zeiss激光共聚焦显微镜 lsm880 with...

Zeiss激光共聚焦显微镜 LSM880 with Airyscan

Reporter: Li Xiaoting (李筱婷） Application Specialist

Date: 9/12/2018

高质量、多维度、大视野显微图像

2018/9/12Carl Zeiss Microscopy

培训内容安排

理论讲座：

共聚焦显微镜的原理及在生命科学领域的应用

上机操作培训：

1. 硬件部件介绍和激光安全介绍

2. 开关机顺序以及注意事项

3. 二维单通道图像的获取

4. 多通道图像的获取

5. 透射光图像的拍摄

6. 图像的保存和导出

7. Z-stack 图像拍摄及 Maximum intensity projection 图像处理

8. Time series 图像拍摄

9. 大视野拼图

10. Airyscan 图像的拍摄

11. 荧光强度分析

用户上机操作：

用户复习上机操作内容，可自带样品进行拍摄，工程师解答用户使用中遇到的问题


9月11日（星期二）

9月12日（星期三）

9月13日（星期四）

9月14日（星期五）

上午

9:00 —— 10:30

理论讲座：共聚焦原理及应用

9:00 —— 12:30

上机操作培训第二组

9:00 —— 12:30

上机操作培训第四组

9:00 —— 12:30

上机操作培训第六组

下午

13:30 —— 17:00

上机操作培训第一组

13:30 —— 17:00

上机操作培训第三组

13:30 —— 17:00

上机操作培训第五组

13:30 —— 17:00

上机操作培训第七组


Agenda


2

3

4

LSM880 激光共聚焦显微镜的光路部件

如何获取一张高质量的图像

激光共聚焦显微镜的原理

1 LSM880 激光共聚焦显微镜的配置及应用范围

共聚焦显微镜：广泛应用于生物、医学各个领域


Nearly all fields of Science...

Agriculturual Research,

Alzheimer, Cancer,

Cell Science, Biochemistry,

Botany, Immunology,

Developmental Biology, Ecology,

Epidemiological Deseases,

Evolutionary Biology,

Food design, Genetics, HIV,

Material Quality Control,

Material Sciences, Medicine,

Membrane Research,

Neurobiology , Parasitology,

Pharmacology, Physics,

Plant Biology, Proteomics,

Signal Transduction, Virology...

基本功能：二维荧光图像采集拍摄高质量的单通道或多通道荧光照片

大鼠肾细胞

Green：GFP融合蛋白，高尔基体

Red：抗体，微管

Blue：DAPI，细胞核

Rat forebrain neurons

Blue: DAPI

Green: anti-beta-tubulin

Red: anti-ActRII

大鼠大脑固定切片，海马区

Blue: DAPI

Green: GFAP (astrocytes)

Red: Phalloidin (F-actin)

基本功能：Z 轴采集果蝇脑 – 不同神经元群体

Z轴采集三维重构

果蝇神经系统大鼠肾小球

• 物镜: Plan-Apo 63x/1.4 Oil

• 最大强度投影

Z轴采集最大强度投影

3D 功能三维结构分析和测量（需3D模块）

Ortho功能： X-Z、Y-Z截面

基本功能：时间序列成像

Mitosis in HeLa-Kyoto cell line during mitosis

Video showing Histone 2B (H2B, red, mCherry) and microtubule end-binding protein 3 (EB3, blue, EGFP)

Sample courtesy of: Jan Ellenberg, EMBL, Heidelberg.

时间序列成像

四维动态结构肿瘤细胞迁移

来源:

Institute of Cancer, Fudan University

Shanghai, China

样品：MDA231细胞胶质 GFP基质金属蛋白酶 Alexa 647

四维动态结构果蝇胚胎发育

Drosophila melanogaster embryo, Jupiter-GFP (microtubules)

Z-stack depth coded: 72 slices, Every 15 min, 11.5 hSample courtesy of Balazs Erdi, Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter

基本功能：大视野拼图视野的扩展

Tilling & Stitching: 227um x 125um

Tilling & Stitching: 227um x 125um

基本功能：大视野拼图视野的扩展

大鼠脑片 (Z-stack, Tiled, MIP )

测量分析：荧光强度分析


测量分析： Profile 功能分析一条线上的荧光强度的分布


可用于分析药物刺激原生质体或者悬浮细胞在加药前后，某特定标记物的在细胞内外的分布

测量分析：共定位分析 co-localization

两种荧光信号的共定位定量分析


直接显示所选区域荧光信号的共定位系数

Picture from Zeiss Campus

http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/introduction.html

扩展功能：光谱扫描，线性拆分最大程度避免荧光串色的影响

光谱扫描和拆分之前光谱扫描和拆分之后

Inte

ns

ity

Wavelength

发射光谱图谱和曲线

扩展功能：光谱扫描，线性拆分最大程度避免荧光串色的影响

Picture from Zeiss Campus

http://zeiss-campus.magnet.fsu.edu /articles/

spectralimaging/considerations.html

果蝇眼

Green: AF488, actin

Red: Cy3, Crumbs

Blue: autofluorescence

光谱拆分去除自发荧光

光谱拆分植物光谱成像

成像要求：

• 自发荧光• 4种荧光标记• 弱激光避免光漂白

Application note: Spectral imaging: a

powerful tool for confocal multicolor

imaging in living plant cells.

www.zeiss.com/microscopy

FRET是指当两个荧光基团满足一定条

件时发生的一种非辐射性的、偶极-偶

极配对过程，借此过程，能量从激发

态荧光供体以非常接近的波长转移到

荧光受体。

FRET发生的条件：

• 供体与受体的距离在2-10nm

• 供体的发射波长与受体的激发波长

一致

• 供体与受体的极性一致

• 供体与受体有足够的荧光寿命

扩展功能：荧光共振能量转移Fluorescence Resonance Energy Transfer （FRET）

荧光共振能量转移 FRET受体发射法 vs 受体漂白法

常用的FRET组合：

CFP/YFP

CFP/dsRED

BFP/GFP

GFP/dsRED

YFP/dsRED

Cy3/Cy5

Alexa488/Alexa555

Alexa488/Cy3

FITC/TRITC

YFP/TRITC

YFP/ Cy3

受体发射法（活样本、固定样本）受体漂白法（固定样本）

1. 分子内结构变化（蛋白质、DNA构象等）

2. 分子间相互作用（酶活力、离子通道结构变化等）

3. 离子浓度测量（通过构建FRET蛋白测量Ca2+, pH, cAMP等）

4. 分子间作用距离测量荧光的效率与距离成6次幂关系，可以灵敏测量1－10nm内的距离变化

荧光共振能量转移 FRETFRET的应用

FRET应用测定Hela活细胞中的Ca2+浓度

Cameleon(变色指示剂)

一种合成蛋白，由4部分组成：CFP

钙调素（CaM）缩氨酸（M13）YFP

CaM结合Ca2+后，蛋白构象发生变化，产生分子折叠，使CFP/YFP相互靠近, 产生FRET

• 胞内Ca 2+浓度↑ , FRET的效率↑

• 485nm荧光减弱, 530nm荧光增强

• CFP 和YFP 间的能量转移与相当的钙成比例

• 细胞内Ca2+浓度通过荧光比率法测定: Ratio=I530/I485

细胞内Ca2+浓度在组氨酸的作用下增加，这种增加被抗组氨酸药物（Cyproheptadine）抑制

FRET应用测定Hela活细胞中的Ca2+浓度

Local irradiation FRAP Experiment

对分子的移动性进行量化

扩展功能：荧光漂白后恢复实验Fluorescence Recovery After Photobleaching （FRAP）

1 生物膜脂质分子的侧向扩散—膜蛋白或膜脂流动性，细胞迁移

2 细胞间隙连接（Gap Junction，GJ）及胞间通讯的研究

3 胞浆及细胞器内小分子物质转移性的观测等

4 细胞骨架构成、核膜结构及大分子组装等

扩展功能：荧光漂白后恢复实验细胞动力学研究

扩展功能：超高分辨率成像Airyscan


AiryscanConfocal

2 µm

有丝分裂中的培养细胞微管蛋白Peter O‘Toole, Ian Morrison (York, UK)

扩展功能：超高分辨率成像Airyscan


AiryscanConfocal

2 µm

扩展功能：超高分辨率成像 Airyscan不仅提高平面分辨率而且提高纵向分辨率1.7倍


Z-stack: 3.98 µm; 步进: 200 nm (Plan-Apochromat 63x/1.4 Oil)

有丝分裂中的培养细胞微管蛋白

Airy

sc

an

Co

nfo

ca

l

46Carl Zeiss Microscopy

Telomere replication without RTEL1: Stalled forks and telomere breakage visualized as doubled

dots using Airyscan. Resolution is meaningless without good SNR.

2 µm2 µm2 µm2 µm

Courtesy: J. Karlseder Ph.D. (Molecular and Cell Biology Laboratory) and

J. Fitzpatrick Ph.D. (Director, Waitt Advanced Biophotonics Core),

The Salk Institute, La Jolla, USA.

Airyscan：高分辨率，高信噪比成像

LSM880 with Airyscan 配置

硬件配置：4个激光器 —— 405，Argon (458/488/514)，561，633

3个检测器 —— 2个PMT + 1个32 channel GaAsP检测器超高分辨检测器 —— Airyscan

软件模块：Tile scan —— 大视野拼图Experiment Designer —— 实验设计


Agenda


2

3

4





宽场荧光显微镜光路

Rat Brain, Double labelling:

Green: Neurons, Blue: Nuclei


荧光光源

CCD

激光共聚焦显微镜只检测焦平面信号


Problem:

Detecting in-focus information

together with out-of-focus

fluorescence signals in wide-field

microscopy

Wide Field

Confocal

Wide Field

ConfocalExcitation

ZEISS

Plan-NEOFLUAR

40x /1,3 Oil

Emission

ZEISS

Plan-NEOFLUAR

40x /1,3 Oil

Excitation

ZEISS

Plan-NEOFLUAR

40x /1,3 Oil

Emission

ZEISS

Plan-NEOFLUAR

40x /1,3 Oil

光学显微镜特性


点光源透镜

x-y

x-z

焦平面

宽场荧光显微镜成像


标本物镜焦平面

共聚焦显微镜成像


标本物镜针孔焦平面

激光共聚焦显微镜光路

PMT

Laser

Rat Brain, Double labelling:

Green: Neurons, Blue: Nuclei


Pinhole （针孔）

PMT

X-Y平面扫描方式逐点扫描点 — 线 — 面


How is a X/Y/Z Stack produced?

Z-Drive

Z轴扫描方式逐层扫描


Agenda


2

3

4





LSM880 光路


efficient handling at laser input

apochromatic

pinhole optics

temp. controlled

fastest linear

scanning

TwinGate: low incident

angle dichroics, high

laser rejection

GaAsP: Single-shot spectral

detection. Cooled and improved

electronics, higher data throughput

Airyscan detector

for superresolution

hexagonal GaAsP

detection array

激光颜色：选择与荧光基团匹配的激光


405 nm DAPI / Hoechst

488nm GFP / Alexa Flour 488 / FITC

561nm RFP / Alexa Flour 568 / Cy3

633nm Alexa Flour 647 / Cy5

激光强度：过高的激光强度导致样品漂白


Sample:

Cultured mammalian cell

expressing GFP (live cell)

Imaging Strategy:

Time series of 1000 images

acquired at low laser power but

relatively high gain setting.

(excitation at 488 nm with just

0.5% of the max. power).

Result:

Hardly any photobleaching !

Intensity Profile

of time series

Time series: 1000 Images (!)

Cultured mammalian cell

expressing GFP

针孔

• 屏蔽非焦平面信号• 针孔增加，光通量增加，非焦平面信

号增加• 针孔减小，光通量减小，非焦平面信

号减小

• 影响光切厚度• 针孔增加，光切厚度增大，Z轴分辨

率变差• 针孔减小，光切厚度减小，Z轴分辨

率增加

一般情况，针孔大小为1AU


Laser

Sample

PMT

Pinhole

检测器：Photomultiplier Tube （PMT，光电倍增管）


PMT

PMT

32-channel array

QUASAR detection unit

PMT Detectors / Photomultiplier Tube Assuming a bright sample

12.09.2018 67

Output

Meter

- +

Power Supply

(Low Gain)

AnodeDynodes

Photoelectric effect:

PhotocathodePhoton

Electron

p

e-

bright Pixel

pp

p

p

p

p

p

p

p

Carl Zeiss Microscopy 67

PMT Detectors / Photomultiplier TubeAssuming a dark sample

12.09.2018 68

Output

Meter

AnodeDynodes


PhotocathodePhoton

Electron

e-

dark Pixel

- +

Power Supply

(Low Gain)

p

p

p


PMT Detectors / Photomultiplier TubeIncrease a dark sample´s signal with more Gain

12.09.2018 69

Output

Meter

AnodeDynodes


Photocathode

Electrons

e-

bright Pixel

Photons p

p

p

- +

Power Supply

(Higher Gain)


Assume a really dimm sample -Extreme Gain values result in Noise

12.09.2018 70

Output

Meter

- +

Power Supply

(Highest Gain)

AnodeDynodes


Photocathode

Electrons

e-

bright Pixel,

but Noise

Photonsp

Red lines indicate electrons that were generated but have not come from

the photons within the sample


GaAsP detector

(schematic illustration)

Typical sensitivity of detectors

GaAsP (Gallium Arsenide

Phosphide) is a semiconductor

material with ideal characteristics

for converting photons into

electrical signals.

Benefits of GaAsP detectors:

Two times better Quantum

efficency than PMTs (resulting in

higher sensitivity, better image

quality, and higher acquisition

speed).

GaAsP detectors can be operated

in integration mode as well as in

photon counting mode.

Gallium-Other Metals

Arsen - Metalloids

Phosphor-non Metal

GaAsP Detector高灵敏度检测器

Better image quality

Higher sensitivity equals better

signal-to-noise ratio (detection

of faint signals)

Faster scanning

Data recording at shorter pixel

times

Need for averaging strategies

largely reduced

Longer imaging

Data recording at lower laser

power (reduced bleaching and

photo-toxic effects in live cell

imaging)

Cultured 2h8 cells labeled with extremely low expressing GFP and

mCherry. Courtesy A. Bruckbauer Cancer Research, London, UK

GaAsP Detector 高灵敏度检测器

LSM 880Operation modes of the GaAsP detection unit

Unmatched flexibility in signal

detection!

Imaging in Lambda Mode

34 detector elements available

for parallel acquisition of

lambda stacks.

Fl. Emission

Spectral Beam

Guides

PMT PMT

Grating

Lambda stack

Inte

nsity

Wavelength

Acquisition of a Lambda StackEach pixel holds the spectral information of the sample

光谱编码图像

拆分通道图像

刚毛海蚯蚓 (环节蠕虫属)

细胞核：DAPI

肌肉：Alexa 488

神经系统：Cy2

光谱扫描，线性拆分分开高度重叠的荧光发射光谱

LSM 880 – Airyscan DetectionZoom optics and a unique 32 GaAsP-PMT design


32 GaAsP-PMT array

adaptive zoom optics

filter wheel

LSM 880 output port

ZEISS

Plan-NEOFLUAR

40x /1,3 Oil

The Confocal PinholePinhole Size Dictates Signal or Resolution


Airyscan detectionFarewell to the pinhole

• 32 GaAsP detectors in hexagonal lattice

• Each detector approx. 0.2 AU in diameter

• Total detection area approx. 1.25 AU in diameter

• Simultaneous improvement in resolution and signal!


Airyscan enhances resolution up to 1.7 foldThe comparison is made with confocal using PH = 1 A.u.

Carl Zeiss Microscopy

120 nm 240 nm

Improvement 1,7x: Measured using 40 nm beads (excitation at 488 nm)

Same acquisition time, same laser power.

Airyscan Confocal (1 A.u.)

LSM 880 – Airyscan: Resolution, SNR, and FlexibilityChoose zoom-in ROI’s, orientations, laser wavelengths, etc.


Use Airyscan just as an additional detector. Scan speeds, ROIs, field orientations,

laser wavelengths, multitracking, z-stacks, time series etc. are setup as always.

Sample handling and fluorophore selection don’t need rethinking

20µm

120 nm

Agenda


2

3

4





什么是高质量的图片？

当我们谈到图像质量，我们说的是……

信号强度信噪比分辨率


Illumination:

Not too bright,

Not too dimm

如何拍摄一张好的图片？合适的图像亮度

2018/9/12 91Carl Zeiss Microscopy

The main tools are:

• Laser power

• Pinhole

• PMT (Gain)

获取一张好的图片，需要关注……激光针孔 PMT

2018/9/12Carl Zeiss Microscopy 92

Look-up table Range Indicator (8bit)

Black

Grey value 0

Grey value 255

White

Grey values

1-255

如何评估一张图像的亮度Range Indicator


= gives

measurements of grey

values along a line

如何测量图像亮度的动态范围Profile


In a good image,

the full dynamic

range is used.

255

0

如何测量图像亮度的动态范围Profile


Loss of

Information

图像过曝Laser power 、Gain （Master）过高


Wasted

dynamic range

图像过暗Laser power 、Gain （Master）不足


“Good” Image

“Bad” Image

扫描策略如何拯救图像的信噪比


To decrease the effect of noise, more

photons (signal) must be collected:

1) Slower Scan Speed

2) Averaging

Scan the image x-times and take

the average signal for each pixel

-> addition of photons from several

scanning runs

Which of the two setups will lead to

better image quality?

扫描策略扫描速度、平均


Resolution okay Resolution too low

如何充分展示图像细节分辨率和 Frame size


Scan Zoom

EC Plan-NEOFLUAR

40x /1,3 Oil DIC

分辨率软件给出匹配最佳分辨率的拍摄参数


How is a X/Y/Z Stack produced?

Z-Drive

Z-stack 拍摄的参数设置


With this setting, the object structures

between the slices cannot be detected. Slice Interval

Slice Interval

At very small intervals a lot of additional data

without additional information is generated.

Z轴步进该设多少光切厚度与Z轴步进


The optimal overlap is fulfilled at “Nyquist” or “Sampling Theorem” conditions.

Sampling frequency (slice interval) must be the double of the information frequency ( z-

resolution or optical slice thickness).

To achieve these conditions just press Optimal Interval in Z-Stack dialog. Then, the slices

overlap by half of their thickness (no missing information @ minimal number of

sections).

Slice Interval

Optical Slice

Thickness

Z轴步进该设多少光切厚度与Z轴步进


共聚焦推荐使用的耗材

Cover Glass:170um

Glass bottom dish

Cover Glass:170um

Cell Chamber Slides

zeiss激光共聚焦显微镜 lsm880 with...

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