Name: laser-induced fluorescence emission (l.i.f.e.) as novel non-invasive tool for in-situ measurements of biomarkers in cryospheric habitats
Uploaded: 2019-10-26T15:00:00.000Z
Duration: 13 min 38 s
Channel: JoVE

K12 Standards

Georgia Standards of Excellence

9-12

Geology

Surface Processes

SG5.c

laser-induced-fluorescence-emission-life-as-novel-non-invasive-tool

Laser-Induced Fluorescence Emission (L.I.F.E.) as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats

The authors gratefully thank Colonel (IL) J.N. Pritzker, the Tawani Foundation, USA, the Austrian Federal Ministry of Science, Research and Economy (Sparkling Science SPA04_149 and SPA05_201), Alpine Forschungsstelle Obergurgl (AFO), Austrian Space Forum (&#214;WF), Roman Erler from the Hintertuxer Natur Eis Palast, the Austrian Federal Forestry and Base Manager Nick Cox from the Arctic Station in Ny Alesund (Svalbard). We are also indebted to Sabrina Obwegeser, Carina Rofner, and Fabian Drewes for their help during filming. Finally, we want to thank James Bradley for giving the voice for the concomitant video.

1. Calibration and validation
NOTE: For the pigment calibration, prepare dilution rows from stock solutions of chla and B-PE. The chla stock solution is diluted with acetone and B-PE is diluted with distilled sterile water. Later, 15 mL of each dilution step will be needed. Protect the pigments from light by wrapping them with aluminum foil. Store the chla in a freezer and the B-PE in a refrigerator until further use. A detailed protocol for the dilution row follows in sections 1.1 for chla and 1.2 for B-PE. Both the chla and the B-PE laboratory calibration for pigment detection and quantification with the L.I.F.E. instrument is described below. A previous calibration24 was made with the same pigments as in this study.
<ol>
	<li>Chlorophylla dilution row
	<ol>
		<li>Dissolve 1 mg of chla (purified from A. nidulans algae) with acetone in a 50 mL sample tube and dilute this chla stock solution with acetone to the following final concentrations: 1,000; 800; 640; 320; 160; 80; 40; 20; 10; 5; 1; and 0.5 ng/mL.</li>
		<li>Transfer 15 mL of each dilution into 50 mL sample tubes and cover them in aluminum foil due to light sensitivity. Store the tubes in a -20 °C freezer until the calibration measurements are taken. 
		NOTE: The protocol can be interrupted here.</li>
		<li>Measure the chla absorption features from each dilution in a double-beam spectrophotometer as triplicates and calculate the chla content as described by Lorenzen25, which will be described in detail in section 2.2.2.</li>
	</ol>
	</li>
	<li>PE dilution row
	<ol>
		<li>Dilute a 4 mg/mL B-PE stock solution with sterile filtered water (pH = 7) to the following final concentrations: 1,000; 800; 640; 320; 160; 80; 40; 20; 10; and 5 ng/mL B-PE. Transfer 15 mL of each dilution in a 50 mL sample tube and cover it with aluminum foil. Store at 4 °C until further use. 
		NOTE: The protocol can be interrupted here.</li>
	</ol>
	</li>
	<li>Setup for calibration
	<ol>
		<li>Build a rack as shown in Figure 5 to establish three measurement platforms, each 1.5 cm higher than the next. 
		NOTE: The rack and column height play an important role for the measurements because the surface of the liquids should remain in the focal point of the L.I.F.E. instrument as indicated in Figure 5.</li>
		<li>Add 5 mL of the highest concentrated dilution in a polyscintillation plastic vial and put it on the highest point of the rack. Measure the fluorescence intensity.</li>
		<li>Place the vial in the middle position of the rack and add another 5 mL (10 mL total volume). Measure the fluorescence intensity. Repeat the procedure at the lowest position on the rack with another 5 mL (15 mL total volume; total of 45 mm in column height). 
		NOTE: Adapt the exposure time for each dilution step to prevent detector saturation (gray values over 255 in 12-bit images) and for a sufficient signal-to-noise ratio of the weak fluorescence signals.</li>
		<li>Repeat steps 1.3.2 and 1.3.3 with all dilutions (steps 1.1.1 and 1.2.1) of chla and B-PE.</li>
		<li>Load the generated data files into the data reduction wizard to automatically count and sum up the gray values from each pixel line along the Y-axis (spatial distribution). 
		NOTE: Varying exposure times are compensated automatically by normalizing the fluorescence intensity to an integration time of 1 s.</li>
		<li>Calculate the pigment area density with a Poisson regression analysis using the known concentrations from the dilution series and the normalized fluorescence intensities that were calculated. Then normalize the fluorescence counts from the three different column heights to 1.5 cm (5 mL) by multiplying the counts from each column height with a factor (factors 1, 0.5, and 0.33, for the 5 mL, 10 mL, and 15 mL concentration solutions, respectively).</li>
	</ol>
	</li>
</ol>
<img alt="Optical setup diagram showing light refraction and focal point analysis." class="xfigimg" src="/files/ftp_upload/60447/60447fig05.jpg" data-altupdatedat="1788282455000" data-alt="Figure 5"> 
Figure 5: Setup for the L.I.F.E. calibration with chla and B-PE under laboratory conditions. 
(A) Lens tube of the instrument. (B) Green laser for B-PE excitation. (C) Blue laser for chla excitation. (D) Scintillation vial. (E) Focal point of the L.I.F.E. instrument. (F) B-PE/water or chla/acetone solution with 5 mL, 10 mL, and 15 mL. (G) Spacers that keep the surface of each solution at the focal plane for three different volumes. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Sampling and sample processing
<ol>
	<li>Collection of snow and ice
	<ol>
		<li>Collect snow and supraglacial ice from a glacier into sterile polyethylene bags and store them frozen until further processing. 
		NOTE: For this study, the samples were collected at Midtre Lovenbreen (MLB), a polythermal glacier near the research village Ny-Ålesund in the high Arctic Archipelago of Svalbard (78°53’ N, 12°03’ E).</li>
		<li>Sample bacterial mats from the glacier forefield into sterile polyethylene bags and transport all samples to a laboratory for further processing.</li>
		<li>Melt frozen material slowly in the dark at 4 °C. Filter liquid samples on GF/F filters (47 mm diameter) and note the filtered volume. Keep the filters frozen until further processing.</li>
	</ol>
	</li>
	<li>Chlorophylla measurements
	<ol>
		<li>Using the L.I.F.E. instrument, measure the filter in four random areas, each in triplicates using the green and blue laser. Calculate the overall pigment concentration by multiplying the area density with the filtered area and filtered volume. Normalize the pigment concentration (µg/L) to a volume of 1 L.</li>
		<li>Assess the chla contents of the GF/F filters with a laboratory standard according to the protocol by Lorenzen25. To do so, put each of the filters in a vial with 13 mL of acetone and store them in the dark at 4 °C overnight. Next, take a vial and place it on ice before sonication for 2 min at 50% power in continuous mode. Squeeze and remove the filter from the vial.</li>
		<li>Attach tygon tubing to a syringe and remove the chla extraction-acetone mix from the vial. Replace the tygon tubing with a GF-5 filter holder. Transfer the solution into a quartz cuvette.</li>
		<li>After calibrating the absorbance spectrometer for acetone, place the sample in the cuvette into the spectrometer and measure the absorbance features between 400–750 nm. Next, remove the cuvette from the spectrometer and add 200 µL of 2 M HCl to the sample. Then, repeat the absorbance measurement to measure the phaeophytin content in the sample.</li>
	</ol>
	</li>
	<li>Measurement of microbial activity via radiolabelled markers and impact of laser intensity and exposure time on productivity rates 
	CAUTION: Beware of the marker radioactivity (β-radiation). Use a laboratory coat, gloves, and goggles, and work under a fume hood in a licensed isotope lab.
	<ol>
		<li>For bacterial production transfer five aliquots of bacterial mats into sterile polyethylene bags. Inactivate the controls with formaldehyde to a 4% final concentration. 
		NOTE: Three aliquots are used for the 3H-labeled leucine uptake and two aliquots are used as controls.</li>
		<li>Expose the mats with a blue laser (405 nm, 5 mW) and with a green laser (532 nm, 5 mW) for 10 s and 30 s each. Repeat the procedure with 50 mW lasers. Then, inactivate the samples that were not treated with formaldehyde. 
		NOTE: One mat is used for only one laser exposure. Use non-exposed microbial mats as controls.</li>
		<li>After the laser treatment, estimate the bacterial and primary production by incorporating 3H-leucine and NaH14CO3, respectively. For bacterial production measurements, use five aliquots per sample (20 mL) and add formaline (4% final concentration) to two of the parallels, which serve as controls for the abiotic incorporation of the marker. Add 3H-leucine (40 nM) to all samples of the various treatments and incubate them for 4 h under in situ conditions (0.1 °C). Terminate the reaction by adding formaline to the remaining live samples.</li>
		<li>For bacterial production of bacterial mats, transfer labelled samples into cryovials. Extract cells with 5% trichloroacetic acid and centrifuge at 10,000 x g for 5 min according to the protocols by Kirchman26 and Bell27. Add scintillation liquid and put the cryovial into a polyscintillation vial. Analyze the samples with a liquid scintillation counter and calculate the uptake rates. 
		NOTE: Three aliquots are used for the 3H-labeled leucine uptake, two aliquots are used as controls.</li>
		<li>For primary production, prepare five replicates of various treatments (100 mL), wrap two of them into aluminum foil to mimic the dark samples, and add NaH14CO3 (1 µCi) to all. Incubate for 4 h in the ambient light and in situ temperature (0.1 °C). Terminate the reaction by darkening the remaining three replicates and filter the sample onto GF/F filters (25 mm diameter).</li>
		<li>Add 100 µL of 2 N HCl to the filters to remove all excess carbon and let it air out under the fume hood. Dry the samples on a heating plate at 80 °C and place samples into polyscintillation vials.</li>
		<li>To measure radioactive disintegrations per minute (dpm) of all treatments of primary and bacterial production, place the cryovials into polyscintillation vials and add 5 mL of scintillation cocktail. Measure dpm with a liquid scintillation counter and calculate the uptake rates.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

Calibration 
There was a linear correlation between pigment concentration and fluorescence intensity after normalizing photon counts to an exposure time of 1 s. Samples with low column height and low pigment concentrations led to an overestimation of the target pigments, compared to higher column heights with the same pigment concentration. Further, weak fluorescence signals required long exposure times for sufficient photon counts on the sensor. However, long integration times also increased the amount of stray light on the sensor, resulting in a decrease of the signal-to-noise ratio. In its current version, the software cannot distinguish between noise and signal during the data reduction process. Hence, low fluorescence intensity measurements led to a pigment overestimation because noise was counted as a signal that derived from the target pigments. Further, fluorescence intensities from more concentrated pigment solutions showed a greater variability than low concentration solutions. This effect could be explained by absorption processes within the pigment solutions that were used for the calibration curve.
Data validation for chlorophylla quantification 
After filtering ice and snow samples, the three-dimensional samples almost appeared as a two-dimensional specimen on the filter. This justified a direct comparison between L.I.F.E (area density) and spectrophotometric data (volumetric measurement).
The data set (Figure 8) indicated that high pigment concentration leads to an underestimation, whereas low pigment concentration leads to an overestimation of the actual value. This effect can be explained by the thickness of the filter cake and hence, the volumetric character of the sample. The laser penetration depth depended on the optical density and thickness of the specimen. High pigment contents were underestimated because the laser could not induce pigment fluorescence in deeper layers. However, in thin filter cakes, low fluorescence signals were captured due to low area densities of pigments. Apparently, the filter itself showed laser-induced signals after passing the 450 nm long-pass filter (Figure 12). This signal was misleadingly counted as fluorescence signal derived from chla. Thus, thin and too thick filter cakes are difficult to measure with the L.I.F.E. instrument.
<img alt="Laser excitation and fluorescence emission diagram with filter reflection setup; optical study." class="xfigimg" src="/files/ftp_upload/60447/60447fig12.jpg" data-altupdatedat="1788282455000" data-alt="Figure 12"> 
Figure 12: Fluorescence signals from thick (A) and thin (B) filter cakes on a GF/F filter. (A) Self-shading prevents laser-induced fluorescence from deeper layers, which results in an underestimate of the actual pigment concentration. (B) Fluorescence emission from filter cake with overlay by filter reflections. (C) Raw data show filter reflection (gray). The spectral property of a laboratory derived chla fluorescence pattern is illustrated in red. Scale bar = 45 mm. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Limitations of the L.I.F.E. prototype 
During data reduction, the MATLAB coded software interpreted the raw images by summing up pixel lines within a given wavelength range. The current version of the software did not distinguish between organic and inorganic derived signals. The presence of multiple signals might lead to an overestimation of the actual pigment content. Long exposure times due to low fluorescence intensities led to a decrease of the signal-to-noise ratio, promoting the effect as described above (see Figure 8 and Figure 12).
A geode rock shown in Figure 13 exhibited red fluorescent light when exposed with green and blue light. Currently, it is not clear whether the fluorescence resulted from minerals or from porphyrin-based molecules. Hence, an overlay of biological and nonbiological signals may limit the application of this method and require the establishment of a fluorescence database specifically made for the L.I.F.E. prototype.
<img alt="Fluorescence emission experiment, microscopy images of samples A, B, C showing varying brightness." class="xfigimg" src="/files/ftp_upload/60447/60447fig13.jpg" data-altupdatedat="1788282455000" data-alt="Figure 13"> 
Figure 13: Mineral fluorescence from a geode rock, found in Ny-Ålesund. The rock was excited with a 532 nm 50 mW laser (A) and a 405 nm 50 mW laser (B). Both pictures were captured with a polarization filter attached on the lens, which led to a falsification of the actual fluorescence colors. (C) True color image without the use of a polarization filter under daylight conditions. Scale bar = 40 mm. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Beutler and others29 concluded that characteristic emission spectra of cyanobacteria in marine ecosystems depend on environmental conditions. Also, the metabolic state has an impact on the fluorescence properties in phototrophic organisms30. The L.I.F.E instrument may distinguish between the algal and cyanobacterial fluorescence pattern by using bio-fingerprint libraries that contain spectral information of the specimen correlated with environmental conditions.
In dark-adapted chla molecules, all reaction centers are fully oxidized and available for photochemistry and no fluorescence yield is quenched31. Using the L.I.F.E. procedure, a specimen is first excited by a 532 nm laser (green) and then with a 405 nm laser (blue). During the second excitation by the blue laser, chla might show a decreased fluorescence response due to prior excitation by the green laser. Chla absorbs energy at 532 nm wavelength, despite its distance from its absorption maximum wavelength32. Before the actual chla measurement at 405 nm, the green laser may cause photochemical reactions, activating quenching mechanisms in the target pigments. Further, pre-illumination of marine phototrophic organisms did not lead to a change in spectral norm curves between 450 nm–600 nm while the standard deviation in fluorescence intensities increased by 25%29. Depending on the species, fluorescence intensities even increased in response to prior excitation. This topic requires further investigation.
Applicability 
We tested the L.I.F.E. instrument in various habitats with an emphasis on cryoconite holes. The laser was successfully applied in soil and biofilm habitats because of the absence of ambient light during the measurement. Cryoconite granules could be measured when sediment layers blocked light from beneath the hole (Figure 14A,C). Thin sediment cryoconite holes were permeable for stray light from beneath (Figure 14B). Stray light interferes with the measurement. Thus, pigment concentration in bare ice surfaces is not measurable under daylight conditions yet. Signal processing efforts are currently underway to enable operation of the system in high ambient light conditions.
<img alt="Cryoconite melting on ice surface; diagram illustrating solar irradiation effects on ice and particulates." class="xfigimg" src="/files/ftp_upload/60447/60447fig14.jpg" data-altupdatedat="1788282455000" data-alt="Figure 14"> 
Figure 14: Cryoconite hole with liquid water on top. (A) Cryoconite on glacier with L.I.F.E. lens tube. Scale bar = 70 mm. (B) The sediment layer (red) is very thin. Stray light bleeds through the cryoconite layer. (C) The sediment layer is thick enough to block the stray light from beneath. This type of cryoconite hole is measurable with the L.I.F.E. instrument. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In conclusion, our L.I.F.E. instrument detected photoautotrophic organisms in terrestrial habitats such as soils, bacterial mats, biofilms, and in cryoconite holes on glacial surfaces. The target molecules were chla and B-PE. The spatial resolution was 30 µm/px. The detection limit for chla was 250 pg/mL and 2 ng/mL for B-PE. After a laboratory calibration we were able to quantify pigment content in samples that were collected at our study site in the Arctic. We applied self-programmed software for an automated data reduction process. The effects of the presence of minerals and changing light conditions during the measurements require further investigation.
With climate warming, increasing temperatures lead to enhanced availability of liquid water, which results in higher biological activity on icy surfaces of autotrophic and heterotrophic nature. Vigorous efforts should be made to detect heterotrophic organisms in situ to give a full picture of active life in the cryosphere. This could be tested with other target pigments and appropriate laser excitation wavelengths. Hence, L.I.F.E. provides a suitable monitoring system providing high temporal and spatial resolution for supraglacial conditions in context with global change as well as possible astrobiological applications.

The cryosphere harbors sea ice, glaciers, high mountain lakes, snow areas, lake ice, melt water streams, and permafrost. These areas cover approximately 11% of the earth’s landmasses1,2 and are overarched by the atmosphere as a recognized cryospheric environment. Recent studies show that massive areas of the cryosphere are quickly retreating3,4. The Antarctic5,6, the Alps7, the Arctic8, and other regions show negative ice mass balances. The retreat of ice caps and glaciers leads to the depletion of our largest freshwater reservoir on Earth. In some areas, glacier retreat is unstoppable5.
For a long time, ice ecosystems were considered sterile environments. However, despite harsh conditions, the presence of active life in the earth’s cryosphere is evident9,10,11,12,13,14,15. Due to the trend towards massive losses of ice by melting, the cryosphere is going through a shift in biological activity, affecting adjacent habitats. To understand those partially irreversible changes we require methods to investigate biological activity in ice under in situ conditions with high spatial and temporal resolution.
In supraglacial environments, life can be found in cryoconite holes, snow covers, melted water, streams, and on bare ice surfaces. However, the most obvious supraglacial habitats are cryoconite holes. They appear globally in glaciated environments and were first described by the Swedish explorer Adolf Erik Nordenskjold during an expedition to Greenland in the 1870’s16,17. The name originates from the Greek words “kryos” (cold) and “konia” (dust). Aeolian-derived dark organic and inorganic debris attach on the ice surface and reduce the albedo locally. Solar radiation promotes melting of the debris into deeper ice layers, forming cylindrical basins with sediment (cryoconite) in the bottom9. Cryoconite holes cover 0.1–10% of glacial ablation zones11.
Cryoconite communities consist of viruses, fungi, bacteria, cyanobacteria, microalgae, and protozoa. Depending on the region, metazoan organisms such as rotifers, nematodes, copepods, tardigrades, and insect larvae can also be found. Edwards and others18 describe cryoconite holes as “ice-cold hotspots”. They also traced functional genes in cryoconite holes that are responsible for N, Fe, S, and P cycling. The mini lake ecosystems respire and photosynthesize at rates found in much warmer and more nutrient-rich habitats11. These findings emphasize the important role of microbial sequestration in supraglacial environments. Beside living communities in cryoconite holes, bare ice surfaces are inhabited by ice algae. Their physiology is well studied19 but their spatial distribution has not been assessed20. Their presence in supraglacial environments decreases the albedo and hence promotes melting that leads to a nutrient outwash and nutrient input into downstream habitats9. Increasing temperatures and hence, a higher availability of liquid water, affects the net ecosystem productivity in these icy ecosystems.
In supraglacial environments, photosynthetically active organisms transform inorganic carbon and nitrogen into organic, available sources for the microbial food web21,22. Until now there are few studies that estimate supraglacial carbon fluxes11,20,23. The discrepancy in proposed rates of carbon flux results from a low spatial and temporal data resolution. Further, the spatial distribution of supraglacial communities outside cryoconite holes is barely assessed. Cook and others20 predicted in their models that supraglacial algal communities fix up to 11x more carbon than contemporary cryoconite holes due to their large surface coverage. The detection of supraglacial algal communities securing sample integrity is still impeded due to missing tools for in situ detection and quantification.
In response to difficulties in logistics, ice ecosystems are less frequently studied than habitats in temperate areas. Data resolution depends on the number of samples assessed and depend on the accessibility of the study sites. Standard sampling methods such as sawing, coring, and subsequent melting involve manipulation of the microbial community. For example, chlorophylla (chla) assessment in solid ice samples is impossible with standard methods without substantial interference. Hence, melting-induced temperature changes within the investigated microbial communities are unavoidable. In response to the thermolability of the photosystem II and other cellular structures in psychrophiles22, laboratory analyses of melted ice samples will always lead to a falsification of in situ conditions.
Non-destructive in situ measurements are the only reasonable way to obtain reliable data. This goal can be achieved using fluorescence-based methods. Due to their light harvesting function, chla and B-phycoerythrin (B-PE) are present in organisms that contribute to the carbon cycle in supraglacial environments, as has been proved by Anesio and others11. Hence, these fluorescent molecules are suitable biomarkers for the quantification of microbial mediated carbon fluxes in ice ecosystems.
In this study, we present the development, calibration, and applicability of a novel non-invasive tool for in situ quantification of chla and B-PE molecules in terrestrial and ice ecosystems. The prototype device is based on laser-induced fluorescent emission, also known as L.I.F.E. The optical instrument (Figure 1) captures fluorescent biomarker signatures after laser-induced fluorescence excitation. The procedure is nondestructive and can be performed at the study site or in a laboratory.
<img alt="Optical excitation setup; diagram; laser spectroscopy system; photonic study, 532nm, 405nm lasers." class="xfigimg" src="/files/ftp_upload/60447/60447fig01.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
Figure 1: The L.I.F.E. prototype. Left: Photo of the instrument without a protective lid. Right: Schematic illustration of the instrument. Total mass = 5.4 kg (laser and optics = 4.025 kg, laptop = 1.37 kg). Aluminum frame = 32.5 cm x 20.3 cm x 6.5 cm. Optical tube: 18.4 cm x 4 cm (diameter). CCD: bluefox mv220g sensor; F: servo-steered long-pass filters (450 nm and 550 nm); L: optical lenses; M1: mirrors; M2: dichroic mirror; MC: microcontroller; P: prism; PBS: polarizing beam splitter; S: slit aperture made of adjustable razor blades. Scale bar = 70 mm. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The portable dual-wavelength kit weighs 4.5 kg and is used on a tripod in combination with an external computer. The field setup is quick and easy. The instrument is attached to the tripod, and the lens tube is attached to the device along with a USB cable and the camera cable. The external computer is connected to the instrument using a USB cable. The tripod legs are adjusted in such a way that the lens tube is directed towards and covers the specimen. Then, a 5 mW green laser hits the sample after passing a polarizing beam splitter that redirects polarized light towards the optical axis of the spectrometer. The specimen exhibits a fluorescent light, illustrated in red in Figure 1. Half of the collimated light passes the polarizing beam splitter and is focused through a servo-steered long-pass filter that removes the laser signals. Next, the signal hits an aperture slit that consists of two adjustable razor blades. A prism spectrally separates the fine line of light orthogonal to the slit aperture before the signal is captured by the sensor. The procedure is repeated with a blue laser. The raw data are transferred automatically to a portable computer that is also used for the software operation.
The instrument is controlled by an external computer using a LabVIEW environment that synchronizes the picture-taking with the CCD camera, switching on/off lasers, and rotating the long-pass filter wheel. The graphical user interface (GUI) is divided into three main sections. The exposure adjustment is done manually. Although the correction between exposure time and signal intensity is linear (Figure 2B), the maximum exposure time is limited to 10 s because longer integration times lead to a significant decrease in the signal-to-noise-ratio. The comment field is used for the description of the sample (Figure 2A). In the right section, raw images are displayed as soon as the measurements are finished. This feature is crucial for immediate data evaluation in the field (Figure 2C–E). Red areas indicate overexposed pixels, which can be avoided by reducing the exposure time.
The subsequent raw data reduction process is decoupled from the image acquisition procedure and can be done at any time after image acquisition.
<img alt="Spectroscopy setup interface; optical excitation measurement; filter settings; spectral analysis." class="xfigimg" src="/files/ftp_upload/60447/60447fig02.jpg" data-altupdatedat="1788282455000" data-alt="Figure 2"> 
Figure 2: L.I.F.E. graphical user interface for the data acquisition and raw data evaluation. (A) The software enables manual text input for sample descriptions. (B) The exposure time can be adjusted before the measurement. (C−E) The raw images are displayed on the right side of the interface. (E) Red colors indicate a saturation of the sensor. (F) The activation of the RUN MEASUREMENT button triggers the data acquisition process. In the array (G), all commands executed automatically during data acquisition are displayed. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Transient absorption spectroscopy diagram showing spatial and spectral separation on CCD." class="xfigimg" src="/files/ftp_upload/60447/60447fig03.jpg" data-altupdatedat="1788282455000" data-alt="Figure 3"> 
Figure 3: Example of a raw image. Left: Raw data of chla standard in acetone solution, recorded with the L.I.F.E instrument. Due to the optical properties of the device, the signal is displayed as a warped line. Right: Interpretation of the raw image per pixel (px). The spectral axis (5 nm/px resolution) is plotted against the spatial axis (30 µm/px resolution). <a href="https://www.jove.com/files/ftp_upload/60447/60447fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The 12-bit gray-scale raw images show a spatial component due to the one-dimensional aperture slit and a spectral component due to the prism in front of the CCD (Figure 3). In response to optical constraints the raw images are distorted. Therefore, they need to be cropped and dewarped by applying a code that recognizes the degree of distortion. This is done with a software wizard (Figure 4). Next, the wavelength calibration is done with the 532 nm laser. The green light is produced by frequency doubling of a 1,064 nm infrared laser. Both wavelengths can be detected by the CCD and therefore, the spectral position of each pixel can be calculated in dewarped images automatically (Figure 4).
The picture is then cropped down to a given wavelength range (550–1,000 nm for green laser measurements and 400–1,000 for blue laser measurements). Gray values from each pixel in a selected pixel line are counted and summed up. A gray value can range from 0–255. After that, every pixel line accounts for one number. Further on-screen software instructions lead to the generation of a plot showing the gray value counts from each pixel line plotted against the spatial coordinates. This allows for a quantitative spatial discrimination of chla and B-PE simultaneously in the sample. Additionally, the spectral properties of a sample can be plotted from selected pixel lines automatically.
<img alt="Optical emission analysis; spectroscopy setup; diagram with spectral fitting results." class="xfigimg" src="/files/ftp_upload/60447/60447fig04.jpg" data-altupdatedat="1788282455000" data-alt="Figure 4"> 
Figure 4: Dewarping raw images. Left: Raw image captured with a green laser. No filter was used. Signals are displayed at 532 nm and 1,064 nm. Exposure time = 0.015 s. Center: The cropped 532 nm signal is used as a reference line to dewarp a set of images. Right: The dewarped image from the raw image source. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>aceton</td><td>Merck</td><td>67-64-1</td><td></td></tr><tr><td>B-Phycoerythrin</td><td>Invirtrogen</td><td>P6305</td><td></td></tr><tr><td>Chlorophyll a standard</td><td>Sigma-Aldrich</td><td>C6144-1MG</td><td></td></tr><tr><td>formaline</td><td>Merck</td><td>HT501128</td><td>36%</td></tr><tr><td>GF/C filters</td><td>Whatman</td><td>WHA1822025</td><td>25mm diameter</td></tr><tr><td>HCl</td><td>Merck</td><td>H1758</td><td>36,5-38%</td></tr><tr><td>L.I.F.E. Prototype</td><td>University of Innsbruck</td><td></td><td>built on demand</td></tr><tr><td>LabView</td><td>National Instruments</td><td></td><td>Software, Laboratory Virtual Instrumentation Engineering Workbench</td></tr><tr><td>Leucine, L-[4,5-3H], 1 mCi</td><td>Perkin Elmer</td><td>NET1166001MC</td><td>radioactive</td></tr><tr><td>Liquid scintillation cocktail Beckman Ready Use</td><td>Beckman</td><td>not more available, can be compensated by Ultra Gold, Packard</td><td></td></tr><tr><td>liquid scintillation counter</td><td>Beckman</td><td>out of stock</td><td>LSC 6000 IC</td></tr><tr><td>NaH14CO3 (4 &amp;micro;Ci/ml)</td><td>DHI Denmark</td><td>4 &amp;mu;Ci/ml, 1 ml</td><td>radioactive</td></tr><tr><td>Osmonics polycarbonate filters</td><td>DHI Denmark</td><td>PCTE</td><td>25mm diameter, 0,2&amp;micro;m pore size</td></tr><tr><td>Polyscintillation vials</td><td>Perkin Elmer</td><td>WHA1825047</td><td>20ml</td></tr><tr><td>sample tubes</td><td>Sigma Aldrich</td><td>T2318-500EA</td><td>Greiner centrifuge tubes, 50ml</td></tr><tr><td>Spectrophotometer</td><td>Hitachi</td><td>NA</td><td>Model U2001, any photometer for absorption spectroscopy measuring at 664nm and 750nm would be appropriate</td></tr><tr><td>trichloric acetic acid (TCA)</td><td>Merck</td><td>T6399</td><td>100%</td></tr><tr><td>ultrasonic probe</td><td>nano lab</td><td>QS1T-2</td><td></td></tr></tbody></table>

laser-induced fluorescence emission (l.i.f.e.) as novel non-invasive tool for in-situ measurements of biomarkers in cryospheric habitats

Global warming affects microbial communities in a variety of ecosystems, especially cryospheric habitats. However, little is known about microbial-mediated carbon fluxes in extreme environments. Hence, the methodology of sample acquisition described in the very few studies available implies two major problems: A) high resolution data require a large number of samples, which is difficult to obtain in remote areas; B) unavoidable sample manipulation such as cutting, sawing, and melting of ice cores that leads to a misunderstanding of in situ conditions. In this study, a prototype device that requires neither sample preparation nor sample destruction is presented. The device can be used for in situ measurements with a high spectral and spatial resolution in terrestrial and ice ecosystems and is based on the Laser-Induced Fluorescence Emission (L.I.F.E.) technique. Photoautotrophic supraglacial communities can be identified by the detection of L.I.F.E. signatures in photopigments. The L.I.F.E. instrument calibration for the porphyrin derivates chlorophylla (chla) (405 nm laser excitation) and B-phycoerythrin (B-PE) (532 nm laser excitation) is demonstrated. For the validation of this methodology, L.I.F.E. data were ratified by a conventional method for chla quantification that involved pigment extraction and subsequent absorption spectroscopy. The prototype applicability in the field was proven in extreme polar environments. Further testing on terrestrial habitats took place during Mars analog simulations in the Moroccan dessert and on an Austrian rock glacier. The L.I.F.E. instrument enables high resolution scans of large areas with acceptable operation logistics and contributes to a better understanding of the ecological potential of supraglacial communities in the context of global change.

Laboratory calibration for B-PE 
The response signals of the B-PE dilution row were measured with the L.I.F.E. instrument in a dark room at 20 °C (Figure 6). The count rate depended on both the concentration and the column height of the measured sample. Low concentration and low column height B-PE specimen fluoresced stronger compared to samples of the same concentration and higher column height.
<img alt="Concentration vs. counts graph; chromatography column data, normalized exposure, scaling factors shown." class="xfigimg" src="/files/ftp_upload/60447/60447fig06.jpg" data-altupdatedat="1788282455000" data-alt="Figure 6"> 
Figure 6: B-PE laboratory calibration. B-PE content and column density calibration is shown. Normalized count rates were calculated for a column height of 1.5 cm. Reprint with permission28. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A Poisson regression was used for the final calibration line fit. There was a linear correlation between the area densities and pixel gray value counts. The function of the curve was y = 81.04x (Figure 7), which means that a gray value count rate of 8,104 in a 1 s exposed sample equaled an area density of 100 ng/cm2 B-PE. The chla calibration is set up in an analogue manner. The function was y = 8.94x.
<img alt="Calibration graph showing data fit, area density vs. counts, for spectroscopic analysis validation." class="xfigimg" src="/files/ftp_upload/60447/60447fig07.jpg" data-altupdatedat="1788282455000" data-alt="Figure 7"> 
Figure 7: Final calibration curve for B-PE. The gray value counts were normalized to an exposure time of 1 s and plotted against the area density. Reprint with permission28. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Application on cryoconite samples from Svalbard and laboratory validation of data 
The mean values of the L.I.F.E. measurements and the single measurements of the same samples derived from conventional extraction using acetone and subsequent analysis with a spectrophotometer are illustrated in Figure 8.
<img alt="Chlorophyll analysis; bar graph; photometry vs. optical method; sample data comparison; experiment result." class="xfigimg" src="/files/ftp_upload/60447/60447fig08.jpg" data-altupdatedat="1788282455000" data-alt="Figure 8"> 
Figure 8: Data validation with natural samples. The samples (MLB) are ranked by chla content, based on the results of a laboratory spectrophotometer (single values) and compared with the chla fluorescence data measured on four random areas per filter. The error bars represent the standard deviation of the L.I.F.E. measurements. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Chla contents ranging from 48 µg/L–67 µg/L were underestimated, and lower chla contents ranging from 0.7 µg/L–7 µg/L were overestimated by the L.I.F.E. prototype. The standard deviations from the L.I.F.E. measurements were low.
Comparison of spectral data from in situ measurements with laboratory standards 
Chla spectra were comparable between cryoconite samples and those from purified from A. nidulans algae. The fluorescence peaks in all samples were located at 700 nm–710 nm. However, spectra derived from cryoconite samples showed higher noise signals between 400 nm–650 nm and from 800 nm–1,000 nm compared to spectra of the chla pigment standard (Figure 9).
<img alt="Fluorescence spectrum graph, 405 nm excitation, relative intensity vs. wavelength, 700 nm peak." class="xfigimg" src="/files/ftp_upload/60447/60447fig09.jpg" data-altupdatedat="1788282455000" data-alt="Figure 9"> 
Figure 9: Spectral data interpretation. Measurements of four cryoconite granules (blue) and a chla standard pigment solution (red) after excitation with 405 nm lasers. The spectra were recorded 1 year after sample collection. The samples were kept frozen and were not exposed to light prior to the measurement. In response to wavelength calibration issues, the fluorescence peak is located at 700 nm–710 nm instead of 680 nm. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Automated cryoconite grain analysis 
In an example of an automated analysis of a cryoconite hole (Figure 10), the highest pigment area densities were observed at pixel line 50. The sample spectrum after excitation with a 532 nm laser showed a peak with a cut off at a gray value of 255 in response to oversaturation of the sensor. This peak derived from the green laser and not from the fluorescent signal.
<img alt="Spectroscopic analysis with raw images at 532nm/405nm excitation, density plots, and spectral graphs." class="xfigimg" src="/files/ftp_upload/60447/60447fig10.jpg" data-altupdatedat="1788282455000" data-alt="Figure 10"> 
Figure 10: Automated data analysis of a single cryoconite granule with a diameter of 1 mm. The sample was collected at Vestre Brøggerbreen (VBB) and measured within 4 h after sampling in a dark laboratory room at the Arctic Station (GB) facility in Ny-Ålesund. The left column shows B-PE measurements and the right column represents chla data. The raw images are displayed on top. Laser-induced fluorescence responses are displayed in gray. Red areas indicate the response from standard pigments. The middle section illustrates the spatial distribution of the target pigments. The spectral properties of the fluorescence signal are displayed in the lower images. <a href="https://www.jove.com/files/ftp_upload/60447/60447fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Impact of laser excitation on productivity in bacterial mats 
Neither primary nor bacterial productivity were affected when increasing the power of the laser and/or the exposure time (Figure 11). No significant differences were detected under laser treatments with increased power.
<img alt="Bacterial carbon production bar chart; excitation wavelengths 532 nm and 405 nm, variable power." class="xfigimg" src="/files/ftp_upload/60447/60447fig11.jpg" data-altupdatedat="1788282455000" data-alt="Figure 11"> 
Figure 11: Productivity measurements of samples from Svalbard. Bacterial mats were exposed with green and blue lasers of varying laser intensities and exposure times. The data are colored according to the laser wavelength source (green and blue). <a href="https://www.jove.com/files/ftp_upload/60447/60447fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Carbon fluxes in the cryosphere are hardly assessed yet but are crucial regarding climate change. Here we show a novel prototype device that captures the phototrophic potential in supraglacial environments based on laser-induced fluorescence emission (L.I.F.E.) technology offering high spectral and spatial resolution data under in situ conditions.

Global warming affects microbial communities in a variety of ecosystems, especially cryospheric habitats. However, little is known about ...

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Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis).  The method involves pruning the panicle prior to a hot water treatment to kill viable pollen.  Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

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Analysis of Fatty Acid Content and Composition in Microalgae

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

surface renewal: an advanced micrometeorological method for measuring and processing field-scale energy flux density data

surface-renewal-an-advanced-micrometeorological-method-for-measuring

Surface Renewal: An Advanced Micrometeorological Method for Measuring and Processing Field-Scale Energy Flux Density Data

Surface renewal is a micrometeorological method that is being used increasingly to determine energy fluxes, but its technical complexity makes it inaccessible to a broad audience. We describe the steps needed to set up and calibrate a surface renewal field station, to acquire and process data, and to correctly interpret results.

a rapid and efficient method for assessing pathogenicity of ustilago maydis on maize and teosinte lines

a-rapid-efficient-method-for-assessing-pathogenicity-ustilago-maydis

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described.  The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

optimize flue gas settings to promote microalgae growth in photobioreactors via computer simulations

optimize-flue-gas-settings-to-promote-microalgae-growth

Optimize Flue Gas Settings to Promote Microalgae Growth in Photobioreactors via Computer Simulations

Flue gas from power plants is a cheap CO2 source for algal growth. We have built prototype "flue gas to algal cultivation" systems and described how to scale up the algal cultivation process. We have demonstrated the use of a mass-transfer bio-reaction model to simulate and to design the optimal operation of flue gas for the growth of Chlorella sp. in algal photo-bioreactors.

technique for studying arthropod and microbial communities within tree tissues

technique-for-studying-arthropod-microbial-communities-within-tree

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

We provide a technique to preserve intact tree phloem and prepare it for observation.  We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

waste water derived electroactive microbial biofilms: growth, maintenance, and basic characterization

waste-water-derived-electroactive-microbial-biofilms-growth

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

single-plant, sterile microcosms for nodulation and growth of the legume plant medicago truncatula with the rhizobial symbiont sinorhizobium meliloti

single-plant-sterile-microcosms-for-nodulation-growth-legume-plant

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Growth of Medicago truncatula plants in symbiosis with the nitrogen-fixing bacteria Sinorhizobium meliloti in individual, sterile microcosms made from standard laboratory plates permits frequent examination of root systems and nodules without compromising sterility. Plants can be maintained in these growth chambers for up to 9 weeks.

high-throughput fluorometric measurement of potential soil extracellular enzyme activities

high-throughput-fluorometric-measurement-potential-soil-extracellular

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples.  Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation.

helminth collection and identification from wildlife

helminth-collection-and-identification-from-wildlife

Helminth Collection and Identification from Wildlife

Wild animals are commonly parasitized by a wide range of helminths.  The four major types of helminths are “roundworms” (nematodes), “thorny-headed worms” (acanthocephalans), “flukes” (trematodes), and “tapeworms” (cestodes).  Here we describe how helminths are collected from a vertebrate animal and how they are preserved and taxonomically...

optimized negative staining: a high-throughput protocol for examining small and asymmetric protein structure by electron microscopy

optimized-negative-staining-high-throughput-protocol-for-examining

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

More than half of proteins are small proteins (molecular mass &lt;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.

design and operation of a continuous 13c and 15n labeling chamber for uniform or differential, metabolic and structural, plant isotope labeling

design-operation-continuous-13c-15n-labeling-chamber-for-uniform-or

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

a venturi effect can help cure our trees

A Venturi Effect Can Help Cure Our Trees

Compared to the more traditional hole-based methods, most of which require the tree to be drilled, tools with lenticular blades transform the basics of endotherapy easing the closure of the wound and allowing the natural uptake of the solutions.

transient gene expression in tobacco using gibson assembly and the gene gun

transient-gene-expression-tobacco-using-gibson-assembly-gene

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

methods for facilitating microbial growth on pulp mill waste streams and characterization of the biodegradation potential of cultured microbes

methods-for-facilitating-microbial-growth-on-pulp-mill-waste-streams

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

transcript and metabolite profiling for the evaluation of tobacco tree and poplar as feedstock for the bio-based industry

transcript-metabolite-profiling-for-evaluation-tobacco-tree-poplar-as

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

tracking microbial contamination in retail environments using fluorescent powder - a retail delicatessen environment example

tracking-microbial-contamination-retail-environments-using

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination.

colorimetric paper-based detection of escherichia coli, salmonella spp., and listeria monocytogenes from large volumes of agricultural water

colorimetric-paper-based-detection-escherichia-coli-salmonella-spp

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here.  Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates.

laboratory estimation of net trophic transfer efficiencies of pcb congeners to lake trout (salvelinus namaycush) from its prey

laboratory-estimation-net-trophic-transfer-efficiencies-pcb-congeners

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

a technique to screen american beech for resistance to the beech scale insect (cryptococcus fagisuga lind.)

a-technique-to-screen-american-beech-for-resistance-to-beech-scale

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

Beech bark disease is initiated by feeding activities of the beech scale insect that create fungal entry points in the bark. Trees that are resistant to the scale insect are also disease resistant. Here we present the protocol we have developed to screen individual beech trees for beech scale resistance.

design and construction of an urban runoff research facility

design-and-construction-of-an-urban-runoff-research-facility

Design and Construction of an Urban Runoff Research Facility

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

experimental protocol for manipulating plant-induced soil heterogeneity

experimental-protocol-for-manipulating-plant-induced-soil

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community’s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

laboratory-determined phosphorus flux from lake sediments as a measure of internal phosphorus loading

laboratory-determined-phosphorus-flux-from-lake-sediments-as-measure

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

a protocol for conducting rainfall simulation to study soil runoff

a-protocol-for-conducting-rainfall-simulation-to-study-soil-runoff

A Protocol for Conducting Rainfall Simulation to Study Soil Runoff

A rainfall simulator was used to apply a consistent rate of uniform rainfall to packed soil boxes in a study of the fate and transport of urea, a nonpoint source environmental contaminant. Under uniform soil and rainfall conditions, antecedent soil moisture content exerted strong control over urea loss in surface runoff.

expression of recombinant cellulase cel5a from trichoderma reesei in tobacco plants

expression-recombinant-cellulase-cel5a-from-trichoderma-reesei

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

evaluation of integrated anaerobic digestion and hydrothermal carbonization for bioenergy production

evaluation-integrated-anaerobic-digestion-hydrothermal-carbonization

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

A novel Upflow Anaerobic Solid State (UASS) reactor was used for biogas production from fibrous feedstock. Digestate from UASS reactor was hydrothermally carbonized into HTC biochar in a pressurized batch reactor. The integration of the two bioenergy concepts was applied in this study to increase overall bioenergy production.

application of two-spotted spider mite tetranychus urticae for plant-pest interaction studies

application-two-spotted-spider-mite-tetranychus-urticae-for-plant

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

Protocols for efficient preparation of homogenous samples of spider mites, infestation of experimental plants, and assessment of plant damage, as required for studies of plant-pest interaction were developed.

a small volume procedure for viral concentration from water

a-small-volume-procedure-for-viral-concentration-from-water

A Small Volume Procedure for Viral Concentration from Water

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

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

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

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, and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

integrated field lysimetry and porewater sampling for evaluation of chemical mobility in soils and established vegetation

integrated-field-lysimetry-porewater-sampling-for-evaluation-chemical

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers

measuring-fluxes-mineral-nutrients-toxicants-plants-with-radioactive

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

epa method 1615. measurement of enterovirus and norovirus occurrence in water by culture and rt-qpcr. i. collection of virus samples

epa-method-1615-measurement-enterovirus-norovirus-occurrence-water

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

soil sampling and isolation of entomopathogenic nematodes (steinernematidae, heterorhabditidae)

soil-sampling-isolation-entomopathogenic-nematodes-steinernematidae

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects.  We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

measurement of greenhouse gas flux from agricultural soils using static chambers

measurement-greenhouse-gas-flux-from-agricultural-soils-using-static

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

unraveling the unseen players in the ocean - a field guide to water chemistry and marine microbiology

unraveling-unseen-players-ocean-field-guide-to-water-chemistry-marine

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

physical, chemical and biological characterization of six biochars produced for the remediation of contaminated sites

physical-chemical-biological-characterization-six-biochars-produced

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

modifying the bank erosion hazard index (behi) protocol for rapid assessment of streambank erosion in northeastern ohio

modifying-bank-erosion-hazard-index-behi-protocol-for-rapid

Modifying the Bank Erosion Hazard Index (BEHI) Protocol for Rapid Assessment of Streambank Erosion in Northeastern Ohio

Streambank erosion potential can be evaluated and ranked using David Rosgen’s Bank Erosion Hazard Index (BEHI), however this protocol has significant limitations. Here we present protocol modifications to address time constraints, allow nonprofessionals to complete accurate assessments, and account for non-alluvial stream conditions in Northeast Ohio.

a whole cell bioreporter approach to assess transport and bioavailability of organic contaminants in water unsaturated systems

a-whole-cell-bioreporter-approach-to-assess-transport-bioavailability

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

a technical perspective in modern tree-ring research - how to overcome dendroecological and wood anatomical challenges

a-technical-perspective-modern-tree-ring-research-how-to-overcome

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

detection of foodborne bacterial pathogens from individual filth flies

detection-of-foodborne-bacterial-pathogens-from-individual-filth-flies

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

a fish-feeding laboratory bioassay to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms

a-fish-feeding-laboratory-bioassay-to-assess-antipredatory-activity

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

epa method 1615. measurement of enterovirus and norovirus occurrence in water by culture and rt-qpcr. ii. total culturable virus assay

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

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