size, thus having an impact on animal welfare.
Zenodo: Sample dataset - cont-3D-snGFR. https://doi.org/10.5281/zenodo.427559616.
This project contains the following underlying data:
- Sample_Dataset_cont-3D-snGFR.lif (Sample file with time series and z-stack of three different glomeruli after injection of LuciferYellow for the analysis of single nephron GFR)
- Results.zip (Sample file for the selection (ROI sets) of the proximal tubulus in the sample dataset, including the resulting measurements (text files) in the time series and 3D modelling of the proximal tubules (tiff files))
- Graphs_2020-09-30.zip (Intermediate results and graphs (png files) as obtained from the sample dataset with selections and measurement data in the results file)
- 2020-09-30-Result_summary.txt (Final summary (text file) of calculated single nephron GFR for the three sample glomeruli based on selections from the results file)
- Dataset1.lif (Image data used for the comparison of previous and extended workflow in Figure 4, includes 15 time series and the corresponding z-stacks)
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).
Source code available from: https://github.com/NephrologieDresden/cont-3D-snGFR
Archived source code at time of publication: https://doi.org/10.5281/zenodo.464242715.
License: GNU General Public License v3.0
The authors acknowlegde the support of the Core Facility Cellular Imaging (CFCI) at the Medical Faculty Carl Gustav Carus, Technical University Dresden. This publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).
Views | Downloads | |
---|---|---|
F1000Research | - | - |
PubMed Central
Data from PMC are received and updated monthly.
|
- | - |
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Multiphoton microscopy and Light-sheet microscopy with application to mouse modes.
Is the work clearly and accurately presented and does it cite the current literature?
No
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Multiphoton microscopy and Light-sheet microscopy with application to mouse modes.
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
No
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Renal pathophysiology, intravital imaging
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Bioimage analysis, Computer vision, Data science
Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
No
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Partly
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Bioimage analysis, Computer vision, Data science
Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
No
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
No source data required
Are the conclusions drawn adequately supported by the results?
Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Nephrology, imaging
Alongside their report, reviewers assign a status to the article:
Invited Reviewers | ||||
---|---|---|---|---|
1 | 2 | 3 | 4 | |
Version 3 (revision) 10 Jun 21 |
read | |||
Version 2 (revision) 12 Apr 21 |
read | read | read | |
Version 1 26 Nov 20 |
read | read |
Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list:
Sign up for content alerts and receive a weekly or monthly email with all newly published articles
Already registered? Sign in
The email address should be the one you originally registered with F1000.
You registered with F1000 via Google, so we cannot reset your password.
To sign in, please click here.
If you still need help with your Google account password, please click here.
You registered with F1000 via Facebook, so we cannot reset your password.
To sign in, please click here.
If you still need help with your Facebook account password, please click here.
If your email address is registered with us, we will email you instructions to reset your password.
If you think you should have received this email but it has not arrived, please check your spam filters and/or contact for further assistance.
Comments on this article Comments (0)