共聚焦原理和样品制备技术

Multi-labelling
³ 合适的激光波长 ³ 不重叠的发射光谱 ³ 相近的荧光强度 ³ 使用AOTF
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CHLAMYDOMONAS You can’t have
too many channels!
Assemblin, DAPI, tubulin &
centrin
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Protecting against 防淬灭
样品制备
2005.7武汉 李 楠
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1. 目的?
. 在成象前,要明确想达到什 么样的目标,这样可以节省 时间同时提高成功率
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2. FLUOROCHROMES ?
对于荧光显微镜和CONFOCAL,选择荧光时应该考虑那 些因素?
激光光源 标记物 细胞器探针 量子产率 光漂白 毒性
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棱镜分光
White light
来自百度文库
blue
BP 470-490 510
LP 515
YFP
blue
BP 480/40 505
BP 527/30 RFP
green
BP 546/14 580
LP 590
CFP
green green
BP 515-560 580 BP 546/12 565
LP 590 BFP/GFP
BP 600/40
FITC/TEXAS RED blue/green
S1 Absorption
Heat
Emission
S0
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Stokes' Law: fluorescence is always at longer wavelength
Absorption
Fluorescence
500
550
600
650
700
Wavelength (nm)
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荧光探针的性质
激λ发ex波（长Stoλcemk和es)发，射根波据长λλeemx 和---λ--e-x--选---择λem激总光是和小滤于片 E=h*m/ λ
Wavelength
12
Bandpass Filter
Transmission (%)
100 80 60 40 20
300
Transmits only this “band”of wavelengths
FWHM
400
500
600
700
800
Wavelength
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Filter sets
Exciter: D470/40x Dichroic 500DCLP Emitter: OG515LP
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Epi-fluorescence
DCLP 520
LP 550
Beyond the dichroic mirror is a barrier filter to ensure that any residual excitation light is blocked. It can also be used to separate the light from different fluorochromes.
BP 480-490
9
Filter 的设置
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Short Wave Pass Filter (SWP)
Transmission (%)
100
80
60 Passes short wavelength
40
20
Absorbs or reflects long wavelength
300
400
500
600
BP 490/20 505 BP 575/30 600
BP 525/20 CFP/YFP
BP 635/40
TEXAS RED/green
BP 560/40 595
DAPI/FITC/TEXAS RED BP 420/30 415
BP 645/75 C/Y/R
BP 465/20
UV/blue/green
500
600
700
800
Wavelength (nm)
18
Double label
Abs 1
Excitation of A1 & A2
Abs 2
Fluor 1 Fluor 2
400
500
600
700
800
Wavelength (nm)
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Double label
Abs 1 Abs 2
Fluor 1
Note that the dichroic also transmits at ~400nm!
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Filter sets
Exciter: D360/40x Dichroic: 400DCLP Emitter: D460/50m
A bandpass barrier filter to exclude other fluorescence (but notice a little transmittance in the far red)
Y3
UV
BP 360/40 400
BP 470/40 Y5
UV + violet
BP 355-425 455
LP 470
Y7
blue
BP 436/7 455
LP 470
GFP
violet + blue
BP 420-490 510
LP 515 FI/RH
blue
BP 450-490 510
LP 515
green
BP 546/12 560
BP 605/75
violet + blue
BP 436/20 455
BP 480/40
UV
BP 385/15 420
BP 460/20
blue
BP 485/20 510
BP 437/45
violet + blue
BP 436/12 445
BP 467/37
blue
There are several ways to protect against the effects of photobleaching:
D You can reduce the light intensity (although this will also reduce your fluorescence intensity).
CY7 red
BP 710/75 750
BP 810/90
GFP-blue
BP 470/40 500
BP 525/50
FITC/rhodamine BP 490/15 500
BP 525/20
blue/green
BP 560/25 580
BP 605/30
blue
BP 500/20 515
BP 535/30
荧光强度--------决定染料的检测灵敏度 荧光寿命--------分子在激发态的平均停留时间 光稳定性--------激发光的强度不可以无限提高 染料分子的相互作用-------浓度依赖式的自身荧光
淬灭现象
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荧光探针的性质
染料分子对环境的敏感性 溶剂的极性 淬灭剂的浓度及其与染料的亲和性 水相的PH值
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常见的商品形式
³ Fluo-3, AM ester (purity: >95% )
10x100 μg $203.00
³ Fluo-3, AM ester (purity: >95% by HPLC)
1 mg
$175.00
³ Fluo-3, AM ester, 1 mM in dry DMSO (purity: >95% by HPLC) 1 ml $203.00
³ Fluo-3, AM ester (purity: >95% by HPLC)
20x50 ug $252.00
³ Fluo-3, pentaammonium salt
1 mg
$161.00
³ Fluo-3, pentasodium salt
1 mg
$161.00
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荧光指示剂的主要种类
1、Protein Conjugates for Biological Detection 2、Nucleic Acid Detection 3、Peptide and Protein Detection, Analysis and Synthesis 4、Enzymes and Enzyme Substrates 5、Probes for Actin, Tubulin and Nucleotide-Binding Proteins 6、Probes for Organelles 7、Probes for Lipids and Membranes 8、Fluorescent Tracers of Cell Morphology and Fluid Flow 9、Assays for Cell Viability, Proliferation and Function 10、Probes for Endocytosis, Receptors and Ion Channels 11、Photoactivatable (Caged) Probes 12、Probes for Signal Transduction 13、Probes for Reactive Oxygen Species 14、Indicators for Ca(2+), Mg(2+), Zn(2+) and Other Metals 15、pH Indicators 16、Indicators for Na(+), K(+), Cl(-) and Other Anions 17、Probes for Membrane Potential
BP 495/15 510
BP 530/30
BP 570/20 590
BP 640/40
Excitation range Exitation Dichroic Suppressi on
CY3 green
BP545/30 565
BP 610/75
CY5 red
BP 620/60 660
BP 700/75
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显微镜的荧光CUBES
Filter cube
A A4 D E4 H3 I3 K3 L5 M2 N2.1 N3 G/R
TX2 B/G/R
Excitation range
Exitation Dichroic Suppressi on
Filter cube
UV
BP 340-380 400
LP 425
400
500
600
700
800
Wavelength (nm)
21
22
‘Bleedthrough’ in fluorescence stained retina
480 + 568nm excitation
568nm alone
Ref: Prof J. Stone, Univ of Sydney 23
700
800
Wavelength
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Long Wave Pass Filter (LWP)
Transmission (%)
100
80
Absorbs or 60 reflects short
wavelength
40
Passes long wavelength
20
0
300
400
500
600
700
800
Longer wavelength = lower energy
}Infrared 700 nm
}Visible } 400 nm
Ultraviolet Shorter wavelength = higher energy
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Absorption / emission
Emission is lower energy = longer wavelength
2 dichroics to separate F1 & F2
Fluor 2
400
500
600
700
800
Wavelength (nm)
20
Double label
Abs 1
Fluor 1
Cannot separate F1 & F2, => excite separately
Abs 2
Fluor 2
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Filter sets
Exciter: 61000v2x Dichroic 61000v2bs Emitter 61000v2m
Some very cunning things are possible with interference filters, such as this triple dichroic set, which excites & detects at 3 wavelengths.
D You can de-oxygenate your sample (O2 is the main reactant in photobleaching).
D You can use an anti-fade agent.
Linan 2004 .
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荧光探针的使用
根据研究目的、研究方法、所用机器类 型选择合适的荧光探针种类和商品形式。
BP 500/20 515
BP 545/45
violet + blue
BP 436/8 445
BP 460/25
blue
BP 495/12
510 BP 535/35
green
BP 580/20
595 BP 630/55
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Double label
Abs 1
Fluor 1
Abs 2
Fluor 2
400