Fluo

optical, electron, and scanning probe microscopy

1) fibrous, structural proteins, mainly members of the collagen family, which are interwoven in a hydrogel consisting of

2) glycosaminoglycans as well as proteoglycans, and

3) adhesion mediating molecules.

The extremely versatile ECM provides mechanical and structural support for the cells and the organ architecture. Moreover, the ECM plays a key role in the regulation of diverse cellular processes such as adhesion,

migration, proliferation, survival and development. A bidirectional interaction between cells and ECM components results in sensitive and quick responses of cells to environmental changes

are the most abundant and fundamental matrix receptors on animal cells. Integrins are transmembrane heterodimeric glycoproteins and fulfill two main functions:

(1) attachment of the cell to the ECM and

(2) bidirectional signal transduction between the ECM and cells. More precise, activated integrin links the intracellular actin filaments to extracellular protein via numerous intracellular adaptor proteins (e.g. talin, vinculin, FAK)

One approach to investigate the impact of extracellular components on cells is to immobilize specific components of the ECM and to incubate the cells on these functionalized surfaces. Consequently, it is possible to study the response to extracellular cues by assessing shape transitions,

metabolic turnover, motility or proliferation, among others

Cells used in this experiment are mouse skeletal muscle cells (C2C12 - immortalized mouse myoblast cell

line)

This can be performed by different methods, for example treatment with crosslinking agents such as paraformaldehyde (PFA) or dehydration of the sample with ethanol or methanol.

• Fixation to halt decomposition and freeze cellular proteins und subcellular structures

• DAPI, Phalloidin oranti-Vinculin antibody staining

• confocal microscopy

• differential-interference-contrast microscopy

• TIRF microscopy

• STED microscopy

• spinning disk microscopy

• why: biological specimens often possess similar optical densities and therefore show low contrast in bright field microscopy

  • to enable the identification of substructures although their transparency is the same as for the surrounding structure

• Phase contrast microcopy is based on the fact that light, which passes a transparent object, still undergoes a phase

  shift

  • dependent on the difference of refractive indices between object and surrounding medium

• separate surrounding light from diffracted light

• if the phase shift is converted in a difference of brightness, a high contrast image can be generated

• illuminated with a ring shaped diaphragm (condenser annulus)

• surrounding light passes and exits the object under a fixed angle, and is focused as a bright ring in the rear focal plane of the objective. However, the light which passes volumes of different refractive indices in the specimen is diffracted, leaves the specimen under a different angle compared to the surrounding light

  bundle, and thus illuminates various locations in the rear focal plane of the objective.

• phase mask is used to alter phase of surrounding light, thus utilizing the different angles of surrounding an difracted light - phase plate ring

• The diffracted light from the sample passes next to the phase mask ring unaltered.

• Finally, both the ring shape surround light and the diffracted light are combined to a parallel light bundle.

• Parts of the specimen which have led to diffraction of the light, thus become dark. Consequently, an image with higher contrast is generated.

• The objectives with a phase mask are typically labeled with Ph and a number (1,2,3 or l) which corresponds to the size of the phase mask. Condenser systems including a

  condenser annulus have to be used with a matching labeling.

the opening of the diaphragm is chosen to have a sufficiently high numerical aperture and thus efficiently high resolution of the image

• noninvasive imaging technique to study living cells, for example during proliferation through cell division or kinetic processes

• Drawbacks of phase contrast images are the characteristic shade-off and halo patterns, in which the observed intensity does not directly correspond to the optical path difference between the

  specimen and the surrounding medium.

• contrast depends on the absorptive and fluorescent characteristics of the samples

• sample has to be stained or cells must produce fluorescent proteins

• In order to illuminate the sample with light matching the excitation wavelength of the fluorophore, the light bundle of a strong light source is filtered through an excitation filter.

• After exciting the fluorophores, the emitted light passes an emission filter, which absorbs all remaining light of the excitation wavelength.

• sample = light source, fluorophore = point emitter of light

• quantitive measurements can be performed when sample is homogenously illuminated

  • subcellular localization and quantification of protein expression, or co-localization of molecules with other cellular structures.

• sample preparation = critical step

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Fluorescent dyes absorb light of a particular wavelength and emit with a characteristic longer wavelength

• experimentally by imaging a calibration slide

• theoretically by using equipment specifications (length [µm]/pixel)

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1 Standby button 2 Left Sideport 3 Focus drive coarse / fine (left side) 4 Control ring, left 5 Objective nosepiece 6 Vertical adjustment knob for condenser 7 Condenser centering screw 8 Condenser (manual or motorized) 9 Microscope stage 10 3-position filter slider slot (diameter 25 mm) 11 Slot for iris stop slider as reflected light aperture stop (motorized) or FL attenuator (motorized) 12 Slot for iris stop slider as reflected light luminous-field stop (motorized) 13 LM set button 14 Drive knobs for controlling XY positioning of the mechanical stage 15 Reflector turret (coded or motorized) 16 Coarse / fine focus drive (motorized) with fine drive, flat (right side) 17 Control ring, right 18 TL button for switching the transmitted light halogen illuminator on and off or for opening and closing the transmitted light shutter 19 RL button for switching the reflected light shutter (fluorescence) on and off 20 TFT display 21 Halogen illumination intensity control 22 Binocular tube 23 Binocular section of the binocular tube 24 Eyepiece 25 Eyepiece adjustment ring 26 Polarizer D with 2-position filter changer

or 3-position filter changer 27 Luminous-field stop control