Synaptic पुटिका Styryl रंगों की Photoconversion का उपयोग ताल का अध्ययन
Summary
एफएम रंजक अमूल्य मदद की synaptic गतिशीलता को समझने में किया गया है. एफएमएस अलग उत्तेजना की स्थिति के दौरान सामान्य फ्लोरोसेंट खुर्दबीन के नीचे पीछा कर रहे हैं. हालांकि, एफएम इलेक्ट्रॉन माइक्रोस्कोपी के साथ संयुक्त रंगों के photoconversion अलग synaptic पुटिका पूल के दृश्य की अनुमति देता है, अन्य ultrastructure घटकों के बीच synaptic BOUTONS में,.
Abstract
The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).
Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.
Protocol
Discussion
कुछ महत्वपूर्ण कदम ध्यान में रखा जाना चाहिए:
- थपका ऊष्मायन तैयारियाँ पूरी तरह से और धोने और शमन के बाद ही किया जाना चाहिए. अन्यथा गैर – प्रतिक्रिया व्यक्त glutaraldehyde थपका के साथ बातचीत और उसके वर्षा के का?…
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| FM 1-43 | Invitrogen | F10317 | ||
| Epon resin | Plano | R1030 | ||
| di-aminobenzidine hydrochloride | Sigma | D5905 | ||
| 50% Glutaraldehyde | AppliChem | A3166 | EM grade | |
| Sylgard | Dow Corning | 104186298 | ||
| Axioskop 2 FS plus | Zeiss | |||
| Objective 20x 0.5 NA | Olympus | Dry objective | ||
| 100W Hg Lamp | Zeiss | |||
| Lamp housing with back mirror | Zeiss | 1007-980 | ||
| MRm camera | Zeiss | 0445-554 | Image acquisition | |
| Ex. Filter (HQ 470/40) | AHF | F49-671 | ||
| Dichroic (495 DCLP) | AHF | F33-100 | ||
| Em. Filter (HQ 500 LP) | AHF | F42-018 | ||
| EM | Zeiss | |||
| Proscan CCD HSS | Proscan Electronic Sys. | 1024 x 1024 |
Referências
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