Name: automated measurement of cryptococcal species polysaccharide capsule and cell body
Uploaded: 2018-01-11T15:00:00
Description: Watch this Scientific Journal Video about Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body at JoVE.com

We would like to acknowledge Anthony Bowen whose slides were used as a second human side-by-side comparison as well as Sabrina Nolan whose slides were used as a third human side-by-side and second microscope comparison.

1. Preparation of India Ink Slide
<ol>
	<li>Pipette 10 &#181;L of cryptococcal sample onto a slide. Any circular yeast strain will work but for this experiment H99 was the only strain used. 
	NOTE: If the sample is directly from culture media, diluting 1:2 with PBS or water can help prevent the India Ink from clumping.</li>
	<li>Pipette 2 &#181;L of India Ink stain onto the sample and mix by physically pushing the pipette tip to the sample and moving in a circular motion until the India Ink appears evenly distribu...

<ol>
	<li>Park, B. J., Wannemuehler, K. A., Marston, B. J., Govender, N., Pappas, P. G., Chiller, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+the+current+global+burden+of+cryptococcal+meningitis+among+persons+living+with+HIV/AIDS.">Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS.</a> AIDS. 23 (4), 525-530 (2009).</li><li>Kwon-Chung, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+neoformans+and+Cryptococcus+gattii,+the+etiologic+agents+of+cryptococcosis.">Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis.</a> Cold Spring Harb Perspect Med. 4 (7), 019760 (2014).</li><li>Granger, D. L., Perfect, J. R., Durack, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Cryptococcus+neoformans.+Regulation+of+capsule+synthesis+by+carbon+dioxide.">Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide.</a> J Clin Invest. 76 (2), 508 (1985).</li><li>Rumbaugh, J., Pool, A., Gainey, L., Forrester, K., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Cryptococcal+Capsule+in+Pathogenesis+of+Cryptococcal+Meningitis.">The Role of Cryptococcal Capsule in Pathogenesis of Cryptococcal Meningitis.</a> Neurology. 80 (7), 007 (2013).</li><li>Bojarczuk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+neoformans+Intracellular+Proliferation+and+Capsule+Size+Determines+Early+Macrophage+Control+of+Infection.">Cryptococcus neoformans Intracellular Proliferation and Capsule Size Determines Early Macrophage Control of Infection.</a> Sci Rep. 6, (2016).</li><li>Robertson, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+neoformans+Ex+Vivo+Capsule+Size+Is+Associated+With+Intracranial+Pressure+and+Host+Immune+Response+in+HIV-associated+Cryptococcal+Meningitis.">Cryptococcus neoformans Ex Vivo Capsule Size Is Associated With Intracranial Pressure and Host Immune Response in HIV-associated Cryptococcal Meningitis.</a> J Infect Dis. 209 (1), 74-82 (2014).</li><li>Araujo, G. d. e. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsules+from+Pathogenic+and+Non-Pathogenic+Cryptococcus+spp.+Manifest+Significant+Differences+in+Structure+and+Ability+to+Protect+against+Phagocytic+Cells.">Capsules from Pathogenic and Non-Pathogenic Cryptococcus spp. Manifest Significant Differences in Structure and Ability to Protect against Phagocytic Cells.</a> PLoS One. 7 (1), 29561 (2012).</li><li>Garc&#237;a-Rivera, J., Chang, Y. C., Kwon-Chung, K. J., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+neoformans+CAP59+(or+Cap59p)+Is+Involved+in+the+Extracellular+Trafficking+of+Capsular+Glucuronoxylomannan.">Cryptococcus neoformans CAP59 (or Cap59p) Is Involved in the Extracellular Trafficking of Capsular Glucuronoxylomannan.</a> Eukaryot Cell. 3 (2), 385-392 (2004).</li><li>Guimar&#227;es, A. J., Frases, S., Cordero, R. J. B., Nimrichter, L., Casadevall, A., Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+neoformans+responds+to+mannitol+by+increasing+capsule+size+in+vitro+and+in+vivo:+Mannitol+impacts+the+structure+of+C.+neoformans+capsule.">Cryptococcus neoformans responds to mannitol by increasing capsule size in vitro and in vivo: Mannitol impacts the structure of C. neoformans capsule.</a> Cell Microbiol. 12 (6), 740-753 (2010).</li><li>Zaragoza, O., Fries, B. C., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Capsule+Growth+in+Cryptococcus+neoformans+by+Mammalian+Serum+and+CO2.">Induction of Capsule Growth in Cryptococcus neoformans by Mammalian Serum and CO2.</a> Infect and Immun. 71 (11), 6155-6164 (2003).</li><li>Rossi, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Resistance+to+Fluconazole+on+Virulence+and+Morphological+Aspects+of+Cryptococcus+neoformans+and+Cryptococcus+gattii+Isolates.">Impact of Resistance to Fluconazole on Virulence and Morphological Aspects of Cryptococcus neoformans and Cryptococcus gattii Isolates.</a> Front Microbiol. 7, (2016).</li></ol>

The critical steps of this technique are preparing the India Ink slide and acquiring the microscope images. While the algorithm has been successfully tested with a variety of slide and image techniques the recommended protocol is described in this manuscript. The polysaccharide capsule is detected based on the exclusion of India Ink particles from the domain of the capsule as these particles are too large to penetrate the polysaccharide fibril network. India Ink exclusion results in a bright circle on top of a dark backg...

Cryptococcus neoformans is a pathogenic yeast found ubiquitously around the globe that is associated with human disease primarily in immunosuppressed populations. C. neoformans most notably accounts for a significant cause of total annual deaths in sub-Saharan Africa due to infectious disease1. The major clinical manifestation of cryptococcal infection is meningoencephalitis, which follows invasion of the central nervous system by transport in infected macrophages (Trojan horse manner) or direct crossing of the blood-brain barrier. C. neoformans expresses several virulence factors including the ability to replicate at human body temperature, urease activity, melanization, and formation of a polysaccharide capsule2. The polysaccharide capsule is composed of repeating glucuronoxylomannan and glucoronoxylomannangalactan polymers and functions as a protective barrier against factors such as environmental stress and host immune responses2.
Although the size of the cryptococcal polysaccharide capsule size has not consistently been associated with virulence, there is evidence that it is a factor in pathogenesis2,3,4,5,6,7. Capsule size is associated with meningitis pathology6, can affect macrophage ability to control Cryptococcus infection5, and can result in loss of virulence if absent8. Hence, capsule size measurements are common in cryptococcal research, but there is no fieldwide standard for a method of capsule measurement.
Currently, C. neoformans polysaccharide capsule measurement is based on manual measurements of microscopy images, and the exact methods of both image and measurement acquisitions vary across laboratories9,10,11. An immediate concern to this method is that some studies require the acquisition of thousands of individual measurements, which makes maintaining accuracy and reliability difficult. Furthermore, even when the results are published, there is often inadequate description of the measurement method. Many publications do not explain how their measurements were obtained, what focal plane was used, how they determined the threshold for capsule identification, whether they used radius or diameter, whether they used one measurement or averaged several, or other details. Some publications only state their method as which program was used, e.g., &#34;Adobe Photoshop CS3 was used to measure the cells&#34;11. This lack of standardization and reporting detail can make reproducibility difficult if not impossible. Differences in human eyesight, computer brightness, microscope settings, slide lighting, and other factors can vary not only between individuals but between samples, whereas calculations based on ratios of pixel intensity values will remain constant and applicable between samples. This technique was generated in the context of providing a standardized, accurate, rapid, and simple technique to measure capsules sizes for a field in which there was none before.
As previously mentioned, the CHT algorithm is long-established, and scripts to automatically detect circles have been written before. This method improves in two areas where other scripts would fall short. First, simply detecting circles is not enough, because with cryptococcal cells two distinct circles must be detected in relation to each other. This method specifically detects cell bodies within capsules, discriminates between the two, and performs calculations only on the relevant body-capsule pairs. Second, even when following the same protocol, different investigators will end up with different acquired images. By allowing the investigator control over every algorithm parameter, this tool can be adjusted to match a broad range of acquisition methods. There is no need for a standardized scope, objective, filter, and so on.
This technique can be readily applied to any situation in which the investigator needs to detect circles within an image that contrast with their background. Both circles lighter and darker than their background can be detected, counted, and measured using this technique.

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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:213px;">India Ink</td>
			<td style="width:99px;">Becton, Dickinson and Co.</td>
			<td style="width:119px;">261194</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Fisherbrand Superfrost Microscope Slides</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-143</td>
			<td>25x75x1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Fisherfinest Premium Cover Glass</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:119px;">12-548-B</td>
			<td>22x22-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Sally Hansen HardasNails Xtreme Wear Nail Polish</td>
			<td style="width:99px;">Sally Hansen</td>
			<td style="width:119px;">N/A</td>
			<td>109 invisible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">SAB Media</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">S3306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Cryptotoccus neoformans</td>
			<td style="width:99px;">ATCC</td>
			<td style="width:119px;">208821</td>
			<td>H99 strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Olympus AX70 Microscope</td>
			<td style="width:99px;">Olympus</td>
			<td style="width:119px;">AX70TRF</td>
			<td>Discontinued ; Bright Field Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Qimaging Retiga 1300</td>
			<td style="width:99px;">Qimaging</td>
			<td style="width:119px;">N/A</td>
			<td>Discontinued ; Camera Microscope Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">MATLAB</td>
			<td style="width:99px;">MathWorks</td>
			<td style="width:119px;">N/A</td>
			<td>Most recent version recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Python Programming Language</td>
			<td style="width:99px;">Python</td>
			<td style="width:119px;">N/A</td>
			<td>Version 2 necessary ; 2.7 recommended</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Microsoft Excel</td>
			<td style="width:99px;">Microsoft</td>
			<td style="width:119px;">N/A</td>
			<td>Most recent version recommended</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:119px;">P3813</td>
			<td></td>
		</tr>
	</tbody>
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automated measurement of cryptococcal species polysaccharide capsule and cell body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Images are first obtained by microscopy of India Ink slides using a bright field microscope coupled with a camera (Figure 1A). It is important to have cells separated and in sufficiently low density not to overwhelm the field of view, as well as to use enough stain to create contrast between cells and background. As stated in the protocol, the exact number of cells for an optimal image will vary depending on the sample, microscope, and object...

Watch this Scientific Journal Video about Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body at JoVE.com

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

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