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applications »Cell Biology - Microscopy

Cell Biology - Microscopy

Cell Biology - Microscopy

Cell biology is the discipline that studies cells to answer scientific questions. All organisms are composed of one or more cells and all vital functions of an organism occur within cells. Cells contain the hereditary information necessary for regulating cell functions. Cells possess DNA, the hereditary material of genes, and RNA, containing the information necessary to build various proteins such as enzymes, the cell's primary machinery. There are also other kinds of biomolecules in cells, e.g. lipids, proteins, macromolecules, etc.
Cell biology research includes learning the physiological properties such as the structure and the organelles of cells, their environment and interactions, their life cycle, division and function and eventual death. This is done both on microscopic and molecular level, and includes the research of single-celled organisms like bacteria as well as specialised cells in multicellular organisms like humans.

Knowing the composition of cells and how cells work is fundamental to all of the biological sciences. Appreciating the similarities and differences between cell types is particularly important to the fields of cell and molecular biology. These fundamental similarities and differences provide a unifying theme, allowing the principles learned from studying one cell type to be extrapolated and generalised to other cell types.

In combination with our products, wide field fluorescence microscopy is used to measure characteristics of fluorescent proteins. Cells, originating from bacteria and insects to mammals, generally are kept in culture and plated at coverslips to do specific experiments. Microscopy enables viewing objects inside cells that are stained or fluorescently tagged. By observing the characteristics, e.g. the fluorescence lifetime, of the fluorescent compounds, not just the localisation of a fluorescent protein, but also the characteristics of its local environment can be imaged. Novel multi-parameter fluorescence imaging systems are being used to study intracellular organisation and inter- and intracellular signalling.
One way to observe the proteins, is by fixation of the cells to the coverslips. Before the cells are fixed, the compounds in the cells can be fluorescently tagged (see living cell-imaging). Also, the compounds inside the cells can be stained after their fixation, for example by use of antibodies. Staining is a biochemical technique of adding a class-specific (DNA, proteins, lipids or carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. The characteristics of the dyes can give answers to specific scientific questions, like whether there is interaction between two different proteins, whether there is a conformational change of the protein after a kind of treatment (see also FLIM and FRET), or whether specific ions have bound to the proteins of interest, etc.
The cells can also be analysed "alive". These living cell-imaging experiments seek to gain information about the localisation and interaction of the desired protein. One way to do this is to replace the wild-type gene with a 'fusion' gene that has a reporting element such as GFP. That will allow easy visualisation of the products of the genetic modification. More sophisticated techniques are in development that can track protein products without mitigating their function, such as the addition of small sequences which will serve as binding motifs to monoclonal antibodies.

Examples of highly photostable fluorescent lifetime images of cells labelled with PURETIME fluorescent dyes can be found at the website of AssayMetrics

 

applications

Biomolecular interactions

Inside cells specific interactions between biomolecules are involved in almost any physiological process. Sensing extracellular signals is a matter of receptor to adapter interactions and an intricate network of structural protein interactions maintains the shape of the cell. Finding interactions between proteins involved in common cellular functions is a way to get a broader view of how they work co-operatively in a cell. One way to observe biomolecular interactions is by doing FRET.
In the text below some examples of different interactions are given, with the link to the paper in question.

More about Biomolecular interactions
 
Confocal FLIM
Confocal FLIM

Confocal imaging on a widefield fluorescence microscope can now be done in combination with frequency domain FLIM. The resulting lifetime image shows enhanced contrast as the detected emission light, coming from planes just above or beneath the focus plane, is reduced significantly. This allows you to see differences in fluorescence lifetime e.g. between the cell membrane and the cytoplasm. 

More about Confocal FLIM
 
TIRF (Total Internal Reflection Fluorescence) - FLIM
TIRF (Total Internal Reflection Fluorescence) - FLIM

TIRF (Total Internal Reflection Fluorescence) microscopy facilitates extremely high-contrast visualisation and thereby high sensitivity of fluorescence near the cover glass. This is done without disturbing cellular activity, thereby enabling tracking of biomolecules, and the study of their dynamic activity and interactions at the molecular level. TIRF enables the selective visualisation of processes and structures of the cell membrane and pre-membrane space like vesicle release and transport, cell adhesion, secretion, membrane protein dynamics and distribution or receptor-ligand interactions. The unique combination of TIRF and frequency domain FLIM makes it possible to measure lifetimes of for instance small focal adhesions near the cover glass.

More about TIRF (Total Internal Reflection Fluorescence) - FLIM
 
Spectrally Resolved FLIM
The spectrally resolved LIFA is an imaging system for fluorescence microscopy that preserves the information required to determine the position, spectrum, and lifetime of the observed fluorescence. This is done by combining several modular components. These consist of the typical LIFA (modulated ICCD camera, modulated LED excitation) and a prism-based imaging spectrograph. 
More about Spectrally Resolved FLIM
 
Ion imaging

For ion imaging, several (fluorescent) indicators are available that have a change in quantum yield upon ion binding. This can mean that they can emit photons with different energy, thus different emission wavelength. Also it can mean that they can have a change in fluorescence lifetime. Therefore, there are two ways in which ion imaging can be done by use of indicators: the ratiometric method and the FLIM method.
Another way in which ion imaging is done, is by using FRET-based indicators that change their conformation upon ion binding. Upon the conformationl change the FRET efficiency changes, which is used as indicator for the ion concentration. E.g., cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM).

More about Ion imaging
 
Probing microenvironment

The fluorescence lifetime is a signature almost insensitive to the fluorophore concentration that provides means of discrimination among molecules with spectrally overlapped emission. A further feature is the sensitivity of the decay time to the microenvironment. This dependence varies between different fluorophores and factors.

More about Probing microenvironment
 
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technology
  • Confocal FLIM
  • FLIM-FRET experiments
  • Non-FRET experiments
  • Notes for FLIM users
  • Papers / Reviews FLIM
  • Papers / Reviews FRET
  • TIRF-FLIM
  • FLIM, Fluorescence Lifetime Imaging Microscopy
  • FRET, Fluorescence Resonance Energy Transfer
downloads
  • Presentation FLIM applications
  • Presentation FRET ratio and FLIM
tutorials
  • Antibody
  • Fixation
  • Fluorescence
  • GFP (and family members)
  • Living cell imaging
  • Widefield fluorescence microscope
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