Name: Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy (Video) | JoVE Journal
Uploaded: 2020-02-02T13:00:00.000+00:00
Duration: 7 min 35 s
Description: Learn to quantify liver size in larval zebrafish using brightfield microscopy for developmental biology research. This method enables precise measurement of organ growth and assessment of genetic and pharmacologic effects.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We would like to acknowledge Maurine Hobbs and the Centralized Zebrafish Animal Resource (CZAR) at the University of Utah for providing zebrafish husbandry, laboratory space, and equipment to carry out portions of this research. Expansion of the CZAR is supported in part by NIH grant # 1G20OD018369-01. We would also like to thank Rodney Stewart, Chloe Lim, Lance Graham, Cody James, Garrett Nickum, and the Huntsman Cancer Institute (HCI) Zebrafish Facility for zebrafish care. We would like to thank Kenneth Kompass for help with R programming. This work was funded in part by grants from the Huntsman Cancer Foundation (in conjunction with grant P30 CA042014 awarded to Huntsman Cancer Institute) (KJE) and NIH/NCI R01CA222570 (KJE).

Animal studies are carried out following procedures approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Utah.
1. Fixing Larvae
<ol>
	<li>At 3&#8211;7 days post fertilization (dpf), euthanize larvae with tricaine methanesulfonate (0.03%) and collect up to 15 larvae in a 2 mL tube using a glass pipette and pipette pump.</li>
	<li>Wash larvae twice with 1 mL of cold (4 &#176;C) 1x phosphate-buffered saline (PBS) on ice. For each wash, remove as much liquid as possible from the tube with a glass pipette and pipette pump, and then add 1 mL of cold PBS to tube.</li>
	<li>Remove as much PBS as possible using a glass pipette and pipette pump, and add 1 mL of cold (4 &#176;C) 4% paraformaldehyde (PFA) in PBS. 
	CAUTION: PFA is an irritant and suspected carcinogen. Gloves should be worn when handling PFA, and concentrated solutions should be handled in a chemical fume hood.</li>
	<li>Incubate at 4 &#176;C at least overnight (but up to several months) with gentle rocking.</li>
</ol>
2. Dissecting Tissues Surrounding Liver
<ol>
	<li>Remove larvae from PFA by rinsing 3x with 1 mL of cold (4 &#176;C) PBS and rocking for 5 min in between rinses. 
	NOTE: It is okay to keep the rinsed larvae in PBS for a day or two at 4 &#176;C.</li>
	<li>Pipette several larvae in PBS into one well of a 9-well round-bottom glass dish.</li>
	<li>Remove skin surrounding liver.
	<ol>
		<li>Use fine forceps to hold larva on its back (belly up), gripping on either side of the head as gently as possible. Then use very fine forceps in your other hand to grab the skin just overlying the heart.</li>
		<li>Pull skin down diagonally towards the tail of the fish and the bottom of the dish on the left or right side of the fish. Repeat for other side (right or left side).</li>
		<li>Continue grabbing flaps of skin and pulling down/back until all of the skin and melanophores overlying or near the liver have been removed.</li>
	</ol>
	</li>
	<li>Remove yolk, if present.
	<ol>
		<li>For 5&#8211;6 dpf larvae, lift yolk off in one piece by holding the fish with fine forceps on its back and using the very fine forceps to prod the yolk gently.</li>
		<li>For 3&#8211;4 dpf larvae, scrape the yolk off in pieces. Hold the fish with fine forceps on its back and use the very fine forceps to stroke the yolk, starting from the ventral side.</li>
	</ol>
	</li>
	<li>Place dissected larvae into fresh cold PBS using a glass pipette and pipette pump.</li>
</ol>
3. Imaging
<ol>
	<li>To mount larvae, pour a few mL of 3% methyl cellulose onto the lid of a clean plastic Petri dish.</li>
	<li>Use a glass pipette and pipette pump to add larvae to the methyl cellulose, adding as little PBS with the larvae as possible.</li>
	<li>Under a dissecting microscope at low magnification, use fine forceps to orient the fish so they are laying on their right side, facing left. 
	NOTE: Make sure the fish are oriented perfectly on their side or the liver measurements may not be accurate.</li>
	<li>Take a picture of each fish.
	<ol>
		<li>Confirm that the fish to be photographed is aligned perfectly, with one eye directly on top of the other eye. If necessary, use fine forceps to tap lightly on head or tail of fish to adjust orientation. If fish&#39;s tail is bent, remove the tail by pinching it forcefully with forceps to remove it so the fish lays flat.</li>
		<li>Zoom in to high magnification and focus on the liver, making sure that the liver&#39;s outline is clearly visible.</li>
		<li>Snap a picture and save the file.</li>
		<li>Repeat for all fish, making sure the magnification is the same for each picture.</li>
		<li>Take a picture of a micrometer using the same magnification (see Figure 2H).</li>
	</ol>
	</li>
</ol>
4. Image Analysis
<ol>
	<li>Measure the area of each fish&#39;s liver using image processing software.
	<ol>
		<li>Blind all liver pictures to avoid potential investigator bias and promote scientific rigor15. This step can be done manually by another lab member or using a computer program (Supplementary Material). Rename files randomly and create a &#34;randomization file&#34; containing a list of the original file names and corresponding blinded file names.</li>
		<li>Open randomized files in order, starting with file 1.</li>
		<li>Choose the freehand selections tool and outline each liver.</li>
		<li>Press Ctrl-M to measure the area of each liver.</li>
		<li>For any livers that cannot be accurately measured, insert a placeholder measurement (very small or very large, so it can be easily excluded later on).</li>
		<li>Save the measurements in a text file (&#34;measurements file&#34;).</li>
	</ol>
	</li>
	<li>Un-blind and analyze data
	<ol>
		<li>Open &#34;measurements file&#34; and &#34;randomization file&#34; in a spreadsheet program.</li>
		<li>Insert a new column in the &#34;measurements file&#34; and add the original file names for the blinded files, using the &#34;randomization file&#34;. Save this file as &#34;unblinded measurements file&#34;.</li>
		<li>Sort data by original file name.</li>
		<li>Be sure to exclude any liver measurements for which pictures were inadequate (see Figure 2A&#8211;G).</li>
		<li>If necessary, convert measurement values into desired scale (mm2, for example).
		<ol>
			<li>Open the scale bar in the image processing software.</li>
			<li>Use the straight line tool to measure 1 mm on the scale bar. The image processing software will measure in the same units as the livers (pixels), giving a conversion factor.</li>
			<li>Use the conversion factor to convert measurements in the &#34;unblinded measurements file&#34;.</li>
		</ol>
		</li>
		<li>Using the spreadsheet program or pasting data into a scientific graphing and statistics software, determine mean and standard deviation and calculate p value(s).</li>
	</ol>
	</li>
</ol>

<ol><li>Lin, D. -C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomic+and+Epigenomic+Heterogeneity+of+Hepatocellular+Carcinoma%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Genomic and Epigenomic Heterogeneity of Hepatocellular Carcinoma.</a> Cancer Research. 77 (9), 2255-2265 (2017).</li>
<li>Ghouri, Y. A., Mian, I., Rowe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+of+hepatocellular+carcinoma%3A+Epidemiology%2C+etiology%2C+and+carcinogenesis%5BTitle%5D)+AND+%22Journal+of+Carcinogenesis%22%5BJournal%5D)">Review of hepatocellular carcinoma: Epidemiology, etiology, and carcinogenesis.</a> Journal of Carcinogenesis. 16, 1(2017).</li>
<li>Evason, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+Chemical+Inhibitors+of+%CE%B2-Catenin-Driven+Liver+Tumorigenesis+in+Zebrafish%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish.</a> PLoS Genetics. 11 (7), 1005305(2015).</li>
<li>Kalasekar, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterogeneous+beta-catenin+activation+is+sufficient+to+cause+hepatocellular+carcinoma+in+zebrafish%5BTitle%5D)+AND+%22Biology+Open%22%5BJournal%5D)">Heterogeneous beta-catenin activation is sufficient to cause hepatocellular carcinoma in zebrafish.</a> Biology Open. 8 (10), (2019).</li>
<li>Nguyen, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high+level+of+liver-specific+expression+of+oncogenic+Kras%28V12%29+drives+robust+liver+tumorigenesis+in+transgenic+zebrafish%5BTitle%5D)+AND+%22Disease+Models+%26+Mechanisms%22%5BJournal%5D)">A high level of liver-specific expression of oncogenic Kras(V12) drives robust liver tumorigenesis in transgenic zebrafish.</a> Disease Models & Mechanisms. 4 (6), 801-813 (2011).</li>
<li>Nguyen, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+inducible+kras%28V12%29+transgenic+zebrafish+model+for+liver+tumorigenesis+and+chemical+drug+screening%5BTitle%5D)+AND+%22Disease+Models+%26+Mechanisms%22%5BJournal%5D)">An inducible kras(V12) transgenic zebrafish model for liver tumorigenesis and chemical drug screening.</a> Disease Models & Mechanisms. 5 (1), 63-72 (2012).</li>
<li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+transgenic+zebrafish+liver+tumor+model+with+inducible+Myc+expression+reveals+conserved+Myc+signatures+with+mammalian+liver+tumors%5BTitle%5D)+AND+%22Disease+Models+%26+Mechanisms%22%5BJournal%5D)">A transgenic zebrafish liver tumor model with inducible Myc expression reveals conserved Myc signatures with mammalian liver tumors.</a> Disease Models & Mechanisms. 6 (2), 414-423 (2013).</li>
<li>Cox, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Yap+reprograms+glutamine+metabolism+to+increase+nucleotide+biosynthesis+and+enable+liver+growth%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth.</a> Nature Cell Biology. 18 (8), 886-896 (2016).</li>
<li>Sadler, K. C., Amsterdam, A., Soroka, C., Boyer, J., Hopkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genetic+screen+in+zebrafish+identifies+the+mutants+vps18%2C+nf2+and+foie+gras+as+models+of+liver+disease%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease.</a> Development. 132 (15), Cambridge, England. 3561-3572 (2005).</li>
<li>Huang, X., Zhou, L., Gong, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liver+tumor+models+in+transgenic+zebrafish%3A+an+alternative+in+vivo+approach+to+study+hepatocarcinogenes%5BTitle%5D)+AND+%22Future+Oncology%22%5BJournal%5D)">Liver tumor models in transgenic zebrafish: an alternative in vivo approach to study hepatocarcinogenes.</a> Future Oncology. 8 (1), London, England. 21-28 (2012).</li>
<li>Yan, C., Yang, Q., Gong, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor-Associated+Neutrophils+and+Macrophages+Promote+Gender+Disparity+in+Hepatocellular+Carcinoma+in+Zebrafish%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Tumor-Associated Neutrophils and Macrophages Promote Gender Disparity in Hepatocellular Carcinoma in Zebrafish.</a> Cancer Research. 77 (6), 1395-1407 (2017).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li>
<li>Rueden, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ImageJ2%3A+ImageJ+for+the+next+generation+of+scientific+image+data%5BTitle%5D)+AND+%22BMC+Bioinformatics%22%5BJournal%5D)">ImageJ2: ImageJ for the next generation of scientific image data.</a> BMC Bioinformatics. 18 (1), 529(2017).</li>
<li>Schindelin, J., Rueden, C. T., Hiner, M. C., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ImageJ+ecosystem%3A+An+open+platform+for+biomedical+image+analysis%5BTitle%5D)+AND+%22Molecular+Reproduction+and+Development%22%5BJournal%5D)">The ImageJ ecosystem: An open platform for biomedical image analysis.</a> Molecular Reproduction and Development. 82 (7-8), 518-529 (2012).</li>
<li>Landis, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+call+for+transparent+reporting+to+optimize+the+predictive+value+of+preclinical+research%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A call for transparent reporting to optimize the predictive value of preclinical research.</a> Nature. 490 (7419), 187-191 (2012).</li>
<li>Dai, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+fat+plus+high+cholesterol+diet+lead+to+hepatic+steatosis+in+zebrafish+larvae%3A+a+novel+model+for+screening+anti-hepatic+steatosis+drugs%5BTitle%5D)+AND+%22Nutrition+%26+Metabolism%22%5BJournal%5D)">High fat plus high cholesterol diet lead to hepatic steatosis in zebrafish larvae: a novel model for screening anti-hepatic steatosis drugs.</a> Nutrition & Metabolism. 12, 42(2015).</li>
<li>Kim, S. -H., Speirs, C. K., Solnica-Krezel, L., Ess, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+model+of+tuberous+sclerosis+complex+reveals+cell-autonomous+and+non-cell-autonomous+functions+of+mutant+tuberin%5BTitle%5D)+AND+%22Disease+Models+%26+Mechanisms%22%5BJournal%5D)">Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin.</a> Disease Models & Mechanisms. 4 (2), 255-267 (2011).</li>
<li>Delous, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sox9b+is+a+key+regulator+of+pancreaticobiliary+ductal+system+development%5BTitle%5D)+AND+%22PLoS+Genetics%22%5BJournal%5D)">Sox9b is a key regulator of pancreaticobiliary ductal system development.</a> PLoS Genetics. 8 (6), 1002754(2012).</li>
<li>Shin, D., Lee, Y., Poss, K. D., Stainier, D. Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Restriction+of+hepatic+competence+by+Fgf+signaling%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Restriction of hepatic competence by Fgf signaling.</a> Development. 138 (7), Cambridge, England. 1339-1348 (2011).</li>
<li>Yin, C., Evason, K. J., Maher, J. J., Stainier, D. Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+basic+helix-loop-helix+transcription+factor%2C+heart+and+neural+crest+derivatives+expressed+transcript+2%2C+marks+hepatic+stellate+cells+in+zebrafish%3A+analysis+of+stellate+cell+entry+into+the+developing+liver%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">The basic helix-loop-helix transcription factor, heart and neural crest derivatives expressed transcript 2, marks hepatic stellate cells in zebrafish: analysis of stellate cell entry into the developing liver.</a> Hepatology. 56 (5), Baltimore, Md. 1958-1970 (2012).</li>
</ol>

The authors have nothing to disclose.

Quantification of liver size is crucial in studies aimed at understanding liver development, regeneration, and oncogenesis. The protocol described here is a relatively quick, easy, and cheap technique for liver size quantification in larval zebrafish. Exercising appropriate caution while performing certain aspects of the protocol can aid in increased accuracy of results and decreased frustration.
Proper fixation of the larvae is crucial towards getting well-preserved biological samples and pre...

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver1 and the third leading cause of cancer-related mortality2. To better understand mechanisms of hepatocarcinogenesis and identify potential HCC therapeutics, we and others have developed transgenic zebrafish in which hepatocyte-specific expression of oncogenes such as &#946;-catenin3,4, Kras(V12)5,6, Myc7, or Yap18 leads to HCC in adult animals. In these zebrafish, liver enlargement is noted as early as 6 days post fertilization (dpf), providing a facile platform for testing the effects of drugs and genetic alterations on oncogene-driven liver overgrowth. Accurate and precise measurement of larval liver size is essential for determining the effects of these manipulations.
Liver size and shape can be assessed semi-quantitatively in fixed zebrafish larvae by CY3-SA labeling9 or in live zebrafish larvae using hepatocyte-specific fluorescent reporters and fluorescence dissecting microscopy5,6. The latter method is relatively quick, and its lack of precision can be addressed by photographing and measuring the area of each liver using image processing software7,10. However, it can be technically challenging to uniformly position all live larvae in an experiment such that two-dimensional liver area is an accurate representation of liver size. A similar technique for quantifying liver size involves using light sheet fluorescence microscopy to quantify larval liver volume8, which may be more accurate for detecting size differences when the liver is expanded non-uniformly in different dimensions. Fluorescence-activated cell sorting (FACS) can be used to count the number of fluorescently labeled hepatocytes and other liver cell types in larval livers8,11. In this method, larval livers are pooled and dissociated, so information about individual liver size and shape is lost. In combination with another liver size determination method, FACS enables differentiation between increased cell number (hyperplasia) and increased cell size (hypertrophy). All of these methods employ expensive fluorescence technology (microscope or cell sorter) and, except for CY3-SA labeling, require labeling of hepatocytes with a fluorescent reporter.
Here we describe in detail a method for quantifying zebrafish larval liver area using bright-field microscopy and image processing software3,12,13,14. This protocol enables precise quantification of the area of individual livers in situ without the use of fluorescence microscopy. While analyzing liver size, we blind the image identity to reduce investigator bias and improve scientific rigor15.

<table><tbody><tr><td>Camera for dissecting microscope</td><td>Leica, for example</td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica, for example</td><td></td><td></td></tr><tr><td>Fine (Dumont #5) forceps</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Glass pipets</td><td>VWR</td><td>14672-608</td><td></td></tr><tr><td>Image analysis software</td><td>Image J/FIJI</td><td></td><td>ImageJ/FIJI can be dowloaded for free: &lt;a href=&quot;https://imagej.net/Welcome&quot; target=&quot;_blank&quot;&gt;https://imagej.net/Welcome&lt;/a&gt;</td></tr><tr><td>Methyl cellulose</td><td>Sigma</td><td>M0387</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Various suppliers</td><td></td><td></td></tr><tr><td>Pipette pump</td><td>VWR</td><td>53502-233</td><td></td></tr><tr><td>Plastic Petri dishes</td><td>USA Scientific Inc</td><td>2906</td><td></td></tr><tr><td>Pyrex 9-well round-bottom glass dish</td><td>VWR</td><td>89090-482</td><td></td></tr><tr><td>Software for blinding files</td><td>R project</td><td></td><td>R can be downloaded for free: &lt;a href=&quot;https://www.r-project.org/&quot; target=&quot;_blank&quot;&gt;https://www.r-project.org/&lt;/a&gt;</td></tr><tr><td>Scientific graphing and statistics software</td><td>GraphPad Prism</td><td></td><td></td></tr><tr><td>Spreadsheet program</td><td>Microsoft Excel</td><td></td><td></td></tr><tr><td>Tricaine methanesulfonate (Tricaine-S)</td><td>Western Chemical</td><td>200-226</td><td></td></tr><tr><td>Very fine (Dumont #55) forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr></tbody></table>

quantifying liver size in larval zebrafish using brightfield microscopy

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Transgenic zebrafish expressing hepatocyte-specific activated β-catenin (Tg(fabp10a:pt-β-cat) zebrafish)3 and non-transgenic control siblings were euthanized at 6 dpf and liver area was quantified using brightfield microscopy and image processing software. Transgenic zebrafish have significantly increased liver size (0.0006 cm2) as compared to their non-transgenic siblings (0.0004 cm2, p &lt; 0.0001; Figure 1).
<p c...

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

quantifying-liver-size-larval-zebrafish-using-brightfield

Research

JoVE Journal

Developmental Biology

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change.

Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

Partial Hepatectomy in Adult Zebrafish

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

Presented here is a protocol for in vivo whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy. The experimental procedure includes sample preparation, image acquisition, and visualization.

In Vivo Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Learn to quantify liver size in larval zebrafish using brightfield microscopy for developmental biology research. This method enables precise measurement of organ growth and assessment of genetic and pharmacologic effects.

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

Explore More Videos