Institute Microscopy Laboratory

Representative of the IBS Director for the Laboratory

Dr hab. Bożena Pawlikowska-Pawlęga, prof. UMCS

Department of Functional Anatomy and Cytobiology

Akademicka 19, 20-033 Lublin

room 133B, tel. 81 5375991

mail: bozena.pawlikowska-pawlega@poczta.umcs.lublin.pl

 

Staff members

Mgr Jarosław Pawelec, room 125B, tel. 660 342 397

Mgr Barbara Zarzyka, room 33B, tel. 81 5375041.

History of the Institute Microscopy Laboratory

The Laboratory was initiated in 2001 at the Department of Comparative Anatomy and Anthropology as a Faculty unit subordinate to the Dean. In 2001-2012, it was headed by prof. dr hab. Antoni Gawron. On December 2019, dr hab. Bożena Pawlikowska-Pawlęga, who was in charge of the Electron Microscopy Laboratory until the end of September 2019, was appointed Representative of the Director of the Institute of Biological Sciences for the Institute Microscopy Laboratory.

 

General information

The laboratory participates in the scientific research of the Faculty of Biology and Biochemistry through work in the field of transmission and scanning electron microscopy. The unit also plays a didactic role in classes on the issues of microscopic techniques. This function is also complemented by annual participation in popularisation and promotion events of the Faculty for schools, such as the Lublin Festival of Science or the Open Door of the Faculty and in individual shows organised for schools.

The Laboratory is equipped with the following devices:

  • Zeiss LEO912AB class 120kV transmission electron microscope (TEM)
  • Tescan VEGA3 LMU scanning electron microscope (SEM)
  • Motorised microscope stand (with a camera and software): Olympus BX-61 and Olympus

The Institute of Biological Sciences is also equipped with

  • Zeiss Axiovert 200M confocal laser microscope with an LSM 5 Pascal scanning head
  • LSM 780 Zeiss laser confocal scanning microscope
  • Langmuir-Blodgett system with a Brewster angle microscope and a computer system
  • FTIR Vertex 70 spectrometer with Hyperion 3000 microscope
  • Spektrometr FTIR  Vertex 70z mikroskopem Hyperion 3000
  • Leica DM4000 B fluorescence optical microscope with a Leica DFC 500 camera and a computer image analysis system

Transmission electron microscope (TEM) Zeiss LEO 912AB class 120kV [at present - out of service for technical reasons]

Premises: Laboratory of Electron Microscopy, room 0101A

Person in charge: mgr Jarosław Pawelec, room 125B, tel. 660 342 397

 

Auxiliary equipment: [ASC1] 

  • EDX microanalysis system for the Bruker Quantax 200 electron microscope
  • Software for the electron microscope camera (with image analysis package) Soft Imaging Solutions I-TEM
  • Leica EM UC 7 Ultramicrotome  (and other equipment required for the preparation of TEM samples, e.g. glass cutter, diamond knife)(premises: Chair of Cell Biology, room 33B, 71B)

 

TEM preparatory procedures:

  • Chemical fixation of samples
  • Dehydration of samples
  • Embedding in resins (routinely in LR White acrylic resin, other options available on request)
  • Trimming and cutting ultrathin sections for TEM grids
  • TEM preparations in the negative contrast technique on grids with formvar/formvar-carbon film

Tescan VEGA3 LMU scanning electron microscope (SEM) equipped with the variable vacuum mode and an optional Peltier freezing stage (up to -500C) for work with non-dehydrated organic samples

Premises: Department of Zoology and Nature Protection, room 152B

Person in charge: mgr Jarosław Pawelec, room 125B, tel. 660 342 397

 

Parameters:[ASC2] 

  • SE, BSE, and LVSTD detectors (type SE for low-vacuum procedures)
  • resolution: up to 3 nm (at 30-kV accelerating voltage with a SE detector in the high-vacuum mode)
  • imaging modes: Resolution (maximum resolution), Depth (increased depth of field), Wide Field (wide field of view at magnifications <100x)

Auxiliary equipment:

  • Quorum Technologies Emitech K550X sputter coater with an Au target
  • Quorum Technologies Emitech K850 critical point dryer (CPD)

SEM preparatory procedures:

  • Chemical fixation (various techniques/protocols as required)
  • Dehydration of preparations (as required)
  • Drying preparations with various methods, including critical point drying (CPD) in CO2
  • Spraying organic preparations with a gold conductive layer (Au) using a sputter coater

SEM examinations:

  • Imaging surface structure of various preparations in the high-vacuum mode (standard)
  • Imaging of non-fixed and non-dehydrated organic preparations by freezing (up to -500 C) in variable vacuum conditions (using a Peltier freezing stage)
  • Standard sample size of approx. 12.5 mm (sample stages with a diameter of 0.5); available option of observation of large-size samples (several dozen mm)

Motorised microscope stand

Premises: Institute Microscopy Laboratory, room 152B

Person in charge: mgr Jarosław Pawelec, room 125B, tel. 660 342 397

Olympus BX-61 biological microscope (with a camera and software)

Parameters: [ASC3] 

  • ocular lens: 10x
  • objective lenses: 4x, 10x, 20x, 40x, 60x, 100x/oil (immersion)
  • differential interference contrast system (DIC/Nomarski)
  • polarising filter in the condenser
  • motorised sample stage, objective revolver, condenser
  • Olympus DP72 camera with software

Microscopic examinations:

  • microscopic examination in bright-field visible light range (VIS) with differential contrast interference (DIC/Nomarski) and in polarised light
  • histological and cellular examinations of animal and plant samples
  • examinations of microbiological preparations

Olympus SZX-16 stereo microscope (with a camera and software)

Parameters:[ASC4] 

  • ocular lens 10x
  • zoom module 16.4x (0.7-11.5x)
  • objective lenses: 0.3x (working distance 141mm), 1.6x (working distance 30mm)
  • zoom range (for the configuration specified above): 2.1-184x
  • ring halogen illuminator (with optical fibre)
  • SZ SZX2-ILLT base with an work option in transmitted light
  • motorised focus column
  • Olympus DP72 camera with software
  • Z-stack function (imaging with high depth of field using a series of
    automatically stacked exposures)

Microscopic examinations:

  • examination of surface/external structures of insects and dissected anatomical structures
  • images of whole insects and their developmental stages
  • documentation images of selected aspects of the biology and behaviour of selected groups of insects
  • examination of plant and fungal materials: seeds, spores, floral structures, shoots,
    images of whole fungi (e.g. plant parasites), plant organs

Zeiss Axiovert 200M confocal laser microscope with an LSM 5 Pascal scanning head

Premises: Department of Functional Anatomy and Cytobiology, room 126B

Person in charge: mgr Jerzy Wydrych, room 126B, tel. 81 537 59 98

Parameters and imaging techniques: [ASC5] 

  • LSM 5 Pascal scanning head
  • laser modules: 405, 458, 488, 514, and 543 nm (imaging in the single- and multi-channel laser fluorescence as well as single- and multi-plane and polarisation contrast modes)
  • UV HBO 50 lamp (imaging in the classic UV fluorescence mode)
  • HAL 100W halogen (imaging in the transmitted light mode
  • AxioCam HR3 HDR camera (recording images in the UV fluorescence and transmitted light modes)
  • PECON Incubator S chamber (in vivo observations)
  • lenses: 5x, 10x, 20x, 40x, 63x Oil, 100x Oil (max. optical magnification 1000x)
  • diaphragms for phase contrast and Nomarski contrast
  • control and recording software: AIM 4.2, ZEN 2007, AxioVision 4.8

Services provided with the use of the device: biological, biophysical, biomaterial, and material examinations:

  • non-invasive imaging of structures in fixed and fresh biological preparations, imaging of biofilms, imaging of cell cultures, including in vivo observations
  • imaging of the structure of microcapsules and other film-forming substances
  • imaging of the structure of crystallising substances
  • morphometric, geometric, and densitometric measurements
  • measurements of fluorescence intensity
  • assessment of margins of structures, skeletonization
  • measurement of changes in intracellular ion concentration
  • observations of the location and co-location of individual cell components using FRET, FRAP, and FLIP techniques
  • creation of three-dimensional images of observed objects

LSM 780 Zeiss laser confocal scanning microscope

Premises: Department of Molecular Biology, room 0106A

Person in charge: prof. Marek Tchórzewski, room 11A, tel. 81 5375956

                                      dr Kamil Deryło, pokój 2A, tel. 81 5375922

The Environmental Laboratory of Intravital Cell Imaging is equipped with the LSM780 Zeiss laser confocal scanning microscopy system based on an Axio-Observer Z.1 microscope with two PMT detectors and a 32-channel GaAsP spectral detector. It has a class 3B laser system with 405, 458, 488, 514, 543, and 633 nm wavelengths. The microscope is equipped with dry lenses: EC Plan-Neofluar 10x/0.3 M27, Plan-Apochromat 20x/0.8 M27, and LD Plan-Neofluar 40x/0.6 Korr M27 as well as immersion lenses: Plan-Apochromat 40x/1.4 Oil DIC M27, LCI Plan-Neofluar 63x/1.3 Imm Korr DIC M27, and Plan-Apochromat 63x/1.4 Oil DIC M27. It also has a thermostatted Plexiglas chamber (controlled conditions - temperature, humidity, and CO2) providing cell lines with an appropriate environment for growth. The system is controlled with Zen2010 software. LSM780 is synchronised with the time-resolved fluorescence spectroscopy PicoQuant system consisting of a TCSPC (Time-Correlated Single-Photon Counting) module - PicoHarp 300, a two-channel SPAD (Single-Photon Avalanche Diodes) detector, and picosecond pulsed diode lasers with 440 and 485 nm wavelengths. Data acquisition and analysis are carried out using SymphoTime (PicoQuant) software. The laboratory is also equipped with an Axio Observer.D1 microscope with an AxioCam MRm Rev. 3Fire Wire digital camera and a 458-, 488-, and 514-nm laser system profiled for TIRF observations. The system is controlled with AxioVision Rel. 4.8.2 software.

The laboratory provides precise imaging of the structure of a living cell and tracking intracellular biological processes in a three-dimensional format, additionally implementing the time element (4D imaging). The system facilitates subcellular location of proteins and other molecules in static and dynamic systems. This research system is mainly intended for analysis of protein-protein, protein-ligand, and protein-nucleic acid interactions inside the living cell, on its surface, and in an in vitro system. The methods implemented in the system include FLIM (Fluorescence Lifetime Imaging Microscopy) imaging, spectral analysis with the use of an ultra-sensitive GaAsP detector, FRAP (Fluorescence Recovery After Photobleaching), FLIP (Fluorescence Loss In Photobleaching), FRET (Förster Resonance Energy Transfer), RICS (Raster-scanning Image Correlation Spectroscopy), FCS (Fluorescence Correlation Spectroscopy), anisotropy, and TIRF/FRET (Total Internal Reflection Fluorescence/Förster Resonance Energy Transfer).

 

Langmuir-Blodgett system coupled with a Brewster angle microscope and computer

Premises: Department of Cell Biology, Laboratory of Biospectroscopy, room 0118B

Person in charge: prof. Mariusz Gagoś, room 43B, tel. 81 5375904

Brewster's Angle Microscopy is one of the basic non-invasive techniques used for investigations of monolayers formed by water-insoluble amphiphilic substances on the water sub-phase surface. This technique is based on changes in the refractive index at the water (aqueous solution)/air interface induced by formation of a Langmuir monolayer.

Brewster angle microscopy (BAM) facilitates:

  • investigation of the dynamic and structural properties of lipid membranes induced by temperature changes
  • investigation of the molecular organisation of bioactive compounds in model lipid membranes
  • determination of the molecular organisation and interactions between such biomolecules as lipids, proteins, drugs, and DNA in the monolayer area

Equipment: a double-barrier Langmuir[ASC6]  KSV trough (Helsinki, Finland) model 2000 with a total area of ​​700 cm2. A platinum Wilhelmy plate is used as a surface pressure sensor. Brewster angle microscope: ultraBAM, Accurion (Göttingen, Germany). The device uses a 50-mW laser emitting light with parallel polarisation (p) and a wavelength of 658 nm, an analyser, and a CCD camera. The module facilitates image detection with a resolution of 2 µm and 10x magnification. Radiation incident on the water/air interface is emitted by the Brewster microscope at an angle of 53.2°.

Ultrabam is a Brewster angle microscopy designed for the air/liquid interface. It provides direct visualisation of Langmuir monolayers or adsorbed layers. It also works with dielectric substrates such as glass, quartz, or similar materials. The nanofilm_ultrabam combines high resolution with focused real-time imaging. Advanced imaging optics provides fully focused images at 20-35 frames per second. The high performance camera and the specific calibration algorithm facilitate quantitative measurements of reflectivity. The adsorption kinetics or thickness alterations can thus be monitored. The device has a motorised analyser for visualisation of optical anisotropy caused by the long-range molecular orientation in monolayers. The powerful software makes operation easy and convenient. As a complete solution, the system consists of a computer, electronics, and all software required for initiation of measurements. The nanofilm_ultrabam is designed as a Brewster Angle Microscope that cannot be upgraded to an ellipsometer.

The Brewster Angle Microscopy (BAM) technique is successfully used for morphological and topological observation of monolayers. It provides information on the state of the monolayer, aggregation and formation of domains, and collapse mechanisms. It facilitates observation of phase separation and three-dimensional structures as well as detection of phase transitions. Before the analysis, a black glass plate is placed at the bottom of the Langmuir trough, which prevents radiation scattering through its Teflon bottom. After application of the monolayer into the surface of the pure subphase, the microscope is calibrated, which involves setting the Brewster angle as well as the image focus and contrast.

 

FTIR Vertex 70 spectrometer with a Hyperion 3000 infrared microscope

Premises: Department of Cell Biology, Laboratory of Biospectroscopy, room 0118B

Person in charge: prof. Mariusz Gagoś, room 43B, tel. 81 5375904

 

Analysis based on spectroscopic and micro-spectroscopic FT-IR measurements:

  • determination of the quality of reagents
  • characterisation of unknown or newly derived compounds
  • analysis of layers and surface components
  • temporal monitoring of chemical and biological reactions
  • identification of substrates, analysis of purity of drugs
  • determination of the structure and quantification of reaction products
  • detection of impurities in products
  • identification and classification of bacteria
  • analyses of biological materials, e.g. cell membranes, protein complexes and their secondary structure, imaging of individual viruses

Basic parameters and measurement potential of the VERTEX 70 spectrometer:[ASC7] 

1. Two detectors with automated selection and switching:

  • DLaTGS detector, spectral range at least 12000-370 cm-1;
  • liquid nitrogen-cooled high-performance MCT detector, spectral range at least 12000 - 450 cm-1.

2. 60° Michelson interferometer:

  • guaranteed spectral resolution: ≤ 0.4 cm-1 with the option to 0.2 cm-1;
  • accuracy of determination of the wavenumber: at least 0.005 cm-1 at 2000 cm-1.

3. Microscope coupled with the FT-IR spectrometer:

Measurement modes: reflection, transmission, and simultaneous measurements and observations in the visible mode, ATR;

Lenses: IR 15x for reflection and transmission measurements, IR 36x for reflection and transmission measurements, ATR 20x (ATR sealed lens with a 5-degree pressure from 0.8 to 8 N and electronic and visual control of the correct position of the crystal)

Detectors:

  • liquid nitrogen-cooled high-performance MCT detector; detector size: at least 0.25 x 0.25 mm;
  • FPA matrix, spectral range min. 4500-900 cm-1, size: min. 64 x 64 (4096) pixels

Thermostatted measurement bench with a microscope:

  • thermostatic range: -196 to 600 ° C (cooled with liquid nitrogen);
  • maximum sample size: 22 mm diameter and 1.5 mm thickness;
  • positioning range: at least 16 x 16 mm

Additional equipment for the VERTEX 70 spectrometer:

1. Multifunctional attachment:

            Measurement modes: ATR, external reflection, diffuse reflection;

            Variable angle-of-attack range of at least 5-85;

            ATR system with a ZnSe hemispherical crystal;

            ATR system with a Ge hemispherical crystal;

            Cell for measurements of liquid samples;

            Thermostatted flow cell for measurement of liquid samples, thermostatic range at least 150°C;

            Controller of the temperature of ATR attachments; temperature range at least 150°C;

            Polariser;

            Software for modelling parameters and digital simulation of spectra

2. Precision polariser:

            Manually adjustable with a resolution of 1°;

            Spectral range of work: from 5000 to 285 cm-1;

            Transmission diameter: at least 22 mm

3. Flow measurement cell:

            Whole windows made of ZnSe without apertures for sample insertion;

            Layer thickness range - one spacer, at least 6-950 µm;

            Operating temperature range from -80 to 200°C;

            Possibility of thermostatting with an electric controller in a range up to at least 200°C;

            Sample inlet - Luer type

4. Multi-angle horizontal ATR attachment ATR:

            Measurement modes – ATR;

            Variable angle-of-attack in the range of at least 20-70° adjustable at 1 ° increments;

            Depth of sample penetration: from 0.5 to 10 µm;

            Number of reflections: from 3 to 12;

            45° ZnSe crystals and 45° Ge crystals in internal and external reflection modules;

            Thermostatted flow cell module with a controller operating in the range of at least up to 120°C

5. Multi-reflection ATR attachment

            Measurement modes – ATR;

            Number of reflections: at least 10;

            45°ZnSe crystal in an external reflection measurement module;

            45° ZnSe thermostatted crystal in an internal reflection measurement module;

            Possibility of measurement of liquid, solid, powder, volatile-solvent samples and samples with large sizes that do not fit in the measurement chamber

 

Leica DM 4000B phase-contrast and fluorescence microscope with LAS V3.1 acquisition, interactive measurement, and image analysis module

Premises: Department of Cell Biology, room 31B

Person in charge: prof. Mariusz Gagoś, room 43B, tel. 81 5375904

 

Analyses carried out with the use of the Leica DM 4000B phase-contrast and fluorescence microscope:

  • analysis of the effects of drugs on normal and cancer cells
  • determination of the diversity and changes in the location of polysaccharides in plant cell walls
  • analysis of the cytoskeleton structure
  • analysis of cell nucleus structure and cellular location of nucleic acids

 

 

Basic parameters of the Leica DM 4000B phase-contrast and fluorescence microscope [ASC8] 

  • Optical system

 

Lenses (Plan Semi-apochromat Fluotar class):

  • PL FL 5x/0.15, working distance 12.0 mm
  • PL FL 10x/0.30 PH1, working distance 11.00 mm
  • PL FL20x/0.50, cover glass correction 0.17, PH2, working distance 1.15 mm
  • PL FL40x/0.75, cover glass correction 0.17, PH2, working distance 0.40 mm
  • PL FL 63x/0.70, cover glass correction range of 0.1-1.3, PH2, working distance 2.6-1.8 mm
  • PL FL 100x/1.30, cover glass correction 0.17, OIL PH3, working distance 0.13 mm

 

  • Condenser: for bright field, dark field, and phase contrast
  • Eyepiece: 2 class HC PLAN 10x/25 with the possibility of mounting eyepiece micrometers
  • Trinocular eyepiece cap with a camera port, light distribution 100/50/0%
  • Focus adjustment: bilateral coaxial micro/macro screws
  • Arm
  • Power supply and communication: a built-in power supply, built-in LCD front information display, 6 programmable function buttons
  • 6-position coded turret
  • Mechanical stage covered with resistant ceramic coating, with the possibility of 110° rotation
  • Illumination:

Light source - a 100W halogen lamp

Control - function of automatic correction of illumination settings after changes in the magnification introduced by the user; manual correction of lighting and automatic saving of the change; motorised spherical and rectangular field aperture (for work with the camera); automatic correction of field aperture settings after changes in the magnification introduced by the user, manual aperture correction and automatic saving of the change; motorised aperture; automatic aperture correction function after changes in the magnification introduced by the user; possibility of manual correction of the aperture and automatic saving of the change

  • Fluorescence:

Illumination module: external light source based on a min. 150W metal-halide lamp with a 5-position motorised filter changer Leica EL6000

Fluorescence filters, broadband classes A, D, I3, N2.1

  • Measurement control, data acquisition, analysis, and presentation

Digital camera: a Peltier actively cooled camera; aluminium housing with a fan; maximum resolution 12 Megapixel (4080 x 3072); min. 2/3 inch CCD matrix; pixel size min. 6.45 x 6.45 µm; active area min. 8.8 x 6.6 mm; exposure time min. from 0.25 ms. to 600 s; S/N ratio 2000:1 (66 dB); a 14-bit AD converter; 42-bit colour depth; 0.70 x C-mount connector; FireWire interface for connection with the Leica DFC500 computer

External PC computer with the Windows operating system; quad-core processor, clock-rating at least 2GHz, 4GB RAM, HDD 500GB, DVD R/RW DL; graphics card compatible with the camera control program at least 512MB, processor clock-rating at least 600MHz; PCI-Express 16x connector; USB 6x; power supply minimum 600W; minimum performance 80%, Fire Wire, Ethernet 1Gbps, Windows XP Pro, or Windows 7 Pro

Control software: program for collecting data from the camera with interactive measurement modules, extensive annotations, and image analysis with automated identification of objects characterised by colour or dimensions - LAS V3.1


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