Techniques

Sample preparation and imaging

In biomedical sciences, the procedure for the sample preparation and imaging using the confocal microscope is mainly taken and adapted from the standard fluorescent microscopy techniques. Besides fluorescence as the signal source, it is possible to record the light reflection from the surface of the optically “thick” bodies such as pollen, chitin insect coverings or various non-biological materials. Regardless of a protocol for sample preparation, the basic advantages of the confocal microscope procedure are:

  • the flexibility of image presentation
  • the possibility of two or three-dimensional analysis of details in cellular and sub-cellular structure
  • work with a collection of a several digital images for 3D reconstruction or for obtaining the temporal series of physiological processes “in vivo”

By using the relatively simple protocols (mostly taken from immunohistochemistry) in a specific way, it is possible to mark certain cellular structures with one or more fluorescent probes – fluorochromes. A large number of such probes is available and they can be used for the visualization of:

  • fixed biological specimen
  • live tissue slices
  • cell cultures

Many fluorescent probes formed around aromatic chemical compounds are made in such a way as to:

  • mark the biological macromolecules (e.g. proteins or nucleic acids) and, thus, visualize the specific structures or cellular compartments such as cytoskeleton, mitochondria, Golgi apparatus, nucleus etc.;
  • enable the monitoring of cellular integrity (live cells in relation to the dead and apoptotic ones), endocitosis, exocitosis, membrane fluidity, protein movement, signal transduction or enzyme activity;
  • enable the monitoring of dynamic processes connected with the concentration change of non-organic metal ions and electrolytes, pH, reactive oxygen species, membrane potential etc.;
  • be applied in genetic mapping and chromosome analysis.

Live cell imaging

Live-cell imaging covers all techniques where live cells are monitored with microscope in real-time and this approach has become an integral part of cell biology studies. Besides the structural organization of cells, dynamic processes can be best observed in living cells using fluorescent dyes or proteins, which allows us to study physiological states of cells or cellular transport.

[excerpts about chemicals are from Thermo Fisher Scientific Inc.]

Fluorescence imaging

  • Parameters of oxidative status:
    • Labeling of mitochondria – potential dependent and independent dyes
      • JC-1 is cationic carbocyanine dye that accumulates in mitochondria. The dye exists as a monomer at low concentrations and yields green fluorescence, similar to fluorescein. At higher concentrations, the dye forms J-aggregates that exhibit a broad excitation spectrum and an emission maximum at ~590 nm.
      • MitoTracker® Orange CM-H2TMRos is a reduced, nonfluorescent version of MitoTracker Orange that fluoresces upon oxidation. MitoTracker Green FM is green-fluorescent mitochondrial stain which appears to localize to mitochondria regardless of mitochondrial membrane potential.
    • Various parameters of oxidative stress:
      • MitoSOX™ Red reagent is fluorogenic dye specifically targeted to mitochondria in live cells. Oxidation of MitoSOX™ Red reagent by superoxide produces red fluorescence.
      • The cell-permeant H2DCFDA is a chemically reduced form of fluorescein used as an indicator for reactive oxygen species (ROS) in cells.
      • DAF-FM is a reagent that is used to detect and quantify low concentrations of nitric oxide (NO).
  • Live/dead quantification (FDA, PI, DAPI, TO-PRO 3)

Calcium dynamics

Labeled calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca2+. Fluo-3 has been used to image the spatial dynamics of Ca2+ signaling, in flow cytometry experiments involving photoactivation of caged chelators, second messengers, and neurotransmitters, and for cell-based pharmacological screening. Fluo-4 is an analog of Fluo-3 with the two chlorine substituents replaced by fluorines, which results in increased fluorescence excitation at 488 nm and consequently higher fluorescence signal levels. Cells may be loaded with the AM ester forms of these calcium indicators by adding the dissolved indicator directly to dishes containing cultured cells.

Fura-2 – a ratiometric and sensitive indicator dye for measuring intracellular calcium. Measuring by ratio considerably reduces the effects of uneven dye loading, leakage of dye, and photobleaching, as well as problems associated with measuring Ca2+ in cells of unequal thickness. Measurements of fura-2 fluorescence can usually be made over a period of an hour without significant loss of fluorescence resulting from either leakage or bleaching.