Pracownie usługowo-badawcze

Dr Anna Anielska-Mazur

Środowiskowa Pracownia Mikroskopii Fluorescencyjnej

Zakres usług i badań

Our facility provides researchers with equipment and expertise that are required for depicting cells and quantitative image analysis. Users are given the opportunity to perform their experiments independently or receive support from facility staff to generate and interpret microscopic data based on light microscopy. Our resources include microscopes with suitable software and a rotary microtome.

Contact person

Anna Anielska-Mazur (PhD), email: aam@ibb.waw.pl, tel.: +48 22 659 57 70
Małgorzata Lichocka (PhD), email: mlichocka@ibb.waw.pl, tel.: +48 22 659 57 70

 

Usługi

Opis usług

The Laboratory of Fluorescence Microscopy provides basic methods and instrumentation background for fluorescence microscopy techniques. We offer access to our facility and technical expertise to all researchers. Our activities mainly focus on providing support with producing microscopic insights that lead to a better understanding of biological processes for many research groups at IBB in their projects and doctoral dissertations. Our daily experience has been gained over 10 years. We have experience in imaging various samples, including plant cells and tissue, nematodes, mammalian cell lines, fungi, yeast, protozoa, and bacteria, reflected by numerous published results.

The laboratory operates on a fee-for-service basis. We recommend that you read our terms and fees. Services include training, imaging, and image analysis and are targeted to all scientists from IBB and scientists from other institutions. Facility staff can deliver technical support, imaging assistance, and consultation. Facility equipment is available after reservation by registered users. For registration, contact our team. We also facilitate independent work. For this reason, we organize individual training. Trained users are allowed to work independently. For advice on experimental designs for microscopy, please contact us by email. We are open to scientific collaboration. For more information, please use email correspondence.

The Laboratory of Fluorescence Microscopy supplies high-quality microscopic imaging of fixed and living cells and tissues. The microscopy systems are set up for imaging stained cells on glass slides or in chambered cover glass. Light microscopy instrumentation includes the following devices: two routine fluorescence microscopes and a confocal microscope.

One widefield microscope is an Eclipse E800 upright fluorescence microscope. It is equipped with multiple objective lenses, DIC optics, and an epi-fluorescence attachment with changeable filter blocks. For more information, see Equipment. A Lumen 200 metal halide fluorescence lamp (Prior) is used as an excitation source. An ORCA ER charge-coupled device camera (Hamamatsu) with a 1344 ´ 1024 pixel resolution is utilized for emission detection. For viewing and capturing microscopic data, Lucia software is employed.

One widefield microscope is a conventional Olympus IX-70 inverted fluorescence microscope. It is equipped with multiple objective lenses, contrast phase optics, and an epi-fluorescence attachment with changeable filter blocks. A mercury pressure lamp is used as an excitation source. The system can be used as a supportive tool for initial testing. Configuration for viewing and capturing microscopic data is under construction.

The Nikon C1 confocal laser scanning microscope is built on a Nikon TE2000 inverted stand and fitted with a laser box that includes four laser lines, a confocal head with substitutive first dichroic mirrors, a 3 PMTs for three fluorescence channels, and a detector for transmitted light, coupled with controlling computers with EZ-C1 software for viewing and capturing microscopic data. The TE2000 is equipped with multiple objective lenses, DIC optics, an epi-fluorescence attachment with changeable filter blocks, a motorized stage, and Nikon DS-U1 color camera. The system is mainly based on Nikon hardware and software. For more information, see Equipment. This confocal microscope enables the simultaneous detection of various fluorescently labelled proteins and their co-localization in examined specimens. It allows the collection of single or several optical sections through thick specimens and the generation of projection and three-dimensional images. The data are commonly processed by EZ-C1 viewer, NIS-Elements, and ImageJ software.

The facility is equipped with a Leica RM2145 semi-motorized rotary microtome, which is used to cut serial sections of large samples. It is designed for paraffin specimens for routine and research applications. Sections can be cut in a range of 0.25-60 µm thickness.

For the last 10 years, the E800 microscope has been used for over 3,500 hours. The confocal system has been used for over 4,500 hours. Microscopic analyses that have been conducted in our laboratory were utilized for many thesis and science projects. In the report period, the facility has been used by several science groups from our institution and outside scientific units (see Customers).

Aparatura

  1. Nikon Eclipse E800 upright fluorescence microscope equipped with:
  • Dia-illuminator for bright field (Philips 12 V 100 W halogen lamp).
  • Epi-illuminator for fluorescence (Prior Lumen 200 metal halide lamp).
  • Range of objectives (see Attachment 1).
  • C-CU universal system condenser (NA 0.90).
  • Range of filters for fluorescence (see Attachment 2).
  • Detector for (epi)-fluorescence and transmitted light (Dia):
    • Hamamatsu ORCA ER Monochrome cooled digital camera (1344 ´ 1024 pixel resolution, 12 bit, 6.45 μm ´45 μm pixel size).
  • Lucia General 4.82 and 5.0 system for image processing and analysis, including software for image capturing by charge-coupled device cameras and simple measurements and a conventional fluorescence microscope.
  1. Nikon EZ-C1 laser scanning confocal system:
  • Eclipse TE2000-E inverted microscope mounted on an antivibration table.
  • PRIOR Scientific 3 H107 Pro Scan II motorized microscope stage.
  • T-RCP remote control pad with joystick (X-Y axis).
  • Dia-illuminator for bright field (Philips 12 V 100 W halogen lamp).
  • Epi-illuminator for fluorescence (HBO 103W/2 OSRAM high-pressure mercury lamp).
  • Range of objectives (see Attachment 3).
  • Universal system condenser (NA 0.52, W.D. 30 mm).
  • Range of filters for fluorescence (see Attachment 4).
  • Detector for (epi)-fluorescence and transmitted light (Dia):
    • Nikon DS-5Mc color camera (2560 ´ 1920 pixel maximal resolution, 12 bit).
  • NIS-Elements system for image processing and analysis, including software for image capturing by charge-couple device cameras and simple measurements.
  • Nikon C1 confocal scanning head with exchangeable first dichroic mirror (408/488/543, 488/543/633).
  • Nikon C1 four-channel confocal system with three independent photomultiplier tube detectors for three fluorescence channels and a detector for transmitted light.
  • Laser lines available in C1 confocal system:
    • Violet diode laser MOD (45.8 mW, 409 nm; Melles Griot, USA).
    • Sapphire 488 nm laser (Coherent, USA)
    • Green He-Ne Laser (1.0 mW, 543 nm; Melles Griot, USA).
    • Red diode laser MOD (10.5 mW, 638 nm; Melles Griot, USA).
  • TE2000 2.33 control software (for TE2000 microscope settings).
  • EZ-C1 3.9 for Nikon C1 confocal (for image acquisition and analysis).
  • Generation of two- and three-dimensional imaging. Applying Z-stacks allows the collection of several optical sections in the specimen to create a three-dimensional image. The confocal system is more suited for thick specimens because it removes out-of-focus light in the specimen.
  • Simultaneous detection of two or three fluorescently tagged proteins and their co-localization in examined specimens.
  • Observation and collection of images during physiological processes using time-lapse recording.
  1. Olympus IX-70 conventional inverted fluorescence microscope (under construction).

Attachment 1. Objectives for Nikon Eclipse E800 fluorescence microscope.

Objective NA WD (mm)
Plan UW 2´ Dry 0.06 7.50 ∞/- BF
CFI Plan Fluor 10´ Dry 0.30 16.0 ∞/0.17 BF, DIC L*
CFI Plan Fluor 20´ Dry 0.50 2.1 ∞/0.17 BF, DIC M*
CFI Plan Fluor 40´ Dry 0.75 0.72 ∞/0.17 BF, DIC M
CFI Plan Apochromat 60´ Oil 1.40 0.21 ∞/0.17 BF, DIC H
CFI Plan Apochromat 100´ Oil 1.40 0.13 ∞/0.17 BF, DIC H

*Unavailable because of a lack of an objective or condenser DIC prism.

NA, numerical aperture; WD, working distance; BF, brightfield; DIC, differential interference contrast (Nomarski contrast); ∞/-, use without coverslip; ∞/0.17, use with cover glass (0.17 mm thickness).

 

Attachment 2. Fluorescence filter cubes for Nikon Eclipse E800 fluorescence microscope.

Cube Excitation Filter Dichroic Mirror Emission Filter
UV-2A (AMCA) 330-380 400 420
UV-2EC (DAPI, Hoechst) 340-380 400 435-485
CFP 436/20 455 480/30
B-2A (Oregon Green) 450-490 505 520
B2-EC (FITC, GFP) 465-495 505 515-555
YFP 500/20 515 535/30
G-2A (Cy3) 510-560 575 590
G2-EC (TRITC, Rodamine, PI) 540/25 565 605/55
FRET (CFP X/YFP M) 436/20 520 540/30

Attachment 3. Objectives for Nikon Eclipse TE2000 inverted microscope.

Objective NA WD (mm)
CFI Plan Apochromat 10´ Dry 0.45 4.0 ∞/0.17 BF, DIC N1
CFI Plan Fluor ELWD 20´ C Dry (correction ring) 0.45 7.4 ∞/0-2 BF, DIC L/N1
CFI Plan Fluor 20´ (Multi-Immersion: Oil, Glycerin, and Water) 0.75 Oil 0.35

Glycerin 0.34

Water 0.33

∞/0.17 BF, DIC N2
CFI Plan Fluor 40´ Oil 1.30 0.20 ∞/0.17 BF, DIC H/N2
CFI Plan Apochromat 60´ Oil 1.40 0.21 ∞/0.17 BF, DIC H/N2
CFI Plan Apochromat VC 100´ Oil 1.40 0.13 ∞/0.17 BF, DIC N2

NA, numerical aperture; WD, working distance; BF, brightfield; DIC, differential interference contrast (Nomarski contrast); ∞/0-2, use with cover glass (0.17 mm thickness) or culture dishes (2 mm thickness(; ∞/0.17, use with coverslip (0.17 mm thickness). CFI Plan Fluor ELWD 20´ Dry objective allows working in culture dishes.

Attachment 4. Fluorescence filter blocks for Nikon Eclipse TE2000-E inverted microscope.

Block Excitation Filter Dichroic Mirror Emission Filter
UV-2EC (DAPI, Hoechst) 340-380 400 435-485
CFP 438/24 455 483/32
B2-EC (FITC, GFP) 465-495 505 515-555
YFP-B (Y-GFP) 500/20 515 535/30
G2-EC (TRITC, Rodamine, PI) 540/25 565 605/55
Y2-EC (Texas Red) 540-580 595 600-660

Współpraca

  • Dobrowolska Grażyna, PhD, Professor, IBB PAS; (Co-localization and BiFC studies of SnRK2 kinases and their interaction partners in living plant cells. Analysis of subcellular localization of GFP-or RFP- tagged proteins involved in response to salt stress in fixed and living Arabidopsis cells.)
  • Hennig Jacek, PhD, Professor, IBB PAS; (Analysis of subcellular localization of FP-tagged bacterial effectors HopQ1, HopBF1, HopAg1, and plant resistance proteins in living plant cells. Co-localization and BiFC study with putative interaction partners. Image analysis for publication of research findings.)
  • Świeżewska Ewa, PhD, Professor, IBB PAS; (Analysis of subcellular localization of FP-tagged polyprenol reductases PPRD1 and PPRD2 in living plant cells, PIN1–GFP, PIN3–GFP, and auxin sensors in Atrgtb1 Arabidopsis thaliana seedling roots. Analysis of endocytosis in root hairs of Atrgtb1 and Atrgtb2 A. thaliana mutants (time-lapse imaging of FM4-64 dye influx)

Klienci

  • Research Institute of Horticulture in Skierniewice, Laboratory of Genetics and Cultivation of Vegetable Plants, Poland
  • University of Warsaw, Faculty of Biology, Institute of Microbiology, Department of Molecular Virusology; Poland
  • Cintamani Poland Majewscy and Koć Sp. J., Piaseczno; Poland
  • Institute of Nuclear Physics, PAS, Krakow; Poland
  • Institute of Biotechnology and Antibiotics in Warsaw; PAS, Poland
  • Botanical Garden – CZRB in Powsin, Poland

Badania

Wybrane publikacje

  • Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1-related protein kinase 2.10 in response to osmotic stress. Maszkowska J, Dębski J, Kulik A, Kistowski M, Bucholc M, Lichocka M, Klimecka M, Sztatelman O, Szymańska KP, Dadlez M, Dobrowolska G. Plant Cell Environ. doi: 10.1111/pce.13465.
  • Alternaria brassicicola– Brassicaceae pathosystem: insights into the infection process and resistance mechanisms under optimized artificial bio-assay. Nowakowska M, Wrzesińska M, Kamiński P, Szczechura W, Lichocka M, Tartanus M, Kozik EU, Nowicki M. European J Plant Path. 2018. online https://doi.org/10.1007/s10658-018-1548-y.
  • Two mutations in mitochondrial ATP6 gene of ATP synthase, related to human cancer, affect ROS, calcium homeostasis and mitochondrial permeability transition in yeast. Niedzwiecka K, Tisi R, Penna S, Lichocka M, Plochocka D, Kucharczyk R. Biochim Biophys Acta Mol Cell Res. Jan;1865(1):117-131 (2018). doi: 10.1016/j.bbamcr.2017.10.003.
  • Phosphatase ABI1 and okadaic acid-sensitive phosphoprotein phosphatases inhibit salt stress-activated SnRK2.4 kinase. Krzywińska E, Kulik A, Ciesielski A, Lichocka M, Dębski J, Ludwików A, Dadlez M, Rodriguez PL, Dobrowolska G. BMC Plant Biol. 2016. doi: 10.1186/s12870-016-0817-1.
  • POLYPRENOL REDUCTASE2 deficiency is lethal in Arabidopsis due to male sterility. Jozwiak A, Gutkowska M, Gawarecka K, Surmacz L, Buczkowska A, Lichocka M, Nowakowska J, Swiezewska E. Plant Cell. 2015. doi: 10.1105/tpc.15.00463.

Publikacje

KULIK A., ANIELSKA-MAZUR A., BUCHOLC M., KOEN E., SZYMAŃSKA K., ŻMIEŃKO A., KRZYWIŃSKA E., WAWER I., McLOUGHLIN F., RUSZKOWSKI D., FIGLEROWICZ M., TESTERINK C., SKŁODOWSKA A., WENDEHENNE D., DOBROWOLSKA G., SNF1-related protein kinases type 2 are involved in plant responses to cadium stress. Plant Physiology (2012) 160(2):868-883 IF 6.535
OLEJNIK K., BUCHOLC M., ANIELSKA-MAZUR A., LIPKO A., KUJAWA M., MODZELAN M., AUGUSTYN A., KRASZEWSKA E., Arabidopsis thaliana Nudix hydrolase AtNUDT7 forms complexes with the regulatory RACK1A protein and Ggamma subunits of the signal transducing heterotrimeric G protein. Acta Biochimica Polonica (2011) 58(4): 609-616 IF 1.234
BUCHOLC M., CIESIELSKI A., GOCH G., ANIELSKA-MAZUR A., KULIK A., KRZYWIŃSKA E., DOBROWOLSKA G., SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor. Journal of Biological Chemistry (2011) 286(5): 3429-41 IF 5.328
WAWER I., BUCHOLC M., ASTIER J., ANIELSKA-MAZUR A., DAHAN J., KULIK A., WYSŁOUCH-CIESZYŃSKA A., ZARĘBA-KOZIOŁ M., KRZYWIŃSKA E., DADLEZ M., DOBROWOLSKA G., WENDEHENNE D., Regulation of Nicotiana tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity. Biochemical Journal (2010) 429(1): 73-83 IF 5,155