Dataset: Epifluorescence Microscopy Cell Size and Biomass
Deployment: TAN1810

Epifluorescence Microscopy Water Column Samples from Subtropical and Subantarctic Waters East of New Zealand

View Data: For data, See Dataset Metadata Page: https://www.bco-dmo.org/dataset/905060

Principal Investigator:

Natalia Yingling (Florida State University, FSU)

Co-Principal Investigator:

Karen E. Selph (University of Hawaii, UH)

Michael Stukel (Florida State University, FSU)

BCO-DMO Data Manager:

Sawyer Newman (Woods Hole Oceanographic Institution, WHOI BCO-DMO)

Project:

Collaborative Research: Quantifying trophic roles and food web ecology of salp blooms of the Chatham Rise (Salp Food Web Ecology)

Version:

Deployment Synonyms:

SalpPOOP

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Description

Methods & Sampling

Dataset acquisition description

Field Collection

Data was collected in the Chatham Rise section of the Southern Ocean, located east of Aotearoa New Zealand, as part of the SALPOOP (‘Salp Particle expOrt and Ocean Production’) voyage during October to November 2018. ). We conducted five Lagrangian experiments (hereafter referred to as “cycles”) that lasted four to eight days (Décima et al., 2023). There were three cycles that were sampled in SA waters (1, 2 and 5) and two cycles in ST waters (3 and 4) while salps were only present in three cycles (1, 2 and 4). Six depths were chosen to span the euphotic zone (based on chlorophyll fluorescence measured during the conductivity-temperature-depth (CTD) downcast profiles).

Epifluorescence Microscopy Sampling

From each depth, two different volumes of water were sampled: 50 mL for nanoplankton- epifluorescence microscopy (filtered through a 0.8-µm pore-size black polycarbonate filter) and 400 mL for microplankton epifluorescence microscopy (filtered through an 8-µm pore-size filter). Utilizing two different sized filters and sampling volumes allowed for appropriate, adjustable filtered volumes and avoid overlapping cells on the slides. 20 µm backing filters were utilized as data has indicated that they support the membrane filters and ensure even dispersal of sample on the filter (Kemp et al., 1993; Taylor et al., 2015). The samples were preserved using buffered formalin, alkaline Lugol’s solution, and sodium thiosulfate then stained using proflavine and 4’, 6-diamidino-2phenylindole (DAPI) (modified protocol from Sherr and Sherr, 1993 in Kemp et al. 1993). During and immediately after filtration, filters were covered with tin foil to prevent photochemical quenching. Filters were mounted onto a glass slide and frozen in a -80º C freezer for later analysis.

Data Processing Description

Dataset Processing Description

Processing Notes from Researcher:

Epifluorescence Microscopy Analysis

Phytoplankton images were captured with an Olympus Microscope DP72 Camera using an Olympus BX51 fluorescence microscope. 20 images were taken under the fluorescence of FITC (green fluorescence) n order to capture the fluorescence of cell proteins. Proflavine overstained cells to the point that we were not able to confidently differentiate heterotrophic cells from autotrophic cells, therefore only biomass and abundance values were able to be calculated. A 60x magnification lens was used to image slides with 0.8 µm filters while 20x magnification was used to image slides with 8 µm filters. Images were then processed using the ImageJ image analysis software (v 1.52a or 1.53c). Cells were manually outlined using the freehand tool and approximate feret cell length and cell area were calculated by ImageJ in pixels and then converted to microns using a calibration scale. To avoid biasing the examined cell size, cells that were roughly greater than 50% out of frame and cells that were broken or fragmented were not included in analysis. Conversions factors were applied to account for volume filtered and percentage of the filter area analyzed. To determine the true filtration diameter, a light microscope was used to examine a 25 mm glass fiber filter (GF/F) filter that had a small amount of dyed water filtered through. It was discovered that the filter funnel blocks roughly 12% of the 25-mm filter and the filtered region had a diameter of 22 mm. Equations 1-5 show the equations used to calculate cell width, biovolume, ESD (equivalent spherical diameter) and biomass, where * implies we assumed that cell height = cell width/2. ESD was used as a consistent measure of mean cell size since many plankton have an irregular shape. The height of a cell was assumed to be roughly equivalent to half of the cell width since cells are often flattened during filtration (Taylor et al., 2011) with the exception of diatoms. The biomass of diatoms (which were the only taxon we could conclusively identify) was estimated allometrically using equation 5 while all other cell biomass (non-diatoms) was estimated allometrically using equation 4. (Menden-Deuer and Lessard, 2000).

Equation 1: Cell width = (4/π) x (Area of the cell/Feret length of the cell)

Equation 2: Biovolume = (4/3)(π) x (Feret Length/2) x (Cell Width/2) x (Cell Height*/2)

Equation 3: ESD = 2 x (3 x Biovolume/4π)^(1/3)

Equation 4: Biomass (non-diatoms) = 0.216 x Biovolume^0.939

Equation 5: Biomass (diatoms) = 0.288 x Biovolume^0.811

More information about this dataset deployment

Funding

Award Number	Funding Source
OCE-1756465	NSF Division of Ocean Sciences
OCE-1756610	NSF Division of Ocean Sciences

Instruments

Camera

Supplied Name: Olympus DP72 camera

Supplied Description:

Slides were calibrated from pixels to microns for both 0.8 µm and 8 µm to determine what length of pixels equates to µm thus only micron area, width and feret length are show in the raw datasheet. Our data also includes conversation ratios in the raw excel sheet. This ratio should be included when determining biomass in units of pg C/mL or abundance in #/mL, along with volume filter, as this ratio incorporates the area of the images taken and gives a value that states the ratio of the cells that were actually counted for 0.8 µm and 8 µm samples.

Instrument Type

Generic Name: Camera

Acronym: camera

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/311/

Generic Description:

All types of photographic equipment including stills, video, film and digital systems.

Fluorescence Microscope

Supplied Name: Epifluorescence microscope: Olympus BX51 microscope with a Olympus DP72 camera and Exfo X-cite Series 120 mercury bulb with a FITC filter for green fluroescence

Supplied Description:

Instrument Type

Generic Name: Fluorescence Microscope

Community Identifier: http://vocab.nerc.ac.uk/collection/L05/current/LAB06/

Generic Description:

Instruments that generate enlarged images of samples using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption of visible light. Includes conventional and inverted instruments.

Parameters

Supplied Name	Supplied description	Supplied Units	Standard Name
ID	Unique number given to each individual cell measured	unitless	sample
Cycle	Lagrangian experiment number	unitless	exp_id
Cast	CTD deployment number	unitless	cast
Particle_Number	Number assigned to each particle imaged in a particular image	unitless	number
Filter_Pore_Size	Pore size of filters that were used to filter the sample	microns (µm)	filter_size
Volume_Filtered	Volume of water filtered for that slide in mL	mL	volume
Depth	Depth the sample originated in meters	meters	depth
Lat	Latitude of sample location in decimal degrees; negative values indicates a Northern coordinate	decimal degrees	lat
Long	Longitude of sample location in decimal degrees; a positive value indicates an Eastern coordinate	decimal degrees	lon
Date	Date in New Zealand Standard Time	unitless	date
Area	Area of the cell	microns squared (µm^2)	area_measured
Width	Width of the cell	microns (µm)	width
Feret_Length	Feret lenth of the cell	microns (µm)	length
Conversion_Ratio	Conversation ratio that incorporates area of the image taken and provides a single value for the ratio of cells actually counted (note: this value changes based on pore size of the filter)	unitless	ratio
Calculated_Width	Calculated width (equation one found within the data processing description of this metadata page)	microns (µm)	width
Biovolume	calculated biovolume (equation two found within the data processing description of this metadata page)	µm^3	biovolume
ESD	Equivalent Spherical diameter (equation three found within the data processing description of this metadata page) in µm	microns (µm)	ESD
Biomass	Biomass in units of pg C (If a non-diatom, use equation four found within the data processing description of this metadata page; if diatom, use equation 5)	picogram of carbon per cell (pg C cell^-1)	wet_weight_biomass
Final_Biomass	Biomass in units of pg C/mL (note: this value is the biomass (pg C) divided by the volume filtered (mL) and the conversion ratio)	picogram of carbon per mL (pg C/mL)	wet_weight_biomass
Diatoms	0 represents that this cell is not a diatom, 1 represents this cell is a diatom	unitless	diatom

Database

Contribute Data

Dataset: Epifluorescence Microscopy Cell Size and Biomass
Deployment: TAN1810

Dataset acquisition description

Dataset Processing Description

Database

Contribute Data

Dataset: Epifluorescence Microscopy Cell Size and BiomassDeployment: TAN1810

Dataset acquisition description

Dataset Processing Description

Dataset: Epifluorescence Microscopy Cell Size and Biomass
Deployment: TAN1810