Name: autofluorescence imaging to evaluate red algae physiology
Uploaded: 2023-02-17T13:25:00
Description: Watch this Scientific Journal Video about Autofluorescence Imaging to Evaluate Red Algae Physiology at JoVE.com

This research was carried out as part of Projects TIN2015-68454-R and 20961/PI/18, financed by the Spanish Ministry of Economy and Competitiveness and the S&#233;neca Foundation of the Murcia Region. Irene Hern&#225;ndez Mart&#237;nez and Francisco Javier Ib&#225;&#241;ez L&#243;pez from the Statistical Support Section of the Scientific and Research Area of Murcia University (Secci&#243;n de Apoyo Estad&#237;stico (SAE), &#193;rea Cient&#237;fica y de Investigaci&#243;n (ACTI), Universidad de Murcia, (Figure 1 were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed with a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/)

The algal species Chroothece mobilis was used for the present study. The species was obtained from the Microalgae Edaphic SE Spain, MAESE 20.29 culture collection. An overview of the protocol is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64533/64533fig01.jpg" /> 
Figure 1: Overview of the study. Chroothece mobilis is i...

<ol>
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Some unicellular or colonial red algae, such as Chroothece,&#160;grow slowly in vitro, but contain multiple autofluorescent compounds that can be analyzed by spectral analysis under a confocal microscope, where differences in pigment emission peaks can be detected. Spectral confocal fluorescence microscopy has allowed us to conduct in vivo studies to evaluate the adaptation or acclimation of photosynthetic organisms8,10,<sup ...

Red algae, such as the genus Chroothece, can grow in extreme habitats, where they frequently have to cope with marked environmental changes1. Floods and droughts are frequent in semi-arid regions where this genus can be found, and some species have been reported in creeks, cliffs, caves, or even thermal waters2. However, most of the time, biological variables, such as competition or grazing, relegate species to non-optimal conditions for their growth. As these organisms are often difficult to culture and either do not grow or grow very slowly under laboratory conditions, one major limitation is the available sample size. Therefore, it is very important to follow non-destructive methods or methods that involve minimal sample manipulation3,4.
The physiological skills needed to survive in these harsh environments can be monitored by following changes in their photosynthetic systems. Metabolic mechanisms, photosynthetic efficiency, and sensitivity to light or culture conditions can be revealed by pigment fluorescence emission profiles, due to accurate changes in their energy transfer or trapping5,6,7,8.
Autofluorescence of cellular compounds can be used as a marker for cytodiagnosis or as a natural indicator of cellular state or metabolism in response to external and internal signals through changes in emission9. It can also be used to discriminate taxonomically different groups of photosynthetic organisms10. Depending on the phylogenetic position of phototrophic microorganisms, one can find different in vivo fluorescence features. Therefore, a taxonomic identification based on the in vivo characteristics of phototrophic fluorescence (including fluorescence absorption and emission spectra) has been attempted on several occasions11,12. Because of the diversity in accessory pigments among phytoplankton taxa, differences in the wavelengths at which chlorophyll a (Chl a) fluorescence is stimulated, or differences in emission spectra, can be used to infer taxonomy13. The in vivo fluorescence excitation and emission spectra of these specimens rely not only on the phyla of algae, but also on photosystem adaptation14. The efficiency of energy transfer to Chl a, or the ratio of Chl a to accessory pigments, and the cellular pigment content are sensitive to growth conditions5.
Red algae, particularly Chroothece, have several accessory fluorescent pigments-phycobiliproteins and carotenoids; the former concentrate in phycobilisomes attached to the thylakoids of chloroplasts. Phycobiliproteins (phycocyanin, phycoerythrin, and allophycocyanin) can capture light at different wavelengths and transmit it to Chl a, which makes photosynthesis in very different light and culture conditions possible15. For instance, Chroothece species can grow inside caves or almost emerge in slightly saline calcareous streams2.
Monochromatic lights affect the growth and pigment composition of photosynthetic organisms, and have been studied to prevent or control the growth of photosynthetic organisms in caves. Mulec et al. showed that red enriched lighting promotes the growth of cyanobacteria, algae, and plants16. Previous studies have also reported that green light affects the pigment composition of cyanobacteria17, while others have revealed that green light prevents the growth of most photosynthetic organisms and some cyanobacteria exhibit a reduction in thylakoids and weaker mean fluorescence intensity18.
To understand the ability of Chroothece as a model organism to overcome harsh conditions, cultured cells have been exposed to increasing light intensities and monochromatic light (green or red)15, to see how it copes with the dim conditions of caves (where red light predominates). The protocol presented herein reproduces the effect of the abovementioned variables on Chroothece&#39;s phycobiliproteins at the cellular level using its own autofluorescence.
Nowadays, fluorescence is commonly used as a tool to study the physiological responses of vascular plants, microalgae, macroalgae, and cyanobacteria13,14,16. Spectral confocal fluorescence microscopy is a superb tool for in vivo studies to evaluate photosynthetic specimens&#39; physiology at the single-cell level10,17,18,19,20, by avoiding problems associated with the low growth rate in the laboratory and the difficulties with obtaining enough biomass for the associated extraction and biochemical methods8. Once cells are treated under different culture conditions for 2 weeks, the lambda scan profile can be measured in vivo. Although there are several publications in which different wavelengths of excitation by confocal imaging have been used3,4,10,17, most phycobiliproteins and Chl a can be detected using a 561 nm wavelength excitation line, and the detected emission ranges from 570 to 760 nm wavelength. These criteria have been based on an analysis previously performed10 with commercial pure pigments (Table 1) by confocal imaging and the obtained results in different algae species20,21,22.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Pigments</td>
			<td rowspan="2">&#955;flmax (nm)</td>
			<td colspan="8">&#955; exc (nm)</td>
		</tr>
		<tr>
			<td>351</td>
			<td>364</td>
			<td>458</td>
			<td>476</td>
			<td>488</td>
			<td>514</td>
			<td>543</td>
			<td>633</td>
		</tr>
		<tr>
			<td>Chl a</td>
			<td>660.9-678.1</td>
			<td>43.4 &#177; 1.8</td>
			<td>11.2 &#177; 0.2</td>
			<td>1.8 &#177; 0.05</td>
			<td>2.0 &#177; 0.08</td>
			<td>12.2 &#177; 0.7</td>
			<td>6.0 &#177; 0.3</td>
			<td>4.2 &#177; 0.16</td>
			<td>80.7 &#177; 1.5</td>
		</tr>
		<tr>
			<td rowspan="2">R-PE</td>
			<td>569.2-583.3</td>
			<td>5.9 &#177; 0.6</td>
			<td>5.9 &#177; 0.16</td>
			<td>11.1 &#177; 0.04</td>
			<td>42.2 &#177; 0.3</td>
			<td>100.0 &#177; 0</td>
			<td>90.0 &#177; 0.3</td>
			<td>99.2 &#177; 0.08</td>
			<td>-</td>
		</tr>
		<tr>
			<td>652.1-668.6</td>
			<td>-</td>
			<td>-</td>
			<td>1.5 &#177; 0.01</td>
			<td>3.7 &#177; 0.04</td>
			<td>26.7 &#177; 0.5</td>
			<td>8.7 &#177; 0.16</td>
			<td>11.1 &#177; 0.16</td>
			<td>11.3 &#177; 0.2</td>
		</tr>
		<tr>
			<td>C-PC</td>
			<td>636.2-676.4</td>
			<td>2.3 &#177; 0.04</td>
			<td>1.0 &#177; 0.01</td>
			<td>0.6 &#177; 0.004</td>
			<td>0.7 &#177; 0.008</td>
			<td>2.0 &#177; 0.08</td>
			<td>2.0 &#177; 0.04</td>
			<td>3.3 &#177; 0.16</td>
			<td>33.6 &#177; 0.9</td>
		</tr>
		<tr>
			<td>APC-XL</td>
			<td>667.3-683.8</td>
			<td>15.1 &#177; 1.5</td>
			<td>9.6 &#177; 0.98</td>
			<td>1.0 &#177; 0.04</td>
			<td>1.2 &#177; 0.08</td>
			<td>5.9 &#177; 0.7</td>
			<td>4.1 &#177; 0.5</td>
			<td>23.2 &#177; 3.5</td>
			<td>91.4 &#177; 2.3</td>
		</tr>
	</tbody>
</table>
Table 1: The pure pigment information used to run the lambda scan analysis. This table shows emission peaks and shoulders/fluorescence band maxima of different fluorochromes/pigments by confocal imaging spectrophotometry for all the excitation wavelengths, and the percentage of light emission by pigments/fluorochromes. Values were calculated by the formula: = MFI*100/255. Each value is the mean &#177; SE (mean &#177; standard error from the mean). Pure pigments were used for calibrating the confocal scanning laser microscope as follows1,2,10. Chlorophyll a was obtained from Spinacia oleracea, R-phycoerythrin (R-PE) from Porphyra tenera, and C-phycocyanin (C-PE) from Spirulina sp. All the species were dissolved in filtered distilled water. Allophycocyanin-XL (APC-XL) was obtained from Mastigocladus laminosus, which was dissolved in ammonium sulfate (60%) and potassium phosphate (pH = 7) to achieve a concentration of 38 mM. The scans were performed with 400 &#181;L of each pigment solution (concentration of 1 mg/mL) using an 8-well covered-glass bottom chamber.
The study of a single excitation wavelength is quite a useful first approximation. In this case, however, it is necessary to elucidate the relative contribution of the different complexes in the fluorescence signal, which is recommended to perform a fluorescence ratio or spectrum analysis at several wavelengths, among other methods.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;-Dish 35 mm, high Glass Bottom</td>
			<td>Ibidi&#160;</td>
			<td>81158</td>
			<td>-</td>
		</tr>
		<tr>
			<td>24 black well plate</td>
			<td>Ibidi</td>
			<td>82406</td>
			<td>&#160;flat and clear bottom for high throughput microscopy</td>
		</tr>
		<tr>
			<td>Algae Incubator</td>
			<td>Panasonic</td>
			<td>MLR-352-PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica Microsystems</td>
			<td>SP8 TCS</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Flask</td>
			<td>Fisher Scientific</td>
			<td>15380591</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>green filter</td>
			<td>PNTA, LEE filters</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>HC PL APO 63X/1.30 GLYC CORR CS2</td>
			<td>Leica Microsystems</td>
			<td>506353</td>
			<td>Glycerol immersion lens</td>
		</tr>
		<tr>
			<td>Image acquisition software. LAS X</td>
			<td>Leica Microsystems</td>
			<td>SP8 TCS</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Panasonic</td>
			<td>FL40SSENW/37MLR-352-PE</td>
			<td></td>
		</tr>
		<tr>
			<td>Quantum photoradiometer</td>
			<td>DeltaOhm&#160;</td>
			<td>DO 9721</td>
			<td>-</td>
		</tr>
		<tr>
			<td>R software</td>
			<td>R Core Team, 2020</td>
			<td>4.0.2.</td>
			<td>-</td>
		</tr>
		<tr>
			<td>red filter</td>
			<td>PNTA, LEE filters</td>
			<td>-</td>
			<td>Can be purchased in a local convenience store or online stores.</td>
		</tr>
		<tr>
			<td>SWES medium</td>
			<td>University of Murcia</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Type G Immersion liquid</td>
			<td>Leica Microsystems</td>
			<td>11513910</td>
			<td>Glycerol&#160;</td>
		</tr>
	</tbody>
</table>

autofluorescence imaging to evaluate red algae physiology

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in full sunshine. Most rhodophytes are red, however some can appear bluish, depending on the proportion of blue and red biliproteins (phycocyanin and phycoerythrin). Different phycobiliproteins can capture light at diverse wavelengths and transmit it to chlorophyll a, which makes photosynthesis under very different light conditions possible. These pigments respond to habitat changes in light, and their autofluorescence can help to study biological processes. Using Chroothece mobilis as a model organism and the spectral lambda scan mode in a confocal microscope, the adaptation of photosynthetic pigments to different monochromatic lights was studied at the cellular level to guess the species' optimal growth conditions. The results showed that, even when the studied strain was isolated from a cave, it adapted to both dim and medium light intensities. The presented method is especially useful for studying photosynthetic organisms that do not grow or grow very slowly under laboratory conditions, which is usually the case for those living in extreme habitats.

Chlorophyll a generally absorbs blue and red wavelengths of visible light, whereas phycobiliproteins use green, yellow, and orange wavelengths7. The autofluorescence of these pigments makes the first approach to study phycobiliproteins and chlorophyll behavior under experimental and field conditions possible.
By comparing the obtained data and plotting on different graphs, significant changes in mean fluorescence intensity (MFI) can be distinguished (<strong class="xfig...

Watch this Scientific Journal Video about Autofluorescence Imaging to Evaluate Red Algae Physiology at JoVE.com

Autofluorescence Imaging to Evaluate Red Algae Physiology

The present protocol describes step-by-step autofluorescence imaging and evaluation of phycobiliprotein changes in red algae based on spectral analysis. This is a label-free and non-destructive method to evaluate cellular adaptation to extreme habitats, when only scarce material is available and cells grow slowly, or not at all, under laboratory conditions.

Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in ...

This confocal microscopic protocol will permit the interpretation of the ecological behavior and adaptations of microalgae from extreme environments with slow growing in the laboratory using autofluorescence of their pigments. This technique is not invasive and minimizes artifacts as chemical compounds are not used. Knowing the performance of pigments of autotrophs and corresponding growth will permit the designing of the methods of control which is the greatest interest for touristic caves and architectural monuments.Begin preparing the inoculum of Chroothece mobilis by transferring it from the agar culture to the seawater medium or SWES liquid medium. Maintain all the cultures for two weeks with a 16 to 8 light dark photo period at 20 degrees Celsius under a low white light intensity and without shaking until the desired cell density is obtained. Use one milliliter of the exponential phase culture having five times 10 to the third cells per milliliter cell density to inoculate in a 24-well plate for different experiments.To reproduce the monochromatic light effect, use the green filter allowing green light to pass through from 470 to 570 nanometers with a peak at the 506 nanometer wavelength and to red light adjusted using a filter between 590 and 720 nanometers and peaks at 678 nanometers. Expose the cell cultures to this light for two weeks. Turn on all the components of the inverted confocal laser scanning microscope including the laser.Mount the cells in the SWES growth medium from each experimental well of the 24-well plate to a 35 millimeter glass bottom dish for imaging. Choose the 63X or 1.30 numerical aperture or NA glycerol immersion objective and place glycerol over the lens. Next, place the well plate on the microscope stage ensuring the specimen does not move during image acquisition.Center the specimen in the light path and focus on the center of the cell by selecting the plane with the highest fluorescence intensity. Once done, open the image acquisition software and choose XY lambda from the dropdown list in the acquisition mode. Select the excitation line of the laser 561 nanometers, eight bits of dynamic range and 1024 by 1024 pixels.Collect the fluorescence emission spectra in the 10 nanometer bandwidth and the lambda step size of four nanometers within the 570 to 760 nanometer range. Set the pinhole at one air unit and run the lambda scan acquisition. After repeating this process in different fields of view and under the different conditions of red and green lights, save all the data.Once the lambda scan is acquired, click on the quantify window at the top of the software to evaluate the collected fluorescence emission spectra. Go to the open project window and select one XY lambda file. Select stacked profile analysis in the imaging software and define a region of interest of four micrometer square in the center of a cell to analyze the mean fluorescence intensity.Export the data and CSV before repeating the process with different cells under different conditions. Next, open the CSV files to select the different fluorescence emission peaks of all the measured regions of interest by selecting the maximum fluorescence data of phycoerythrin phycocyanobilin, C.phycocyanin, allophycocyanin, and chlorophyll A respectively in the CSV file. Once done, create a new table with all the maximum fluorescence values obtained from each phycobiliprotein and chlorophyll peak and plot the data on a graph.Significant differences in mean fluorescence intensity were observed. The mean fluorescence intensity of phycoerythrin phycocyanobilin significantly decreased when the cells were exposed to monochromatic green light. In contrast, no difference was observed in red light compared to the control.The green light had the opposite effect on allophycocyanin and chlorophyll A in which the mean fluorescence intensity significantly increased due to the adaptation of antenna complexes due to increased quantity or improved connectivity of the antenna complexes among others. The red light produced a non-significant increase in allophycocyanin and chlorophyll fluorescence. To study the effect of different growth conditions on cells in culture, it's crucial to perform the measurements with exactly the same microscope settings.It's possible to analyze other environmental parameters relevant to the culture of autofluorescence organisms, as well as the fluorescence signal within the visible spectrum. This technique is a very powerful approach to a study life organisms with several autofluorescence pigments.

autofluorescence-imaging-to-evaluate-red-algae-physiology

Research

JoVE Journal

Biology

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing

An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The carotid bodies (CB), a key organ of peripheral hypoxic sensitivity, express the long functional isoform of leptin receptor (LepRb) but the role of leptin signaling in control of breathing has not been fully elucidated. We examined the hypoxic ventilatory response (HVR) (1) in C57BL/6J mice before and after leptin infusion at baseline and after CB denervation; (2) in LepRb-deficient obese db/db mice at baseline and after LepRb overexpression in CBs. In C57BL/6J mice, leptin increased HVR and effects of leptin on HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB augmented the HVR. Therefore, we conclude that leptin acts in CB to augment responses to hypoxia.

Our study focuses on the effects of leptin signaling in carotid body (CB) on the hypoxic ventilatory response (HVR). We performed &#39;loss of function&#39; experiments measuring the effect of leptin on HVR after CB denervation and &#39;gain of function&#39; experiments measuring HVR after overexpression of the leptin receptor in CB.

An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The ...