共聚焦原理和样品制备技术

样品制备
2005.7武汉 李 楠
1

.
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
2

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
3

棱镜分光
Longer wavelength = lower energy White light
} } }
Infrared
700 nm
Visible
400 nm
Ultraviolet
Shorter wavelength = higher energy
4

Absorption / emission
Emission is lower energy = longer wavelength
Heat
S1 Absorption
Emission
S0
5

Stokes' Law: fluorescence is always at longer wavelength
Absorption
Fluorescence
500
550
600
650
700
Wavelength (nm)
6

荧光探针的性质
激发波长λem和发射波长λex -----------λem总是小于 λex（Stockes) ， 根据 λem 和λex 选择激光和滤片 E=h*m/ λ 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
7

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
8

LP 550 DCLP 520
Epi-fluorescence
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
10

Short Wave Pass Filter (SWP)
100 80 Transmission (%) 60 40 20
Passes short wavelength
Absorbs or reflects long wavelength
300
400
500 600 Wavelength
700
800
11

Long Wave Pass Filter (LWP)
100 80 Transmission (%) 60 40 20 0 300
Absorbs or reflects short wavelength
Passes long wavelength
400
500 600 Wavelength
700
800
12

Bandpass Filter
100 80 Transmission (%) 60 40 20
Transmits only this “band”of wavelengths
FWHM
300
400
500 600 Wavelength
700
800
13

Filter sets
Exciter: D470/40x Dichroic 500DCLP Emitter: OG515LP
Note that the dichroic also transmits at ~400nm!
14

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)
15

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.
16

显微镜的荧光CUBES
Filter cube
Excitation range UV UV UV + violet blue violet + blue blue blue blue green green green FITC/TEXAS RED blue/green TEXAS RED/green UV/blue/green
Exitation
Dichroic Suppressi on LP 425 BP 470/40 LP 470 LP 470 LP 515 LP 515 LP 515 400 455
Filter cube
Excitation range CY3 green CY5 red CY7 red GFP-blue FITC/rhodamine blue/green blue green violet + blue UV blue violet + blue blue violet + blue blue green
Exitation BP545/30 BP 620/60 BP 710/75 BP 470/40 BP 490/15 BP 560/25 BP 500/20 BP 546/12 BP 436/20 BP 385/15 BP 485/20 BP 436/12 BP 500/20 BP 436/8 BP 495/12 BP 580/20
Dichroic Suppressi on 565 660 750 500 500 580 515 560 455 420 510 445 515 445 510 595 BP 610/75 BP 700/75 BP 810/90 BP 525/50 BP 525/20 BP 605/30 BP 535/30 BP 605/75 BP 480/40 BP 460/20 BP 437/45 BP 467/37 BP 545/45 BP 460/25 BP 535/35 BP 630/55
A A4 D E4 H3 I3 K3 L5 M2 N2.1 N3 G/R TX2 B/G/R
BP 340-380 400 BP 360/40 BP 436/7 BP 355-425 455 BP 420-490 510 BP 450-490 510 BP 470-490 510 BP 480/40 BP 546/14 BP 546/12 BP 490/20 BP 575/30 BP 560/40 BP 495/15 BP 570/20 505 580 565 505 600 595 415 510 590
Y3 Y5 Y7 GFP FI/RH
BP 515-560 580
YFP BP 527/30 RFP LP 590 CFP LP 590 BFP/GFP
BP 600/40 BP 525/20 BP 635/40 BP 645/75 BP 465/20 BP 530/30 BP 640/40
CFP/YFP C/Y/R
DAPI/FITC/TEXAS RED BP 420/30
17

Double label
Abs 1 Fluor 1
Abs 2
Fluor 2
400
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)
19

Double label
Abs 1 Fluor 1 2 dichroics to separate F1 & F2 Fluor 2
Abs 2
400
500
600
700
800
Wavelength (nm)
20