Automated sample area definition for high‐throughput microscopy

Zeder, Michael; Ellrott, Andreas; Amann, Rudolf

doi:10.1002/cyto.a.21034

Cited by 30 publications

(31 citation statements)

References 12 publications

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“…Triplicate filters from water depths of 20, 150, 400, 550, and 700 m were stained with 4=,6-diamidino-2-phenylindole (DAPI) subsequently to the CARD-FISH procedure, and the DAPI and CARD-FISH counts were done with a Zeiss AxioImager.Z2 microscope (Carl Zeiss AG, Jena, Germany) with an automated stage. Image acquisition was done using the software package AxioVision, release 7.6 (Carl Zeiss AG, Jena, Germany), comprising an automated focusing routine, an automated sample area definition using the macro MPISYS, and a manual image quality assessment (22)(23)(24). The software took pictures of the DAPI channel (350 nm) and the FISH channel (488 nm) at different fields of view along a track on the sample.…”

Section: Fig 2 (Top)mentioning

confidence: 99%

“…A single representative picture was made from each z-stack for each probe to calculate the total cell counts per sample. To do this, the average biovolume of single CARD-FISH-stained bacterial cells was measured from each of the extracted representative single pictures using the YABBA software (22). The total cell counts were calculated by dividing the total cell biovolume with the average biovolume of a single cell.…”

Section: Fig 2 (Top)mentioning

confidence: 99%

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Colonization in the Photic Zone and Subsequent Changes during Sinking Determine Bacterial Community Composition in Marine Snow

Thiele

Fuchs

Amann

et al. 2015

Appl Environ Microbiol

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Due to sampling difficulties, little is known about microbial communities associated with sinking marine snow in the twilight zone. A drifting sediment trap was equipped with a viscous cryogel and deployed to collect intact marine snow from depths of 100 and 400 m off Cape Blanc (Mauritania). Marine snow aggregates were fixed and washed in situ to prevent changes in microbial community composition and to enable subsequent analysis using catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). The attached microbial communities collected at 100 m were similar to the free-living community at the depth of the fluorescence maximum (20 m) but different from those at other depths (150, 400, 550, and 700 m). Therefore, the attached microbial community seemed to be "inherited" from that at the fluorescence maximum. The attached microbial community structure at 400 m differed from that of the attached community at 100 m and from that of any free-living community at the tested depths, except that collected near the sediment at 700 m. The differences between the particle-associated communities at 400 m and 100 m appeared to be due to internal changes in the attached microbial community rather than de novo colonization, detachment, or grazing during the sinking of marine snow. The new sampling method presented here will facilitate future investigations into the mechanisms that shape the bacterial community within sinking marine snow, leading to better understanding of the mechanisms which regulate biogeochemical cycling of settling organic matter.T he formation of macroscopic organic aggregates such as marine snow is an important process for the removal of photosynthetically fixed carbon from the surface ocean to the deep sea (1). The export of organic matter is the main driver for the oceanic sequestering of atmospheric carbon dioxide on long time scales (2).Settling marine snow aggregates harbor diverse microbial communities that play an important role in the degradation of the organic compounds and release of dissolved nutrients and organic matter to the water column (3-5). Therefore, the attached microbial communities may play an important role in the regulation of the magnitude and efficiency of the biological carbon pump. However, little is known about bacterial community composition and dynamics within marine snow collected below the depths reached by scuba divers. This is because the fragile nature of marine snow makes the collection of individual, intact marine snow particles nearly impossible.Previous investigations have relied on size fractionation of water samples to observe and compare clustering of free-living bacteria to that of bacteria associated with particles (5-8). These studies observed that the composition of the microbial communities associated with particles differed from that of free-living communities in the surrounding water column. However, other studies have found similarities between the free-living and attached microbial communities (9-11), potentially indicating methodolo...

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Section: Fig 2 (Top)mentioning

confidence: 99%

Section: Fig 2 (Top)mentioning

confidence: 99%

Colonization in the Photic Zone and Subsequent Changes during Sinking Determine Bacterial Community Composition in Marine Snow

Thiele

Fuchs

Amann

et al. 2015

Appl Environ Microbiol

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“…S1A to G in the supplemental material) on a Zeiss AxioImager.Z2 microscopic stand (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) with a cooled charged-coupled-device Fig. S1B to D in the supplemental material) (SamLoc [15]). Subsequently, the SamLoc software was used to define coordinates for image acquisition based on user-defined grids with FOVs, with a minimum distance of 250 m between FOVs (see Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The pixel size of this microscope and the associated objectives was 0.1068 m pixel Ϫ1 . The acquisition of overview images was done via webcam and using the SamLoc software (15). The previously described automated image acquisition software MPISYS was modified for the different hardware requirements and was extended with new functionality to account for the ship's movements.…”

Section: Methodsmentioning

confidence: 99%

“…It was applied to several thousands of sample preparations for high-throughput analysis of fluorescence in situ hybridization (FISH) assays, along a transect across the Atlantic Ocean (14). Further development of this system enabled, for example, the autonomous recognition of the shape of the filter pieces (SamLoc [15]) or sped up the image acquisition time by using light-emitting diode (LED) illumination, which removed the need for manual shutter opening and closing during optical filter changes.…”

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Modification of a High-Throughput Automatic Microbial Cell Enumeration System for Shipboard Analyses

Bennke

Reintjes

Schattenhofer

et al. 2016

Appl Environ Microbiol

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In the age of ever-increasing "-omics" studies, the accurate and statistically robust determination of microbial cell numbers within often-complex samples remains a key task in microbial ecology. Microscopic quantification is still the only method to enumerate specific subgroups of microbial clades within complex communities by, for example, fluorescence in situ hybridization (FISH). In this study, we improved an existing automatic image acquisition and cell enumeration system and adapted it for usage at high seas on board an oceanographic research ship. The system was evaluated by testing settings such as minimal pixel area and image exposure times ashore under stable laboratory conditions before being brought on board and tested under various wind and wave conditions. The system was robust enough to produce high-quality images even with ship heaves of up to 3 m and pitch and roll angles of up to 6.3°. On board the research ship, on average, 25% of the images acquired from plankton samples on filter membranes could be used for cell enumeration. Automated enumeration was highly correlated with manual counts (r 2 > 0.9). Even the smallest of microbial cells in the open ocean, members of the alphaproteobacterial SAR11 clade, could be confidently detected and enumerated. The automated image acquisition and cell enumeration system developed here enables an accurate and reproducible determination of microbial cell counts in planktonic samples and allows insight into the abundance and distribution of specific microorganisms already on board within a few hours. IMPORTANCEIn this research article, we report on a new system and software pipeline, which allows for an easy and quick image acquisition and the subsequent enumeration of cells in the acquired images. We put this pipeline through vigorous testing and compared it to manual microscopy counts of microbial cells on membrane filters. Furthermore, we tested this system at sea on board a marine research vessel and counted bacteria on board within a few hours after the retrieval of water samples. The imaging and counting system described here has been successfully applied to a number of laboratory-based studies and allowed the quantifi- T he exact quantification of cells is fundamental to microbial ecology and hence for understanding the interaction of microorganisms with biotic and abiotic factors. Still, the counting of microbial cells on membrane filters using an epifluorescence microscope remains the method of choice (1-3) for the enumeration of picoplankton cells, although it is rather time-consuming and relies on the experience of the individual person counting. Additionally, manual counting of an entire membrane filter is not practical within a given time frame, and therefore, only a small part is analyzed (usually 12 to 20 fields of view [FOVs]) (3, 4). Consequently, several tools have been developed over the past 2 decades to automatically enumerate microbial cells by means of image acquisition and subsequent image analysis (e.g., references 5-13). Mos...

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MRT letter: Micro‐ to nanoscale sample collection for high throughput microscopy

Cheong

Liew

2013

Microscopy Res & Technique

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In high throughput microscopy, it is often assumed that the objects under investigation are fixed spatially. In addition, it is also presumed that the objects are sufficiently populated, otherwise there will be need to search through vast tracks of field of views before any recording can be done. The ability to collect objects at one location in the hydrated state is thus desirable and this is a challenge when the density of target objects in a sample is very low. In this work, we report that the generation of a squeezing flow from a circular coverslip compressing on suspensions is able to collect particulate (microbeads, fluorescent nanobeads and live algal cells) and non-particulate (EGFP) objects at the rim region of the coverslip. With a coverslip of 13 mm diameter, volumes between 2 µL and 4 µL were found to completely fill the coverslip without breaching the rims. Sample compression speeds between 100 µm/s and 1000 µm/s did not have any effect on object collection outcomes. In effect, the simple placement of coverslips on top the drop of sample by hand without a motorized translator was found to produce similar collection outcomes. Quantitative measurements confirmed that all the objects investigated were displaced and relocated at the rim regions to a very high degree.

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Automated sample area definition for high‐throughput microscopy

Cited by 30 publications

References 12 publications

Colonization in the Photic Zone and Subsequent Changes during Sinking Determine Bacterial Community Composition in Marine Snow

Colonization in the Photic Zone and Subsequent Changes during Sinking Determine Bacterial Community Composition in Marine Snow

Modification of a High-Throughput Automatic Microbial Cell Enumeration System for Shipboard Analyses

MRT letter: Micro‐ to nanoscale sample collection for high throughput microscopy

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