प्रांतस्था संगठन और गतिशीलता के सूक्ष्मजीवों में दृश्य, कुल आंतरिक प्रतिबिंब प्रतिदीप्ति माइक्रोस्कोपी का उपयोग
Summary
कुल आंतरिक प्रतिबिंब प्रतिदीप्ति माइक्रोस्कोपी (TIRF) संरचनाओं उच्च विपरीत और लौकिक संकल्प में कोशिका की सतह के करीब निरीक्षण के लिए एक शक्तिशाली तरीका है. हम प्रदर्शन कैसे TIRF दीवार संलग्न सेल बैक्टीरियल और फंगल कोशिकाओं के प्रांतस्था में प्रोटीन की गतिशीलता का अध्ययन करने के लिए नियोजित किया जा सकता है.
Abstract
TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.
Protocol
Discussion
TIRF माइक्रोस्कोपी छवि परिधीय प्रोटीन के लिए पसंद की तकनीक है. क्षणभंगुर क्षेत्र की कम गहराई में प्रवेश ध्यान प्रकाश है, जो शोर अनुपात करने के लिए एक बहुत अच्छा संकेत होता है और उच्च फ्रेम दर, या बहुत दुर्?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम के मैक्स प्लैंक सोसायटी द्वारा वित्त पोषित किया गया था.
Materials
| Name of the tool/reagent | Type | Company | Catalogue number |
| Orbital Shaker | Tool | UniEquip | UNITWIST 3-D ROCKER SHAKER |
| TIRF microscope | Till | Customized setup | |
| Glass container | Tool | Vitlab | 340-232880353 |
| Ceramic staining rack | Tool | Thomas Sci. | 8542E40 |
| Concanavalin A | Reagent | Sigma | L7647 |
| Coverslips #1(18 x 18 mm) | Microscope | Menzel Gläser | BB018018A1 |
| Microscope Slides | Microscope | Menzel Gläser | AA00000102E |
| Immersion Oil | Microscope | Zeiss | Immersol 518F |
| Agarose | Reagent | Invitrogen | 16500-500 |
| FluoSpheres | Reagent | Invitrogen | F8795 |
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