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| Laser TIRF 3 - Applications |
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Exocytosis, endocytosis or single molecule detection, explore details at the limit of visibility. Laser TIRF 3 offers you a maximum of flexibility – with up to four laser lines and a wide variety of applications.
| Multi-Color TIRF with up to three laser lines used simultaneously |
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To see interactions of several proteins in processes near the cell membrane, you need a different label for each protein.
Here is an example: You observe molecular aspects of cell movement to find out how the motors that drive the cells work. With Multi-Color TIRF you can visualize the distribution of stress fibers, which shows which part of the cell is stationary and which is moving.
With the new laser module in Laser TIRF 3, this application is greatly simplified. Up to three different tags can be excited and detected simultaneously. During sequential excitation, the penetration depth can be kept constant with all wavelengths involved.
HeLa Cells
Objective: αPlan-Apochromat 100x/1.46 Oil
Natalia Andreyeva and Vic Small, IMBA Vienna, Austria
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HeLa Cells – Cytoplasmic vesicles
in red (excitation with 561 nm),
focal adhesions in Green
(excitation with 488 nm) | HeLa Cells – Cytoplasmic vesicles
in red (excitation with 561 nm),
focal adhesions in green
(excitation with 488 nm) |
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The combination of TIRF and epifluorescence is used if, in addition to the TIRF plane, it is also of interest to see where in the cell's interior the molecules to be observed originate from.
For example, you can follow the traffic of vesicles from the Golgi apparatus in the depths of the cell to their site of release on the membrane. If you use the motorized TIRF slider, you only need to vary the angle of illumination via the software.
| TIRF/transmitted-light contrasting methods |
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Combination of TIRF with brightfield contrasting methods such as differential interference or phase contrast while maintaining laser safety.
With TIRF alone, only the structures immediately above the coverslip are visible, but never the entire depth of the cell, nor its entire supporting surface. Often, however, you may need to overlay the TIRF fluorescence signals with the cell morphology to extract more spatial information.
B16/F1 melanoma cells(mouse) – Multicolor TIRF combined with DIC
Objective: αPlan-Fluar 100x/1.45 Oil. Specimen: Oberbanscheidt, van den Boom, Bähler, Institute of General Zoology and Genetics, University of Münster, Germany
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| Green: CFP-actin, excitation 458 nm; red: YFP-myrpalm, excitation 514 nm | Single channel display, green: CFP-actin, excitation 458 nm | Single channel display, red: YFP-myrpalm, excitation 514 nm |
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| TIRF and high-speed imaging |
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With high time resolution you can register fast processes or detect several tags or fluorescent proteins simultaneously.
The colocalization of signals is possible only if the frame rate is limited neither by the switching of the excitation wavelength or illumination angle nor by the camera's shutter speed.
With Laser TIRF 3 integrated in the concept of rapid image acquisition, it is possible for the first time to analyze processes near the membrane simultaneously and with high resolution.
Thanks to new filter sets and complete integration into the AxioVision system software, you can combine TIRF with FRET.
For high temporal and spatial resolution, we specially recommend the new dual camera setup. Two cameras detect, simultaneously and with full resolution, the various fluorescence signals that are excited simultaneously by two laser wavelengths in TIRF. FRET analysis can be done conveniently with the aid of the Physiology package of the AxioVision software.
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The plus-end binding protein Bim1p (green – excitation 488 nm) of Saccharomyces cerevisiae labels the ends of the rhodamine-labelled microtubules (red – excitation 561nm). TIRF microscopy enables the visualization of the interaction of microtubule-associated proteins with dynamic microtubules in highly purified in-vitro assays.
Objective: αPlan-Apochromat 100x/1.46 Oil
Tomasz Zimniak and Stefan Westermann, IMP Vienna, Austria |
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