This work was supported by Natural Sciences and Engineering Research Council of Canada (grant 402105), Canadian Foundation for Innovation (grant 129087) and Alberta Education and Training (grant AAETRCP-12-026-SEG) to RJC.

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The authors have nothing to disclose.

소형 형식으로 조류 성장. 현재 실험에서 복제가 증가 될 수 있도록 마이크로 타이 터 플레이트에서 1ml의 배양 부피 조류 배양의 소형화를 허용한다. 이 녹조류는 실험 내내 건강 보장하는 것이 중요하다; 조류의 영양 요건이 충족 될 수 있도록, 다양한 조류 매체를 평가하기 위해 마이크로 타이 터 플레이트 포맷을 사용하여, 성장 곡선 (도 2)를 수행한다. 또한,이?...

식물 플랑크톤의 생리는 전통적으로 원뿔 플라스크에 20 ㎖에서 carboys 1-7에서 5 L에 이르는 메조 규모의 실험에서 연구되어왔다. 각 시점에 대한 복제 샘플을 희생하는 것이 관리하기 어려운 실험 장치를 만들어으로이 실험 규모는 실험 모니터링을위한 서브 샘플링을 필요로한다. 줄이거 나 대량의 서브 샘플링 및 의사 - 복제의 제한을 제거한다 조류 생리학 실험 실험 용적을 소형화하여 일주 동일한 인큐베이터 공간을 사용하면서 기능은 독립적 인 실험의 수를 증가시키는. 마이크로 타이 터 플레이트 포맷은 가변 실험적 조건에서 조류를 조작 1ml의 배양 부피를 사용하여 조류 생물 검정을 위해 개발되었다. 실험이 작은 부피 복제물의 수가 증가 될 수 있도록 허용 복제에 의한 샘플 간의 감소 가변성 실험의 재현성을 증가(그림 2) 12 1백40일에 대한 실험 컨트롤 (즉, 무균 조류 배양)를 유지하면서 실험하고, 진정한 복제를 할 수 있습니다. 이 마이크로 타이 터 플레이트 형식은 쉽게 같은 실험 다양한 질문에 대한 적응 : 박테리아가 조류 호스트와 공생 중성 또는 병원성 상호 작용을해야합니까? 조류의 복합 자극의 추가 또는 독성인가? 이들 및 다른 문제는이 새로운 포맷을 사용하여 9-11 빠른 높은 처리량 방법으로 해결할 수있다. 48 웰 마이크로 타이 배양 플레이트는 각 1 ㎖를 잘 단일 시간 시점에서 샘플링 독립적 인 실험 장치가 될 수 있습니다. 다양한 매개 변수를 포함하여 1 ml의 볼륨에서 샘플링, 이에 국한되지 수 있습니다 WATER-펄스 진폭 변조 (WATER-PAM) 형광 측정 (재료 및 장비 표 참조) 1을 사용 엽록소 형광 및 광 화학적 매개 변수3. WATER-PAM의 형광 측정 조류 13의 실험을 모니터링하는 데 사용할 수있는 신속하고 비 침습적 방법이다. 14, 15 (WATER-PAM 4 ml의 볼륨 - - 2 중간에 희석 문화의 300 μL 150) 그것은 작은 문화 볼륨에서 광합성 효율과 광계 건강을 측정 할 수 있습니다. WATER-PAM의 형광 측정에 더하여,이 설정을 포함한 다른 다양한 파라미터를 측정하는데 사용될 수 있으나, 이에 제한되지 : 조류 세포 및 조류 세포 형태의 변화에​​ 부착 된 박테리아를 시각화하는 현미경; 단위 (CFU) 수를 형성하는 박테리아 식민지; 및 조류 세포 수 및 식별 주민들을위한 유동 세포 계측법.

<table><tbody><tr><td>10 cu. ft. Diurnal incubator (6012-1)</td><td>Caron Corporate</td><td>112310-6012-1-11</td><td>&lt;a href=&quot;http://www.caronproducts.com&quot;&gt;www.caronproducts.com&lt;/a&gt;</td></tr><tr><td>Nunc EasYFlask 25 cm&lt;sup&gt;2&lt;/sup&gt;, Vent/Close Cap, 7 ml working volume, 200/cs</td><td>Thermo Fisher Scientific</td><td>N156340</td><td>&lt;a href=&quot;http://www.fishersci.ca&quot;&gt;www.fishersci.ca&lt;/a&gt;</td></tr><tr><td>Multiwell TC Plates &amp;ndash; 48-well</td><td>BD Biosciences Discovery Labware</td><td>353078</td><td>&lt;a href=&quot;http://www.bdbiosciences.com/&quot;&gt;www.bdbiosciences.com&lt;/a&gt;</td></tr><tr><td>P1000 Gilson The Pipetting Standard&amp;mdash;Gilson&#039;s Pipetman</td><td>Mandel Scientific Company Inc.</td><td>GF-F123602</td><td>&lt;a href=&quot;http://www.mandel.ca&quot;&gt;www.mandel.ca&lt;/a&gt;</td></tr><tr><td>P10mL Gilson The Pipetting Standard&amp;mdash;Gilson&#039;s Pipetman</td><td>Mandel Scientific Company Inc.</td><td>GF-F161201</td><td>&lt;a href=&quot;http://www.mandel.ca&quot;&gt;www.mandel.ca&lt;/a&gt;</td></tr><tr><td>Wide Orifice Tips nonsterile [100&amp;ndash;1,250 &amp;micro;l]</td><td>VWR International</td><td>89079-468</td><td>&lt;a href=&quot;http://www.ca.vwr.com&quot;&gt;www.ca.vwr.com&lt;/a&gt;</td></tr><tr><td>Ultrafine Tips nonsterile [100&amp;ndash;1,250 &amp;micro;l]</td><td>VWR International</td><td>89079-470</td><td>&lt;a href=&quot;http://www.ca.vwr.com&quot;&gt;www.ca.vwr.com&lt;/a&gt;</td></tr><tr><td>Finntip 10 ml [Vol: 1 - 10 ml]</td><td>Thermo Fisher Scientific</td><td>9402151</td><td>&lt;a href=&quot;http://www.fishersci.ca&quot;&gt;www.fishersci.ca&lt;/a&gt;</td></tr><tr><td>WATER-Pulse Amplitude Modulation (Water-ED)</td><td>Heinz Walz GmbH, Effeltrich, Germany</td><td>EDEE0232</td><td>&lt;a href=&quot;http://www.walz.com&quot;&gt;www.walz.com&lt;/a&gt;</td></tr><tr><td>15 mm diameter quartz glass cuvette (WATER-K)</td><td>Caron Corporate</td><td></td><td>&lt;a href=&quot;http://www.caronproducts.com&quot;&gt;www.caronproducts.com&lt;/a&gt;</td></tr><tr><td>Sodium chloride (crystalline/certified ACS), Fisher Chemical</td><td>Thermo Fisher Scientific</td><td>Thermo Fisher Scientific</td><td>&lt;a href=&quot;http://www.fishersci.ca&quot;&gt;www.fishersci.ca&lt;/a&gt;</td></tr><tr><td>BD Difco Marine Broth 2216</td><td>BD Biosciences Discovery Labware</td><td>BD Biosciences Discovery Labware</td><td>&lt;a href=&quot;http://www.bdbiosciences.com&quot;&gt;www.bdbiosciences.com&lt;/a&gt;</td></tr><tr><td>BD Bacto Agar</td><td>BD Biosciences Discovery Labware</td><td>BD Biosciences Discovery Labware</td><td>&lt;a href=&quot;http://www.bdbiosciences.com&quot;&gt;www.bdbiosciences.com&lt;/a&gt;</td></tr><tr><td>L1 Medium Kit, 50 L</td><td>NCMA [National Center for Marine Algae and Microbiota</td><td>NCMA [National Center for Marine Algae and Microbiota</td><td>&lt;a href=&quot;http://www.ncma.bigelow.org&quot;&gt;www.ncma.bigelow.org&lt;/a&gt;</td></tr></tbody></table>

a small volume bioassay to assess bacterial/phytoplankton co-culture using water-pulse-amplitude-modulated (water-pam) fluorometry

배양 실험 1-7에 걸쳐 서브 샘플링 될 수 있도록 미세 조류의 실험 조작을위한 종래의 방법은 대량 배양 (5 L 내지 20 ml)에 사용했다. 대량의 서브 샘플링 다양한 이유로 문제가 될 수있다 : 1)는 총 부피와 표면적의 변화 원인은 다음 실험 기간 동안 배양 체적비; 2) 의사 복제 (즉, 동일한 치료 플라스크 8에서 샘플)는 종종 오히려 진정한 복제물 (복제 치료에서 즉, 샘플링)보다 채용을 복제; 3) 실험 기간은 총 부피에 의해 제한된다; 4) 배양 무균 또는 일반적인 세​​균 미생물 오염은 일반적으로 서브 샘플링 동안 발생 장기 실험 동안 유지하기 어렵다. 미세 적정 플레이트의 사용은 최대 48 내의 별도의 처리 1 ㎖의 각각의 배양에 사용되는 복제 볼륨을 가능12.65 X 8.5 X 2.2 cm 판함으로써 실험 볼륨을 감소하고 치료를 서브 샘플링하지 않고 광범위한 복제를 허용. 9-11 스크리닝 세균성 조류 공동 배양, 조류 생리 테스트 및 독소 : 또한,이 기술은 실험을 포함한 다양한 포맷에 맞게 수정 될 수있다. 조류, 박테리아 및 / 또는 공동 문화와 개인 우물로 제한 등 다양한 실험 절차에 대한 샘플링,하지만 할 수 있습니다 WATER-펄스 진폭 변조 (WATER-PAM) 형광 측정, 현미경, 세균 집락 형성 단위 (CFU) 수와는 유동 세포 계측법. 마이크로 타이 터 플레이트 포맷 WATER-PAM 형광 측정의 조합은 광 화학적 수율 및 샘플, 높은 재현성과 낮은 가변성과 다른 광 화학적 파라미터의 다수의 신속한 측정이 가능하며 실험의 과정 속에 든 대형 유리 병 또는 원추형 플라스크를 서브 샘플링 많은 함정을 피할 .

WATER-PAM의 형광 측정 수치. 물 - 펄스 진폭 변조 (PAM) 형광 측정은 형광 (엽록소 콘텐츠에 대한 프록시) 및 조류 문화의 광합성 수율 (PSII 건강)를 결정하는 빠르고 효율적인 방법입니다. PAM WinControl 소프트웨어는 원시 데이터 값의 스프레드 시트 (다음 어두운 적응 조류 시료 기본적인 파라미터이다)를 생성한다 : 어두운 적응 세포의 F 0 = 형광 <p cl...

Watch this Scientific Journal Video about A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry at JoVE.com

A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

작은 볼륨 생물 검정은 물 - 펄스 진폭 변조 (WATER-PAM) 형광 측정을 사용하여 세균 / 식물 플랑크톤의 공동 문화를 평가하는

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be ...

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A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry

10.5K 조회수.  University of Alberta. The goal of this procedure is to demonstrate the reproducibility and adaptability of using a microtiter plate format for microalgal screening. This rapid screen combines WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry to measure photosynthetic yield as an indicator of Photosystem II (PSII) health with small volume bacterial-algal co-cultures.

비디오: A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated WATER-PAM Fluorometry

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

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

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.

물 화학 및 해양 미생물에 필드 가이드 - 바다에서 보이지 않는 선수를 해명

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

연구

환경과학

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Cytometry와 수생 Biofilms의 특성

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

특성화 미생물 역학-Cytometry 워크플로 순수 문화에서 자연 커뮤니티를 기반으로

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

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

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

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

수생 생태계에서 유기 탄소 흐름의 새로운 경로를 밝히기 위해 추적자로 형광으로 분류 된 박테리아

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

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

천연 화합물 라이브러리의 탐험에 적합 안티 바이오 필름 분석 실험의 플랫폼

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

면역학 및 감염병학

Operation of Laboratory Photobioreactors with Online Growth Measurements and Customizable Light Regimes

The laboratory study of microalgae can be experimentally challenging. In addition to the cultivation requirements of non-photosynthetic microorganisms, phototrophs also require illumination. Routinely, researchers seek to provide custom light supplies, i.e., vary the light intensity and time over which it is delivered. Such flexibility is difficult with standard benchtop lights. Usually, cultivation studies also require growth comparisons between experimental treatments. Frequently, growth is assessed over an extended duration, e.g., multiple times a day over a week-long trial. Manual measurements can be time-consuming and lack data resolution. Therefore, photobioreactors (PBRs) with automatic growth monitoring and customizable light supply are useful for replicated experiments with multiple treatments. The current work presents the design, construction, and operation of laboratory PBRs. The materials are easily sourced and relatively inexpensive. The design could be duplicated with moderate skill. Each structure has a footprint of ~40 cm2 and hosts three 1 L glass bottles for triplicate replication. Bottles rest upon platforms containing magnetic stirrers and are arranged vertically within a 1 m high and 15 cm diameter polyvinyl chloride (PVC) pipe. The pipe interior is lined with light-emitting diodes (LEDs). These LEDs produce continuous light intensities from 0&#8211;2400 &#181;mol photons m-2 s-1 of photosynthetically active radiation (PAR). Users design a custom lighting program. The light intensity can be adjusted each second or held constant for longer durations. Oxygen produced from photosynthesis exits each bottle via a one-way volumetric gas sensor. Software is used to record gas sensor data. The amount of oxygen produced can be correlated to biomass growth. If biomass samples are required, a syringe can be used to extract culture. The method is suited for microalgae grown with bicarbonate as the carbon source. These PBRs are valuable to a laboratory that requires replicated experiments, light regime flexibility, and continuous high-resolution growth data.

This publication describes the design of laboratory photobioreactors (PBRs) with customizable light regimes. The growth of cyanobacteria or microalgae, using bicarbonate as their carbon source, is monitored continuously by measuring volumetric oxygen production. These PBRs facilitate rapid, replicated laboratory growth comparisons with little user intervention during experiments.

온라인 성장 측정 및 사용자 정의 가능한 조명 체계를 갖춘 실험실 광생물 반응기 운영

The laboratory study of microalgae can be experimentally challenging. In addition to the cultivation requirements of non-photosynthetic microorganisms, ...

생물학

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.

Cuvette-type Fast Repetition Rate Fluorometer에 의한 Colacium sp.의 부착 단계의 광생리학 측정

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of non-photochemical quenching (NPQ) has proven to be an effective strategy to increase photosynthetic performance. However, the potential to breed for improved NPQ and a complete understanding of the genetic basis of NPQ relaxation is lacking due to limitations of oversampling and data collection from field-grown crop plants. Building on previous reports, we present a high-throughput assay for analysis of NPQ relaxation rates in Glycine max (soybean) using pulse amplitude modulated (PAM) chlorophyll fluorometry. Leaf disks are sampled from field-grown soybeans before transportation to a laboratory where NPQ relaxation is measured in a closed PAM-fluorometer. NPQ relaxation parameters are calculated by fitting a bi-exponential function to the measured NPQ values following a transition from high to low light. Using this method, it is possible to test hundreds of genotypes within a day. The procedure has the potential to screen mutant and diversity panels for variation in NPQ relaxation, and can therefore be applied to both fundamental and applied research questions.

The protocol introduces a high-throughput method for measuring the relaxation of non-photochemical quenching by pulse amplitude modulated chlorophyll fluorometry. The method is applied to field-grown Glycine max and can be adapted to other species to screen for genetic diversity or breeding populations.

펄스 진폭 변조된 엽록소 형광계를 이용한 작물에서의 비광화학적 담금질의 고처리량 분석

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of ...

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.

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.

홍조류 생리학을 평가하기 위한 자가형광 이미징

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

Immunocytochemical Visualization of Proteins from Cyanobacterial Cells with High Autofluorescence of Phycoerythrin and Phycourobilin

Presented is a protocol for visualizing and quantifying a specific protein in cells at the cellular level for the marine cyanobacterium Crocosphaera watsonii, a crucial primary producer and nitrogen fixer in oligotrophic oceans. One of the challenges for marine autotrophic N2 fixers (diazotrophs) is distinguishing probe-derived fluorescence signals from autofluorescence. C. watsonii was selected to represent chlorophyll-, phycoerythrin- and phycourobilin-containing cyanobacteria. The protocol allows for simple and semi-quantitative visualization of proteins in C. watsonii at a single-cell level, enabling investigation of protein production under different environmental conditions to evaluate the metabolic activities of the target cyanobacteria. Furthermore, the fixation and permeabilization methods are optimized to enhance the fluorescence signals from target proteins to distinguish them from autofluorescence, especially from phycoerythrin and phycourobilin. The enhanced signal can be visualized using confocal or widefield fluorescence microscopy. Additionally, fluorescence intensity was semi-quantified using Fiji software. This single-cell analysis workflow allows the evaluation of cell-to-cell variations of specific protein content. The protocol can be performed in any life science laboratory as it requires only standard equipment and can also be easily adapted to other phycoerythrin-containing cyanobacterial cells.

This protocol outlines the visualization and quantification of a particular protein within cells at the cellular level for the phycoerythrin-containing cyanobacterium, Crocosphaera watsonii.

Presented is a protocol for visualizing and quantifying a specific protein in cells at the cellular level for the marine cyanobacterium Crocosphaera ...