Equipment

Confocal microscope

LSM 510 system with Axioskop 2FS mot microscope

Developer & country : Carl Zeiss GmbH, Germany
Year of purchase : 2004

Main characteristics : The principles of confocal microscopy were set by the physicist and constructor Marvin Minski who the patent rights has belonged to since 1957. The term confocal is a coining with an origin in the English phrase CONjugated FOCAL plane.

During the 1980s the confocal microscopic systems were constructed by combining improved computers, lasers and optics and have been used more due to their exquisite characteristics in spite of their high price. The Bio-rad company constructed and presented the first microscope.

The microscopic system designed by the Carl Zeiss company, which has been a leading manufacturer of optical systems since the 19th century, is placed in our Centre. Recently, Zeiss has purchased the part of Bio-rad company and, thus, become an owner of their patents in the field of the laser technology.

The LSM 510 family represents the advanced generation of confocal microscopic systems with wide areas of use. Our Centre is a host of the system based on Axioskop 2FS mot upright microscope. This optical device of excellent quality can be used as a light broad-field microscope, an epiflourescent microscope and, primarily, as the confocal system basis. The obtaining of high-resolution confocal images is enabled by laser illumination as well as by the analogue to digital conversion supported by the last-generation computer system. The laser block contains an Argon-laser (with several spectral lines: 457 nm, 488 nm, 514 nm) and two Helium-Neon lasers (543 nm and 633 nm). In this way, the fluorochrome (fluorescent indicator) excitation is enabled, which absorbs the light in the blue and green part of the visible spectrum. Three-channel registration of the emitted light is in the range from 457 nm through the green and red part of the visible spectrum close to the IR light. By using the mercury lamp light, it is also possible to obtain the signal from the fluorochrome, which needs the UV excitation such as a DNK marker DAPI.

The confocal microscope is applied in many areas of biomedical research since it provides the gaining of the clearer images of the cells and tissue structures marked by the fluorochrome.The precise laser beam direction enables the light intensity diminution in front of and behind the focal plane. Z-filter i.e.“pinhole” determines the thickness of the optical section, where the light signal originates from, and which regulates the confocality of the obtained image. Thus, the combining of the laser, controlled optics and z-filter enables the illumination and the recording of the emitted light from one point determined precisely in any part of the specimen volume. The laser beam scanning and computer processing give an optical section of the sample. The series of optical sections using LSM software make a three-dimensional image of the specimen (3D reconstruction).

At the same time, the background blurriness hampering epiflourescent microscopy is reduced and a high-resolution image is obtained.

The popularity of the confocal microscope is based on the simplicity of obtaining extemely high quality images from samples prepared for the conventional microscopy and, also, on the possibility of applications in many areas of the contemporary biomedical and biotechnological research.

Multiflourescence gained by the confocal microscopy is ideal for the direct visualisation of the relationship and interaction between molecules and other cellular components in their natural context.

The advantages of the confocal microscope, especially LSM 510 (Carl Zaiss):

  • obtaining a clearer image, when compared to the conventional fluorescent microscope;
  • the production of thin sections of specimen is not necessary;
  • computer-controlled microscope with minute focus control as well as the image digitalization;
  • data set collection for three dimensional object reconstruction is available;
  • the possibility of acquisition in “the fourth dimension” – time, for monitoring the kinetics of biochemical and physiological processes “in vivo”
  • the possibility of collecting two images simultaneously from the sample marked by more fluorchromes (up to four indicators in the same specimen);
  • micrographies are obtainable even from the specimens with low fluorescence.

Applications in:

  • all biomedical research areas;
  • pharmaceutical industry;
  • medical and dental clinical practice and diagnostics;
  • examination and analysis of materials and other different substances in industry and biotechnology.

Monochrome camera AxioCam ICm1

Developer & country : Carl Zeiss GmbH, Germany
Year of purchase : 2010

Application field : Routine examination in morphological studies and recording of various biological samples suited for confocal microscope.

Main characteristics : Microscope digital camera with 1.4 megapixel resolution for recording in real-time with ring adapter for mounting on confocal microscope; accompanied with software for recording and analysis.

High temporal resolution video microscopy

Developer & country :

  • Carl Zeiss Gmbh, Germany (Axio Observer microscope with anti-vibration table)
  • VisiFluor® High Performance Ca2+, ion and FRET imaging (VisiChrome® High Speed Polychromatic Illumination System)
  • Photometrics’ Evolve 512 “Intelligent” EMCCD camera, US
  • VisiView® high performance imaging software, Visitron Systems GmbH
  • ALA scientific, US (High Speed Solution Exchange)

Year of purchase : 2012

Application fields : The main purpose of this system is dynamic video microscopy with high temporal resolution for investigating changes in intracellular ion concentration and production of free radicals in biological samples like cell cultures and tissue biopsy specimens. This system is compatible with system for electrophysiological measurements and confocal microscopy.

Main characteristics :

  • Inverted microscope for studies in bright field with phase contrast and epifluorescence accompanied with high performance objectives for live cell imaging.
  • Fast monochromatic source of fluorescence for illumination during fluorescence photometry, fluorescence imaging, scanning of optical wavelength and photolysis.
  • System for fast exchange of perfusion solutions.
  • EM – CCD camera for recording low fluorescence signals, TIRF microscopy, fluorescence from single molecules, spectral imaging and luminescence.
  • Accompanying software connected to monochromator for illumination control and to camera for recording as well as analysis of recorded material.

System for electrophysiological measurements

(“Patch-Clamp” system)

Developer & country : Heka GmbH, Germany
Year of purchase : 2008

Application field : Electrophysiological measurements on performed on bilogical systems (cell cultures, plants or fungi, organotypic slice culture as well as acute tissue slices).

Main characteristics : The system is comprised of feedback amplifier HEKA EPC8, AD/DA converter Heka LIH 1600 and HEKA MIM4 Eppendorf micromanipulator. For preparation of electrodes we have PIP5 pipette/micro-electrode vertical puller, while polishing could be performed at ALA CPM-2 (Coating and Polishing Microforge) device mounted on accompanying microscope.

Cell culture equipment

Main components :

Research microscope Axiovert 40 CFL (Carl Zeiss GmbH, Germany, 2008) :
inverted microscope with stage for observing cell cultures. Objectives and accompanying filters offer imaging in bright field with phase and differential interference contrast, as well as observation of epifluorescence. The main purpose of this microscope is observation of live cells in culture. It can be also used for examination of fixed cells and tissues on microscope slides.

Laminar Flow Hood for aseptic lab work, cell incubator NAPCO CO2 5400, Simplicity® water purification system, VAPRO® vapor pressure osmometer, set of manual pipettes (adjustable-volume, variable).