We thank Donna Epps, histologist at Institute for Genomic Biology, University of Illinois Urbana-Champaign (UIUC), for her capable technical assistance in sample preparation and sectioning. This work was supported by a research grant to B.W. Fouke from the Office of Naval Research (N00014-00-1-0609). In addition, C.A.H. Miller received grants from the UIUC Department of Geology Wanless Fellowship, UIUC Department of Geology Leighton fund and UIUC Department of Geology Roscoe Jackson fieldwork fund. Interpretations presented in this manuscript are those of the authors and may not necessarily represent those of the granting institutions. We also thank the Caribbean Research and Management of Biodiversity (Carmabi) laboratory on Cura&#231;ao for their support and collaboration in collecting the coral tissue biopsy samples. We thank Claudia Lutz, IGB Media Communication Specialist for her able language correction.

NOTE: Reagents to be prepared for Serial Block Face Imaging of Coral Samples
1. Preinfiltration Wax
<ol>
	<li>Melt 3.6 g of STEARIN flakes in a glass beaker. Mix well on a hot plate (60-70 &#176;C).</li>
	<li>Add 400 mg of Sudan IV (to minimize wax background fluorescence). Mix well and wait until a red translucent solution is achieved.</li>
	<li>Add 96 ml&#160;hot molten paraffin (100%) and mix well.</li>
</ol>
1.2) Embedding Wax
<ol>
	<li>Melt 7.2 g of STEARIN flakes in a glass beaker and mix well on a hot plate (60-70 &#176;C).</li>
	<li>Add 0.8 g of Sudan IV. Mix well and wait until a red translucent solution is achieved.</li>
	<li>Add paraffin granules (162 g) and mix until paraffin melts completely.</li>
	<li>Add 30 g of white granular Vybar and melt completely in the same beaker; once melted, mix.</li>
	<li>Loosely close the glass bottle with a lid. Place the glass bottle in a 60 &#176;C convection oven to keep the ingredients in a liquid state. Carry out all infiltrations in this oven.</li>
	<li>Split the total volume of the 200 ml&#160;red wax in to two glass bottles of 100 ml&#160;each. Use one aliquot for infiltration and the other for final embedding.</li>
</ol>
1.3) Embedding Coral Tissues for Serial Block Face Imaging
<ol>
	<li>Wash the coral polyps collected in the field (SI Video 1) and stored (3-6 months at 4-5 &#176;C in paraformaldehyde) in phosphate buffered saline (3x 5 min) and decalcify when ready to be imaged. Decalcify the polyps in ExCal solution&#160;for 24 hr or until as the polyps are totally devoid of CaCO3. Incubate several decalcified coral polyps as a single block in a 25, 50, 75, and 100% ethanol series, followed by 1x xylene substitute&#160;to dehydrate the samples.</li>
	<li>Place the processed polyps in a preheated 65 &#176;C oven containing 100% xylene substitute. Incubate for 30 min twice by changing to fresh solution. Orient the polyps in such a way that the top of the polyps faces down and the top surface is as flat as possible.</li>
	<li>Make solutions of 2:1, 1:1, and 1:2 xylene substitute and preinfiltration wax (step 1.1) in 50 ml Falcon tubes.</li>
	<li>Incubate coral polyps with these three increasing concentrations of preinfiltration wax, followed by 3x&#160;incubation in 100% preinfiltration wax for 30-60 min&#160;each time.</li>
	<li>Note: Depending on the thickness of the samples, steps 1.3.1-1.3.5 could be increased with longer periods of time.</li>
	<li>Move the samples to embedding wax (see step 1.2.6) after 3x incubation in 100% preinfiltration wax.</li>
	<li>Remove the embedding wax after 30 min. Replace with fresh embedding wax and continue incubation for a minimum of 4 hr at 65 &#176;C.</li>
</ol>
1.4) Embedding in Red Wax
<ol>
	<li>Photograph the block (the stainless steel tray where the white wax is placed and on top of which the sample is placed). This is necessary because once embedded in the wax, the location of the sample will become invisible as the embedding red wax is opaque.</li>
	<li>Place small drops of high melting point wax around the new preheated stainless steel embedding mold and allow it to cool. Pour a small volume of freshly melted embedding wax from second container as stated in step 1.2.6.</li>
	<li>Position the coral polyp facing down quickly over the white wax dot, then place a plastic sample holder on the stainless steel tray and pour more embedding wax so that the wax comes up to the surface of the plastic mold.</li>
	<li>Take the entire setup out of 65 &#176;C oven and allow it to cool on a bench or a cool surface until the wax completely hardens.</li>
	<li>This may take 6 hr up to day or two. Place the block desiccated in a refrigerator at 4 &#176;C, protected from light, for long term storage.</li>
</ol>
1.5) Sectioning at the Serial Block Face Setup
<ol>
	<li>Trim the block and cut 1 &#956;m sections using a microtome. Do not collect the sections as they will become like powder. Remember, we are imaging only the block face.&#160;The sample appears when the white wax begins to disappear.</li>
	<li>Capture images of the smooth block face which contains the sample every time a section is removed. Continue until the coral polyp disappears as in SI Video 2.</li>
	<li>Record/Capture the images with a monochrome camera using a FITC fluorescent filter to pick up the auto-fluorescence of the chromatophores/coral polyp. Image 3-4 decalcified cores from each species.</li>
</ol>
2. Imaging Corals Under Two-photon Fluorescence Microscopy
<ol>
	<li>Fix coral polyp cores, each containing around 10-12 polyps about an inch in diameter, in 4% paraformaldehyde at the site of collection (sea shore) as soon as they are harvested under water.</li>
	<li>Keep samples at 4 &#176;C until imaging. Washed cores are placed in the same solution upside down in a cover glass bottom dish (0.17 mm thick).</li>
	<li>Using a two photon laser at 780 nm excitation, image 3-4 polyps at two different magnifications (digital zoom) using a 10X&#160;(0.3 NA) objective. The coral polyp shape and height varies between samples (usually 1-2 mm) and the imaging depth is also limited by the imaging objective&#8217;s working distance.</li>
	<li>Use the tile scan mode to collect approximately 25-100 (5 x 5 or 10 x 10 tiles) images per focal plane in xy&#160;and 50-100 images through the z&#160;axis at 10 or 20 &#181;m interval, totaling around 5,000-10,000 images/coral polyp.</li>
	<li>Image three to four polyp areas to represent a core and coral species. NOTE: Image acquisition time varies between 2-5 hr/polyp area.</li>
	<li>Store all images in raw data format in the system&#8217;s hard disk as LSM 5. Render 3D in a 3D image analysis and rendering software.</li>
</ol>
3. 3D Volume Rendering and Visualization of SBFI and Two-photon Spectral Fluorescence Data
<ol>
	<li>Crop the 2D data to reduce the file size by focusing on a single polyp using the square cropping tool in the program and compile as a single tiff file (reduce the file size also by saving the file in 8 bit format) in the acquisition software.</li>
	<li>Open the assembled files of the SBFI data (collected in step 1.5.1) or the tiled multiple z-stacks of two photon optical sections (collected in step 2.1.4) in the program Imaris Surpass module under volume algorithm.</li>
	<li>Project the SBFI data rendered in 3D using a shadow projection. Create an isosurface mode where the voxels are thresholded to create a solid surface pattern (SI Video 3).</li>
	<li>Visualize the 3D projections using a clipping plane algorithm at xy, xz, and yz&#160;orthogonal modes to reveal 3D structure and shape of corals.</li>
	<li>Animate the projections using a key frame animation module in the same Imaris program (SI Videos 3-7).</li>
	<li>Generate video files at 5% compression and generate a movie clips in avi format using volume (SI Videos 4 and 6), 3D and Isosurface modalities (SI Videos 5 and 7).</li>
</ol>

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The authors declare no conflict of interests.

Coral reef research is a highly interdisciplinary research effort, involving analysis of the simultaneous physical, chemical, and biological phenomena that operate in the marine environment. The study of complex coral reef ecosystems is therefore best completed within a &#8216;Powers of Ten&#8217; contextual framework (Figure&#160;10). This graphic compilation illustrates that the coral ecosystem covers a wide range of spatial dimensions (10-9 to 105 m). Furthermore, this exercise i...

Global warming and accompanying environmental change are directly affecting the health and distribution of tropical marine corals1-4. Multiple impacts are being observed, including coral bleaching and the emergence of infectious diseases5-6. However, more accurate prediction of future coral response to these environmental threats will require that a histological &#8220;baseline&#8221; be established, which defines tissue morphology and cell composition and distribution for &#8220;apparently healthy&#8221; corals. In turn, &#8220;impacted&#8221; corals can then be quantitatively compared. Furthermore, this baseline should be established for apparently healthy corals under a variety of environmental conditions, so that &#8220;healthy response&#8221; can also be gauged across environmental gradients. As an initial step toward establishing this baseline, a high-resolution 3D study has been undertaken of how apparently healthy coral polyp tissue morphology and cellular composition responds to increases in water depth (WD) and accompanying decreases in sunlight irradiance. Results can then be used to establish a more comprehensive mechanistic understanding of coral adaptation, as well as to gain insight into coral-symbiont evolution and the enhancement of light harvesting.
Stony corals (Scleractinia) are colonial marine invertebrate animals that play host to a complex assemblage of other microorganisms, collectively referred to as the coral holobiont7-10. The research undertaken in the present study seeks to use a suite of cutting-edge imaging technologies to simultaneously track changes with increasing water depth in the tissue pigments and symbiotic zooxanthellae of apparently healthy host corals. This will establish the required comparative tissue cell &#8220;baseline&#8221; across a bathymetric gradient for apparently healthy corals and act as indicators of coral health10. Coral pigments, called chromatophores, act to absorb, reflect, scatter, refract, diffract, or otherwise interfere with incident solar radiation11. The zooxanthellae-chromatophore endosymbiotic relationship has enabled the coevolution of strategically advantageous light-harvesting optimization and skeletal growth strategies, as well as trophic plasticity (shifting feeding strategies back-and-forth from autotrophy to heterotrophy) for the coral animal12.
The southern Caribbean island nation of Cura&#231;ao (formerly part of the Netherlands Antilles) lies approximately 65 km north of Venezuela within the east-west trending Aruba-La Blanquilla archipelago (Figure 1A). The 70 km long southern coast of Cura&#231;ao contains a continuous modern and Miocene-Pliocene-Pleistocene-Holocene ancient fringing coral reef tract13,14. Mean annual SST on Cura&#231;ao varies approximately 3 &#176;C annually, ranging from a minimum of 26 &#176;C in late January to a maximum of 29 &#176;C in early September, with a mean annual temperature of 27.5&#177;0.5 &#186;C (NOAA SST Data Sets, 2000-2010). The coral reef at Playa Kalki (12&#176;22&#8217;31.63&#8221;N, 69&#176;09&#8217;29.62&#8221;W), lying near the northwestern tip of Cura&#231;ao (Figure 1A), was chosen for sampling because it has been previously well-studied and the marine ecosystem at this location is bathed in fresh nonpolluted seawater7,15-19. Two closely related scleractinian coral species, M. annularis and M. faveolata, were chosen for experimentation and analysis in this study because each species: (1) exhibits distinctly different and nonoverlapping bathymetric distributions on the reef tract with respect to the shelf break and the associated carbonate sedimentary depositional environments (M. annularis range = 0-10 m WD; M. faveolata range = 10-20 m WD20; Figures 1B, 2A, and 2B); (2) is a common coral reef framework builder throughout the Caribbean Sea21; and (3) has well-studied ecological, physiological, and evolutionary relationships22.
Field sampling for the present study was conducted using standard SCUBA diving techniques offshore of Playa Kalki on Cura&#231;ao. A shallow-to-deep water bathymetric transect was established that ran across the shelf, over the shelf break, and into the deep water fore reef environments. Apparently healthy coral heads were then identified for sampling along this bathymetric transect, including: (1) three individual ~ 1 m diameter coral heads of M. annularis, all of which were at 5 m water depth (WD); and (2) three individual ~ 1 m diameter coral heads of M. faveolata, all of which were at 12 m WD. Photosynthetically active radiation (PAR) was measured as 33-36% PAR at 5 m WD and 18-22% PAR at 10 m WD. Sampling was conducted in January when the SST was 26 &#176;C at the water depths of both the 5 m and 12 m. Each of these six coral heads was sampled in triplicate at equivalent spatial positions (i.e., approximately 45&#176; N latitude on each of the six hemispherical coral heads). Each individual sample consisted of a 2.5 cm diameter coral tissue-skeleton core biopsy that was collected with a cleaned arch punch. Three coral tissue-skeleton biopsies were sampled on standard SCUBA with gloved hands from each of the coral heads (9 from M. annularis colonies at 5 m WD and 9 from M. faveolata at 12 m WD). Immediately upon collection at depth, each biopsy core sample was placed in a sterile 50 ml polypropylene centrifuge tube, screw-top sealed, and returned to the surface. The seawater was decanted from each centrifuge tube and each core biopsy was then immersed, stored, and transported in 4% paraformaldehyde.
SBFI imaging has previously been performed on a wide range of biological samples, including whole-brain and whole-heart human tissues, intact mouse embryos, zebra fish embryos, and multiple types of animal samples with intact bones23-30. Most of these studies utilized optical/light microscopy with either fluorescence or bright field techniques. However, studies have been conducted at ultra-high magnifications using scanning electron serial block face imaging in the past31. In the present study, a modified SBFI protocol has been developed for and applied to corals for the first time. Because M. annularis and M. faveolata coral polyps are 1-2 mm in thickness, none of the routine light microscopy techniques would be capable of penetrating the entire thickness of coral polyp tissue. Therefore, we have SBFI sample preparation protocol specifically designed for coral samples. In addition, we have custom designed a stereomicroscope holder, which is motorized to move in both x and y directions. This apparatus takes images of the block face of the sample rather than collecting the sections using a regular microtome in front of the microscope. We also introduced another nonlinear optical two-photon microscopic technique to image the same coral polyps across the entire thickness of the coral tissues. This overcomes the limitations imposed by SBFI in terms of decalcification and the possibility of changes in tissue morphology and volume (shrinking) that may be induced by sample preparation (dehydration) and processing protocols. Furthermore, the emission profiles from the corals were spectrally resolved to identify their peak emissions and variations between the chromatophores and the photosynthetic zooxanthellae. These results were evaluated in the context of the method used and their individual advantages regarding acquisition time, analysis time, and the ability to resolve fine structural details without compromising structural integrity of the coral tissue.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Coral Tissue Skeleton</td>
			<td>None</td>
			<td>None</td>
			<td>2.5 cm Biopsy from natural habitat</td>
		</tr>
		<tr>
			<td>Arch Punch Coring Device</td>
			<td>C.S. Osborne and Company</td>
			<td>No. 149</td>
			<td>For Coral biopsy collection</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 15700</td>
			<td>16% Pre-diluted</td>
		</tr>
		<tr>
			<td>Histoclear/Safeclear II</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 64111-04</td>
			<td>Non-Toxic alternate to Xylene, Dehydration and Deparafinization</td>
		</tr>
		<tr>
			<td>Xylene and Ethanol</td>
			<td>Fisher Scientific</td>
			<td>Fisher Scientific</td>
			<td>Dehydration</td>
		</tr>
		<tr>
			<td>Paraffin Wax</td>
			<td>Richard Allen Scientific</td>
			<td>Type H REF 8338</td>
			<td>Infiltration solution</td>
		</tr>
		<tr>
			<td>Vybar</td>
			<td>The Candle Maker</td>
			<td>None</td>
			<td>Component of Red Wax</td>
		</tr>
		<tr>
			<td>Stearin</td>
			<td>The Candle Maker</td>
			<td>None</td>
			<td>Component of Red Wax</td>
		</tr>
		<tr>
			<td>Sudan IV</td>
			<td>Fisher Chemical</td>
			<td>S667-25</td>
			<td>Red Wax-Opaque background</td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin (WGA)</td>
			<td>Life Technologies</td>
			<td>W32466</td>
			<td>For labeling&#160; Coral Mucus</td>
		</tr>
		<tr>
			<td>Prolong Gold</td>
			<td>Life Technologies</td>
			<td>P36095</td>
			<td>Anti-fade mounting media</td>
		</tr>
		<tr>
			<td>Fluoro Dish</td>
			<td>World Precision Instruments</td>
			<td>FD-35-100</td>
			<td>For two-photon imaging</td>
		</tr>
		<tr>
			<td>XY Motor, Driver and Controller</td>
			<td>Lin Engineering</td>
			<td>211-13-01R0, R325, R256-RO</td>
			<td>XY Translational Movement</td>
		</tr>
		<tr>
			<td>Hot Plate</td>
			<td>Corning</td>
			<td>DC-220</td>
			<td>Melting all wax</td>
		</tr>
		<tr>
			<td>Convection Oven</td>
			<td>Yamato</td>
			<td>DX-600</td>
			<td>Infiltration and Embedding</td>
		</tr>
		<tr>
			<td>Tissue Processor</td>
			<td>Leica</td>
			<td>ASP 300</td>
			<td>Dehydration, Infiltration</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>RM2055</td>
			<td>Disposable knifes</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Carl Zeiss</td>
			<td>Stereolumar V 12</td>
			<td>1.5x (30 mm WD) Objective</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope with ApoTome</td>
			<td>Carl Zeiss</td>
			<td>Axiovert M 200, ApoTome I System</td>
			<td>Imaging thin section of a polyp: Zooxanthellae</td>
		</tr>
		<tr>
			<td>Axiocam camera</td>
			<td>Carl Zeiss</td>
			<td>MRm</td>
			<td>Monochrome camera 1388x1040 pixels</td>
		</tr>
		<tr>
			<td>Axiovision Software</td>
			<td>Carl Zeiss</td>
			<td>Version 4.8</td>
			<td>Image acquisition program</td>
		</tr>
		<tr>
			<td>Two-Photon Laser</td>
			<td>Spectraphysics</td>
			<td>Maitai eHP, pulsed laser (70 fs)</td>
			<td>With DeepSee module</td>
		</tr>
		<tr>
			<td>Laser Scanning Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 710 with Spectral Detector</td>
			<td>34 channel PMT detection</td>
		</tr>
		<tr>
			<td>Zen Software</td>
			<td>Carl Zeiss</td>
			<td>2010 or above</td>
			<td>for two-photon and spectral image acquisition</td>
		</tr>
		<tr>
			<td>Imaris Suite Software</td>
			<td>Bitplane, Inc.,</td>
			<td>Version 7.0 or above</td>
			<td>3D Volume, Iso-surface Rendering, Visualization</td>
		</tr>
	</tbody>
</table>

multimodal optical microscopy methods reveal polyp tissue morphology and structure in caribbean reef building corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

A custom designed SBFI apparatus (manufactured specifically for the present study; Figure 3) produced the first detailed 3D digital elevation maps (DEMs) of the outer surface texture and morphology of the M. annularis and M. faveolature coral polyps (Figure 4 and SI Videos 1-2). This yielded images of previously undescribed stacked lobes of coral tissue concentrically radiating outward from the center of each polyp (Figures 4B, 4D, and ...

Watch this Scientific Journal Video about Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals at JoVE.com

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

multimodal-optical-microscopy-methods-reveal-polyp-tissue-morphology

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12.2K 조회수.  University of Illinois at Urbana-Champaign.  영상 기술의 통합 제품군은 카리브해 산호 Montastraea의 annularis와 M.에서 용종의 형태와 조직 구조를 결정하기 위해 적용되었습니다 faveolata. 형광, 직렬 블록 얼​​굴, 및이 광자 공 초점 레이저 주사 현미경 lobate 구조 용종 벽 및 예상 chromatophore 및 주산 셀러 밀도 및 분포를 확인했다. 

비디오: Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Methods of Soil Resampling to Monitor Changes in the Chemical Concentrations of Forest Soils

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental changes. Repeated sampling to monitor these changes in forest soils is a relatively new practice that is not well documented in the literature and has only recently been broadly embraced by the scientific community. The objective of this protocol is therefore to synthesize the latest information on methods of soil resampling in a format that can be used to design and implement a soil monitoring program. Successful monitoring of forest soils requires that a study unit be defined within an area of forested land that can be characterized with replicate sampling locations. A resampling interval of 5 years is recommended, but if monitoring is done to evaluate a specific environmental driver, the rate of change expected in that driver should be taken into consideration. Here, we show that the sampling of the profile can be done by horizon where boundaries can be clearly identified and horizons are sufficiently thick to remove soil without contamination from horizons above or below. Otherwise, sampling can be done by depth interval. Archiving of sample for future reanalysis is a key step in avoiding analytical bias and providing the opportunity for additional analyses as new questions arise.

Repeated soil sampling has recently been shown to be an effective way to monitor forest soil change over years and decades. To support its use, a protocol is presented that synthesizes the latest information on soil resampling methods to aid in the design and implementation of successful soil monitoring programs.

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental ...

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.
To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for &#34;off-the-shelf&#34; immunotherapies.

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm morphogenesis, that is the structural diversification of biofilms during community assembly, represents a remarkable challenge across spatial and temporal scales. Here, we present an automated biofilm imaging system based on optical coherence tomography (OCT). OCT is an emerging imaging technique in biofilm research. However, the amount of data that currently can be acquired and processed hampers the statistical inference of large scale patterns in biofilm morphology. The automated OCT imaging system allows covering large spatial and extended temporal scales of biofilm growth. It combines a commercially available OCT system with a robotic positioning platform and a suite of software solutions to control the positioning of the OCT scanning probe, as well as the acquisition and processing of 3D biofilm imaging datasets. This setup allows the in situ and non-invasive automated&#160;monitoring of biofilm development and may be further developed to couple OCT imaging with macrophotography and microsensor profiling.

Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time.

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm ...

Split Point Analysis and Uncertainty Quantification of Thermal-Optical Organic/Elemental Carbon Measurements

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon (EC). This is commonly accomplished using thermal-optical OC/EC analyzers (TOAs), which enable measurement via controlled thermal pyrolysis and oxidation under specific temperature protocols and within constrained atmospheres. Several commercial TOAs exist, including a semi-continuous instrument that enables on-line analyses in the field. This instrument employs an in-test calibration procedure that requires relatively frequent calibration. This article details a calibration protocol for this semi-continuous TOA and presents an open-source software tool for data analysis and rigorous Monte Carlo quantification of uncertainties. Notably, the software tool includes novel means to correct for instrument drift and identify and quantify the uncertainty in the OC/EC split point. This is a significant improvement on the uncertainty estimation in the manufacturer&#8217;s software, which ignores split point uncertainty and otherwise uses fixed equations for relative and absolute errors (generally leading to under-estimated uncertainties and often yielding non-physical results as demonstrated in several example data sets). The demonstrated calibration protocol and new software tool enabling accurate quantification of combined uncertainties from calibration, repeatability, and OC/EC split point are shared with the intent of assisting other researchers in achieving better measurements of OC, EC, and total carbon mass in aerosol samples.

This article presents a protocol and software tool for the quantification of uncertainties in the calibration and data analysis of a semi-continuous thermal-optical organic/elemental carbon analyzer.

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is&#160;presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Here, standardized protocols are presented to assess induction of the heat shock response (HSR) in Caenorhabditis elegans using RT-qPCR at the molecular level, fluorescent reporters at the cellular level, and thermorecovery at the organismal level.

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...