激光共聚焦显微镜分析技术

Multicolour Fluorescence
Drosophila Stardust
Tritonea buccal ganglia Drosophila Muscle attachment
Application: EYFP in living cells
HeLa cells expressing pure EYFP which is expected to be freely mobile
现代激光扫描共聚焦显微镜 各部分的设计应用了先进的激光 技术、光学显微镜技术、计算机 控制及图像处理技术、精密的机 械技术;整个系统由共聚焦显微镜 专用的计算机软件统一精确地控 制,从而保证各部分协调地工作。
How a Confocal Image is Formed
Pinhole 1
Pinhole 2
Excitation Filter Fixed characteristics, low selectivity
Specimen
Principle Leica TCS SP2 AOBS
Programmable spectral detector (Leica 1997):
Programmable beam splitter (AOBS, Leica 2002): Programmable excitation filter (AOTF, Leica 1993):
• Seamless recording of full spectrum
• Up to 4 PMT´s with individual gain adjustments to compensate for emission properties of stains
(PMT=Photomultiplier tube)
Confocal Microscope
Arc Lamp Excitation Diaphragm Excitation Filter Ocular
Laser Excitation Pinhole Excitation Filter PMT
Objective
Objective
Emwk.baidu.comssion Filter
From Scanhead
To Scanhead
Scanning Galvanometers
Point Scanning
x
y
Laser out
To Microscope
Laser in
棱镜分光,狭缝扫描
• Prism disperses with high transmission • Efficient coupling of PMT´s
狭分缝光谱扫描
光谱扫描带宽 精度 2 纳米
滤光片系统 OTE
灵敏度较光谱式差
光学能量转移效率
94%
98% 88.5% 86.7% 90.3% 88.5% 100%
光谱式探头
棱镜分光,狭缝扫描,全无滤片
• 第一优势:提高灵敏度 • 光谱式与滤光片比较： 88% 对 61% • 为了达到相同样品质量 (相同探测发射 能量), 较低OTE 的仪器需要较高激光能 量作为激发以致更大机会做成样品漂白 ，干 扰 PSF, 因此降低图象质量
Specimen
Detector Condenser Lens Objective Lens
Modified from: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989
Fluorescent Microscope
激光共聚焦显微镜技术 第2章
激光扫描共聚焦显微镜的基本结构、 工作原理及基本功能
一 、激光扫描共聚焦显微镜的基本结构和工作 原理 激光扫描共聚焦显微镜是一种用于图像采集 和分析的大型精密仪器。其主要由以下几部分组 成:激光光源、扫描器（内装有针孔光栏、分光 镜、发射荧光单色器及检测器)荧光显微镜（装 有微米步进马达）系统、光学装置、计算机图像 存储与处理及控制系统。
(cells courtesy of T. A. Knoch, K. Rippe, German Cancer Research Center and KIP, University of Heidelberg)
~11 m
cell too bright!
FCS measurement spot same image with 5fold increased values (offline)
Optical Mixer - MRC 1024 UV
Argon Laser 353,361 nm UV Fast Shutter Visib le UV Correction Optics
Filter Wheels
ArgonKrypton Laser
488, 514 nm
488,568,647 nm Beam Expander To Scanhead
Leica FLIM: overview
Emission Filter Emission Pinhole
Principle of a standard Confocal
Detector
Barrier Filter Fixed characteristics Pinholes
Laser
Beam splitter Fixed characteristics, low selectivity, transmission losses