高階多維度影像分析平台
High-Dimensional Advanced Imaging Core Facility
I10-白光雷射倒立型共軛焦顯微鏡-STED系統
Leica SP8-gSTED /STED
Leica SP8-gSTED /STED
儀器描述
STED顯微鏡應用壓制雷射形成donut以壓制螢光，產生比共軛焦顯微影像更高解析度的gated STED影像，其光學解析力至50 nm以下，是為不經運算之純光學超解析技術。STED技術中，解析度可隨壓制雷射之強度而變化。壓制雷射強度越強，可達到越高的解析度，搭配LightGate技術移除雜訊，同樣雷射強度下解析度可提升1.4倍。
主要特點
- 多色高解析度觀察
- 多點位置應用及樣品大面積拼圖
- 光譜分析功能
- 弱螢光影像
- 螢光共振能量轉移 (FRET)
- 螢光漂白恢復術 (FRAP) 及光漂白螢光損失 (FLIP) 實驗
- 活細胞時間序共軛焦影像
- 螢光生命週期顯微影像 (FLIM)
- 超解析顯微影像 (STED)
儀器規格
- 電動倒立型顯微鏡：Leica DMI 6000
- 雷射系統：
■ 共軛焦系統：405 nm,及白光雷射系統WLL 470-670 nm連續可調
■ 超解析系統：雙STED壓制雷射系統 (Depletion laser)：592 nm, 775 nm - 螢光濾片：DAPI, EGFP, TexasRed
- 物鏡：10x, 20x Imm Corr 甘油鏡, 40x油鏡, 63x油鏡, 93x Glyc (STED)甘油鏡, 100x (STED)油鏡
- 感測器：兩組光電倍增管(PMT)感測器, 兩組光譜型高感度偵測器(HyD)
- DIC 微分干涉相位差觀察模式
- 電動載物台
- LightGate偵測技術
- 3D gSTED 模組
- 活細胞觀察系統：
■ 方盤密閉式培養盒，加熱控制器，二氧化碳控制器，濕度控制用加熱器 - 控制軟體：LAS-X SP8
- 離線分析軟體：LAS-X SP8
使用注意事項
- 建議使用1.5H (0.17 mm) 蓋玻片
- 建議使用Confocal專用玻璃底培養皿
- 若須使用STED超高解析圖像擷取，請使用精度高之蓋玻片或玻璃底培養皿
- 活細胞長時間培養觀察，可使用玻璃底培養皿或多孔盤
- 使用STED功能須避免會受592 nm雷射激發之染劑，例如DAPI或HOECHST等 (DAPI可使用TO-PRO-3, YOYO-3, DRAQ 5替代)
- STED Sample製備參考：(超解析顯微技術有其限制，委託上機樣品須符合STED樣品備製需求)
- https://webcdn.leica-microsystems.com/fileadmin/academy/2019/Quick_Guide_STED_Sample_Preparation/STED_Sample_Preparation_Guide_Online_20190705.pdf
- https://webcdn.leica-microsystems.com/fileadmin/academy/2019/Quick_Guide_STED_Sample_Preparation/The_Guide_to_STED_Sample_Preparation_MC-0002370_25032021.pdf
- 以下功能應用不提供自行操作：
■ 1. 非一般標準玻片型式之樣品，非乾式樣品，具有溶液或細胞培養液樣品。
■ 2. 使用STED模組
■ 3. Photon manipulation實驗 - 使用前請先與管理員討論
放置地點
國家生技研究園區A棟4樓407b室
管理人員
羅麗紋 博士
電話：02-7750-5554 或 02-7750-5556
Description
Nanoscopy has revolutionized the study of subcellular architecture and dynamics and is on its way to becoming the new gold standard in fluorescence imaging (Nobel Prize for Chemistry 2014).
The fully integrated STED (STimulated Emission Depletion) systems Leica TCS SP8 STED meet the requirements of daily research and provide fast, intuitive, and purely optical access to structural details far beyond the diffraction limit. Resolution becomes tunable in x, y and z.
Application
- Multi-color high-resolution scanning –LIGHTENING
- Fluorescence multiple position and large area merge imaging
- Spectrum scan
- Low-fluorescence imaging
- FRET experiment
- FRAP and FLIP experiment
- Living cell time-lapse confocal imaging
- FLIM scanning and analysis
- STED super-resolution imaging
Specification
- Microscope main body：Leica DMI 6000
- Laser system：
■ Confocal system：405 nm, WLL 470-670 nm
■ Depletion laser：592 nm, 775 nm - Filter：DAPI, EGFP, TexasRed
- Objective：10x, 20x Imm Corr Glyc, 40x oil, 63x oil, 93x Glyc (STED), 100x (STED) oil
- detector：2, PMT detector；1, HyD SMD detector；1, HyD detector
- DIC module
- Automatic sample stage
- LightGate scanning
- 3D gSTED module
- Living cell incubation system：Stage-top box incubator, O2 and CO2, temperature and humility controller
- Control software：LAS-X SP8
- OFF-Line software：LAS-X SP8
Precautions for user
- Recommend to use “1.5H (0.17 mm)” coverslip
- Recommend to use glass bottom dish or microplate
- Recommend to use high-grade coverslip or glass bottom dish for STED application
- Some functional application is only technician operation：
■ 1. Non-common used slice, Wet-type sample which contain culture medium and buffer
■ 2. Photon manipulation - To avoid the 592 nm laser excitation dyes, eg. DAPI / Hoechst, by using STED system (DAPI replacement dyes: TO-PRO-3, YOYO-3, DRAQ 5)
- Reference for STED sample preparation: Suggestion from Leica website (STED technology has the limitation, and user must be understood the requirement of sample preparation)
Facility location
National Biotechnology Research Park Building A, 4th Floor, room 407b
Manager
Li-Wen Lo, PhD
E-mail：llw@gate.sinica.edu.tw
電話：02-7750-5554 或 02-7750-5556