Optical microscopy as no other method has been able to demonstrate for biology researches the relationship between available research methods and scientific results obtained. The ability to explore the biological object in vivo, directly observe the processes, this all makes this method irreplaceable in a lot of researches. The resource center is represented by modern microscopic technique including confocal and two-photon microscopy, super resolution techniques, special optical methods such as the detection of individual fluorescent molecules, determining the concentration of analytes in living cells, the determination of the rate of diffusion of molecules of nutrients, etc. An important component is the availability of all necessary equipment for work with living cells including micromanipulation, the massive research and cell sorting by flow cytometer sorter. In addition to the optical equipment in research center, there are modern software for morphometric and image processing.
Confocal Laser Scanning Microscopy
Multiphoton excitation microscopy
Confocal Laser Scanning Microscopy
Equipment:
Confocal laser scanning microscopes Leica SP5 & Leica SP5 MP
Confocal Laser Scanning Microscopy (CLSM) is one of a series of methods to generate slices from microscopic samples by means of optics. The sample stays intact, and the slicing may be repeated many times. True Confocal Scanning (TCS) is a technique, where only a single, diffraction limited spot is illuminated and observed at a time. The benefit of confocal imaging is a dramatically increased contrast by removal of out-of-focus haze. Z-sequences of optical slices (3D image stacks) are sources for subsequent rendering as anaglyphes, depth-coded maps or 3D movies.
Leica SP5 confocal microscope based on Leica DMI-6000 inverted microscope allows visualization of the spatial structure of the living and the fixed material to a depth of 200 microns, but for the depth more than of 80-100 microns is recommended to use a method of two-photon microscopy. Confocal microscopy method can be used to study living state cells and tissues, to obtain data on distribution of labeled fluorescent probe substance in several dimensions (x, y, z, time, wavelength emission), as well as special techniques including FLIM, FRAP , FLIP, and FCS.
Available lasers:
- Argon laser 458, 476, 488, 496, 514 nm.
- Helium–neon lasers 543 & 633 nm.
- Semiconductor laser 405 nm (SP5 only).
- Multiphoton laser 690-1040 nm(SP5 MP only ).
- WLL 470-670 nm (SP5 MP only).
Detection wavelengths: 400-800 nm.
Scan rate: 1 Hz - 16 kHz.
Maximal frame size: 8192x8192.
http://www.leica-microsystems.com/science-lab/topics/confocal-microscopy/
Multiphoton excitation microscopy
Equipment:
Confocal laser scanning microscope Leica SP5 MP, laser SpectraPhysics MaiTai HP (14 W Millennia + Tsunami).
Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to about one mm in depth. It differs from traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, as the wavelengths of the two exciting photons are longer than the wavelength of the resulting emitted light. For multiphoton microscopy a pulsed infrared laser with a tunable wavelength from 690 nm to 1040 nm is used. The advantages of multiphoton microscopy include: the use of infrared laser for excitation, which reduces scattering and allows you to explore thicker biological objects; excitation strictly limited area that prevents fluorochrome fading upstream and downstream of the place; less phototoxicity; the ability to detect all of the light from the spot, not only the light transmitted through the confocal aperture that makes possible to collect more light.
http://www.leica-microsystems.com/science-lab/topics/multiphoton-microscopy/
Laser microdissection
Equipment:
Leica LMD7000 Laser Microdissection System.
Laser Microdissection, also known as LMD or LCM (Laser Capture Microdissection), is a contact- and contamination-free method for isolating specific single cells or entire areas of tissue from a wide variety of tissue samples. The thickness, texture and preparation technique of the original tissue are relatively unimportant. The dissectate is then available for further molecular biological methods such as PCR, real-time PCR, proteomics and other analytical techniques. Laser microdissection is now used in a large number of research fields, e.g. neurology, cancer research, plant analysis, forensics or climate research. The method is meanwhile also applied for manipulation of cell cultures or for microengraving of coverslips.
Laser:
- Maximal pulse power - 120 µJ.
- Pulse frequency - 10-5000 Hz.
- Wavelength - 349 nm.
Application-specific Consumables:
Specimen carriers:
- Glass membrane slides.
- PEN and PPS membrane slides.
- Large PEN and PPS slides.
- PEN coverslip slides.
- Frame slides.
- Petri dishes with PEN membrane.
- Ibidi slides.
Collection devices:
- Caps/microcentrifuge tubes.
- Universal Holder for 8-well strips, 8-well strip caps and multi-well slides like 18-well Ibidi slides or LOC (Lab on a Chip) slides.
- 8-well strip caps.
- Petri dishes.
http://www.leica-microsystems.com/science-lab/topics/laser-microdissection/
Quantitative microscopy
Equipment:
Fluorescence Lifetime Correlation Spectroscopy System Leica TCS SMD FLCS
Leica TCS SMD FLCS is the complementary device for the confocal microscope providing FLIM (Fluorescence Lifetime Imaging) measurements in the combination with the FCS (Fluorescence Correlation Spectroscopy). The device allows single molecule detection (SMD), fluorescence attenuation with FLIM, fluorescence anisotropy measuring, fluorescence correlation and cross-correlation spectroscopy measuring, etc.
The fusion of Time-Correlated Single Photon Counting and Fluorescence Correlation Spectroscopy, called Fluorescence Lifetime Correlation Spectroscopy (FLCS), is a method that uses picosecond time-resolved fluorescence detection for separating different FCS-contributions.
FLCS is of particular advantage when using spectrally inseparable fluorochromes that differ in their lifetime for Fluorescence Cross-Correlation Spectroscopy (FCCS) because it offers elimination of spectral cross talk and background. It also offers a way around detector afterpulsing artifacts.
In FLCS, a separate autocorrelation function is calculated for each fluorochrome component determined by its decay pattern, emitted, for example, by various species in the sample. The only assumption is that various emissions have different TCSPC histograms (i.e., different fluorescence lifetimes), which is practically always the case. The core of the method is a statistical separation of different intensity contributions with similar lifetimes, performed on a single photon level.
Quantitative methods in fluorescent microscopy:
- Fluorescence-lifetime imaging microscopy or FLIM is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. The lifetime of the fluorochrome signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample.
- Fluorescence correlation spectroscopy (FCS) is a correlation analysis of fluctuation of the fluorescence intensity.
FCS can be used to obtain quantitative information such as
- Diffusion coefficients.
- Hydrodynamic radii.
- Average concentrations.
- Kinetic chemical reaction rates.
- Singlet-triplet dynamics.
- Single Molecule Detection microscopy (SMD).
- Photon Counting Histogram (PCH).
Förster resonance energy transfer or fluorescence resonance energy transfer (FRET) is a mechanism describing energy transfer between two fluorochrome molecules. A donor fluorochrome, initially in its electronic excited state, may transfer energy to an acceptor fluorochrome through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. Measurements of FRET efficiency can be used to determine if two fluorochromes are within a certain distance of each other.
http://www.leica-microsystems.com/science-lab/topics/quantitative-fluorescence/
