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![Page 1: PowerPoint-bemutató - · PDF fileBarkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010 1.2 mM actin 0 100 200 300 400 500 0 2 4 6 8 1.2 mM G-actin time, s 3 0s v = 12.68](https://reader035.vdocuments.pub/reader035/viewer/2022062906/5a76b42f7f8b9a63638d7462/html5/thumbnails/1.jpg)
![Page 2: PowerPoint-bemutató - · PDF fileBarkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010 1.2 mM actin 0 100 200 300 400 500 0 2 4 6 8 1.2 mM G-actin time, s 3 0s v = 12.68](https://reader035.vdocuments.pub/reader035/viewer/2022062906/5a76b42f7f8b9a63638d7462/html5/thumbnails/2.jpg)
![Page 3: PowerPoint-bemutató - · PDF fileBarkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010 1.2 mM actin 0 100 200 300 400 500 0 2 4 6 8 1.2 mM G-actin time, s 3 0s v = 12.68](https://reader035.vdocuments.pub/reader035/viewer/2022062906/5a76b42f7f8b9a63638d7462/html5/thumbnails/3.jpg)
(𝑑𝑥 , 𝑑𝑦) = ∆= 0.61𝜆
𝑁𝐴~200 𝑛𝑚
𝑑𝑧 = 2𝑛𝜆
𝑁𝐴 2~800 𝑛𝑚
𝑁𝐴 = 𝑛𝑠𝑖𝑛𝛼
![Page 4: PowerPoint-bemutató - · PDF fileBarkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010 1.2 mM actin 0 100 200 300 400 500 0 2 4 6 8 1.2 mM G-actin time, s 3 0s v = 12.68](https://reader035.vdocuments.pub/reader035/viewer/2022062906/5a76b42f7f8b9a63638d7462/html5/thumbnails/4.jpg)
![Page 5: PowerPoint-bemutató - · PDF fileBarkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010 1.2 mM actin 0 100 200 300 400 500 0 2 4 6 8 1.2 mM G-actin time, s 3 0s v = 12.68](https://reader035.vdocuments.pub/reader035/viewer/2022062906/5a76b42f7f8b9a63638d7462/html5/thumbnails/5.jpg)
http://www.nature.com/nature/journal/v178/n4543/abs/1781194a0.html
![Page 6: PowerPoint-bemutató - · PDF fileBarkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010 1.2 mM actin 0 100 200 300 400 500 0 2 4 6 8 1.2 mM G-actin time, s 3 0s v = 12.68](https://reader035.vdocuments.pub/reader035/viewer/2022062906/5a76b42f7f8b9a63638d7462/html5/thumbnails/6.jpg)
![Page 7: PowerPoint-bemutató - · PDF fileBarkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010 1.2 mM actin 0 100 200 300 400 500 0 2 4 6 8 1.2 mM G-actin time, s 3 0s v = 12.68](https://reader035.vdocuments.pub/reader035/viewer/2022062906/5a76b42f7f8b9a63638d7462/html5/thumbnails/7.jpg)
![Page 8: PowerPoint-bemutató - · PDF fileBarkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010 1.2 mM actin 0 100 200 300 400 500 0 2 4 6 8 1.2 mM G-actin time, s 3 0s v = 12.68](https://reader035.vdocuments.pub/reader035/viewer/2022062906/5a76b42f7f8b9a63638d7462/html5/thumbnails/8.jpg)
𝛼1 = 𝛼𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙
𝑛1
𝑛2𝛼2 = 900
𝛼1 < 𝛼𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙
𝛼1 > 𝛼𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙
αcritical ≡ α1 𝛼2 = 900
𝒏𝟏𝒏𝟐
𝒔𝒊𝒏𝜶𝒄𝒓𝒊𝒕𝒊𝒄𝒂𝒍 = 𝟏
𝑛1𝑠𝑖𝑛𝛼1 = 𝑛2𝑠𝑖𝑛𝛼2
𝒏𝟏 > 𝒏𝟐
𝛼1 < 𝛼2
α1
α2
![Page 9: PowerPoint-bemutató - · PDF fileBarkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010 1.2 mM actin 0 100 200 300 400 500 0 2 4 6 8 1.2 mM G-actin time, s 3 0s v = 12.68](https://reader035.vdocuments.pub/reader035/viewer/2022062906/5a76b42f7f8b9a63638d7462/html5/thumbnails/9.jpg)
![Page 10: PowerPoint-bemutató - · PDF fileBarkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010 1.2 mM actin 0 100 200 300 400 500 0 2 4 6 8 1.2 mM G-actin time, s 3 0s v = 12.68](https://reader035.vdocuments.pub/reader035/viewer/2022062906/5a76b42f7f8b9a63638d7462/html5/thumbnails/10.jpg)
INCIDENT𝐸1(Ԧ𝑟, t) = 𝐸1exp [𝑖(𝑘1 Ԧ𝑟 − 𝜔𝑡)]
TRANSMITTED
𝐄𝟐 Ԧ𝐫, 𝐭 = 𝐄𝟐𝐞𝐱𝐩 𝐢 Ԧ𝐤𝟐 Ԧ𝐫 − 𝛚𝐭 =
= 𝐸2exp [𝑖(𝑘2𝑠𝑖𝑛𝛼2𝑥 + 𝑘2𝑐𝑜𝑠𝛼2𝑧 − 𝜔𝑡)]
𝑖𝑘2 𝑠𝑖𝑛𝛼2𝑥 + 𝑐𝑜𝑠𝛼2𝑧 = 𝑖𝑘2(𝑠𝑖𝑛𝛼1𝑛
𝑥 + 𝑖𝑠𝑖𝑛2𝛼1𝑛2
− 1𝑧)
𝛂𝟏 𝛂𝐜 𝐄𝟐 Ԧ𝐫, 𝐭 == 𝑬𝟐𝒆𝒙𝒑[𝒊(𝒌𝟐𝒙 − 𝝎𝒕)]
𝛂𝟏 𝛂𝐜, 𝑬𝟐 𝒓, 𝒕 =
= 𝑬𝟐 𝒆𝒙𝒑 𝒊 𝒌𝟐𝒔𝒊𝒏𝜶𝟏
𝒏𝒙 − 𝝎𝒕 ∙ 𝒆𝒙𝒑[−𝒌𝟐
𝒔𝒊𝒏𝟐𝜶𝟏
𝒏𝟐− 𝟏𝒛] 𝐸1(Ԧ𝑟, t)
𝐸2(Ԧ𝑟, t)
𝑛1
𝑛2
k2
k1
α1
α2
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𝐸evanescent Ԧ𝑟, t =
𝐸𝑒𝑥𝑝[−𝑘2𝑠𝑖𝑛2𝛼1𝑛2
− 1𝑧]
= 𝑬𝒆𝒙𝒑 −𝟐𝝅
𝝀
𝟏
𝒏𝟐𝒔𝒊𝒏𝟐𝜶𝟏𝒏𝟏
𝟐 − 𝒏𝟐𝟐𝒛
𝐼 𝑧 = 𝐸2= 𝐈𝟎𝐞𝐱𝐩 −
𝐳
𝐝
𝐝 =𝛌
𝟒𝛑𝐧𝟏(𝐬𝐢𝐧𝟐𝛂𝟏 − (
𝐧𝟐𝐧𝟏)𝟐)−
𝟏𝟐
𝐼 𝑑 =𝐼0𝑒
0 100 200 300 400 5000.0
0.2
0.4
0.6
0.8
1.0
d
WFevanescent - TIRF
n2 = 1.333
n1 = 1.515
0 = 491 nm
incidence
= 65o
rela
tive
fie
ld in
ten
sity
z = distance from the interface (nm)
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𝒔𝒊𝒏𝜶𝒄𝒓𝒊𝒕𝒊𝒄𝒂𝒍 =𝒏𝟐𝒏𝟏
n2
n1= 0.879
αcritical = 61.62𝑜
0.5 0.6 0.7 0.8 0.9 1.0 1.10
10
20
30
40
50
60
70
80
90
100n2 = 1.333
n1 = 2.400
cri
tical =
critical a
ng
le (
o)
refractive index ratio = n2/n
1
n1 = 1.333
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1.30 1.35 1.40 1.450
200
400
600
800
1000
d =
de
ca
y le
ng
th (
nm
)n
2 = refractive index of the sample
angle of incidence
i (
o)
68.86
70.63
73.57
78.46
n1 = 1.515
= 491 nm
𝐝 =𝛌
𝟒𝛑𝐧𝟏(𝐬𝐢𝐧𝟐𝛂𝟏 − (
𝐧𝟐𝐧𝟏)𝟐)−
𝟏𝟐
60 65 70 75 800
50
100
150
200
250
300
350
400
critical
= 61.62o
(nm)
405
491
568
640
n2 = 1.333
n1 = 1.515
d =
de
ca
y le
ng
th (
nm
)
i = angle of incidence (o)
𝜶𝟏 → 𝜶𝒄𝒓𝒊𝒕𝒊𝒄𝒂𝒍: 𝒅 → ∞
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B16/F1 melanoma cell
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𝑛1 > 𝑛2𝑛𝑤𝑎𝑡𝑒𝑟 2 = 1.333, 𝑛𝑐𝑒𝑙𝑙 2 = 1.36
𝑛𝑔𝑙𝑎𝑠𝑠(1) = 1.515
𝑛 =𝑛2
𝑛1= 0.879 𝑛 =
𝑛2
𝑛1= 0.8
𝛼𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑐𝑒 > 𝛼𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙
for glass : water interface: 𝛼𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙 = 61.62𝑜
for glass : cell interface: 𝛼𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙 = 63.86𝑜
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𝑟 = 𝑛𝑓𝑠𝑖𝑛𝛼
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𝑛𝑤𝑎𝑡𝑒𝑟 (2) = 1.333
𝑛𝑔𝑙𝑎𝑠𝑠(1) = 1.515
𝑛𝑖𝑚𝑚𝑒𝑟𝑠𝑖𝑜𝑛(1) = 1.515
𝑛𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙 = 61.62𝑜
𝑵𝑨 𝒎𝒊𝒏𝒊𝒎𝒖𝒎 = 1.515 ∗ 𝑠𝑖𝑛61.62𝑜 = 𝟏. 𝟑𝟑𝑵𝑨~𝟏. 𝟒
𝑛𝑐𝑒𝑙𝑙(2) = 1.38
𝑛𝑔𝑙𝑎𝑠𝑠(1) = 1.515
𝑛𝑖𝑚𝑚𝑒𝑟𝑠𝑖𝑜𝑛(1) = 1.515
𝑛𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙 = 65.63𝑜
𝑵𝑨 𝒎𝒊𝒏𝒊𝒎𝒖𝒎 = 1.515 ∗ 𝑠𝑖𝑛65.63𝑜 = 𝟏. 𝟑𝟖𝑵𝑨 > 𝟏. 𝟒!
𝑵𝑨 ↑→ 𝑾𝑫 ↓
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1.30 1.35 1.40 1.450
200
400
600
800
1000
d =
de
ca
y le
ng
th (
nm
)n
2 = refractive index of the sample
angle of incidence
i (
o)
68.86
70.63
73.57
78.46
n1 = 1.515
= 491 nm
𝐝 =𝛌
𝟒𝛑𝐧𝟏(𝐬𝐢𝐧𝟐𝛂𝟏 − (
𝐧𝟐𝐧𝟏)𝟐)−
𝟏𝟐
60 65 70 75 800
50
100
150
200
250
300
350
400
critical
= 61.62o
(nm)
405
491
568
640
n2 = 1.333
n1 = 1.515
d =
de
ca
y le
ng
th (
nm
)
i = angle of incidence (o)
𝜶𝟏 → 𝜶𝒄𝒓𝒊𝒕𝒊𝒄𝒂𝒍: 𝒅 → ∞
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Inner life of the cell
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0 2000 4000 60000.0
0.2
0.4
0.6
0.8
1.0
1.2
re
lative
pyre
nyl flu
ore
sce
nce
time (s)
𝑣 = 𝑘+ 𝐺0 − 𝑐𝑐 𝐹 − 𝑘− 𝐹
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0 2000 4000 60000.0
0.2
0.4
0.6
0.8
1.0
1.2
re
lative
pyre
nyl flu
ore
sce
nce
time (s)
𝑣 = 𝑘+ 𝐺0 − 𝑐𝑐 𝐹 − 𝑘− 𝐹
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0 2000 4000 60000.0
0.2
0.4
0.6
0.8
1.0
1.2
re
lative
pyre
nyl flu
ore
sce
nce
time (s)
𝑣 = 𝑘+ 𝐺0 − 𝑐𝑐 𝐹 − 𝑘− 𝐹
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m
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Prokop A. et al. Journal of Cell Science 2013
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Barkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010Goncalves-Pimentel et al. PLOS One 2011
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ACTIN
DAAM FH2
DAAM FH1-FH2
Barkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010
1.2 mM actin
0 100 200 300 400 5000
2
4
6
8
1.2 mM G-actin
time, s
fila
me
nt
len
gth
, 1
03 s
ub
un
its
v = 12.68 ± 0.96 su/s
1.2 mM actin + 2.4 mM DAAM FH1-FH2
0 100 200 300 400 5000,0
0,2
0,4
0,6
0,8
1,0
1.2 mM G-actin
+ 2.4 mM DAAM FH1-FH2
time, s
fila
ment
len
gth
, 1
03 s
ubunits
v = 0.99 ± 0.32 su/s
1.2 mM actin + 2.34 mM DAAM FH2
0 100 200 300 400 5000,0
0,2
0,4
0,6
0,8
1,01.2 mM G-actin
+ 2.34 mM DAAM FH2
fila
ment
len
gth
, 1
03 s
ubunits
time, s
v = 0.59 ± 0.22 su/s
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0.3 mM actin + 0.72 mM profilin
v = 2.91 ± 0.50 su/s
500 s
10
mm
0.3 mM actin + 0.72 mM profilin + 2.6 mM DAAM FH2
v = 0.20 ± 0.05 su/s
0.3 mM actin + 0.72 mM profilin + 2.4 mM DAAM FH1-FH2
v = 2.35 ± 0.28 su/s
ACTIN
DAAM FH2
DAAM FH1-FH2
Barkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010
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Vig A. Bugyi B. et al. manuscript in preparation 2016
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Vig A. Bugyi B. unpublished
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Vig A. Bugyi B. unpublished
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Nanometer targeting of microtubules to focal adhesions.
model IX2; Olympus
100x NA 1.65
high refractive index immersion oil (diodomethane; Sigma-Aldrich)
special high NA coverslips (n = 1.788; Olympus)
multi-line laser (Innova 70C; Coherent) CCD camera (MicroMAX 1024B; Princeton Instruments).
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100x 1.46 NA objective 100x (Carl Zeiss, Inc.)488- and 568-nm laser lines (Laser Physics USA)rear-illuminated CCD camera (Cascade 512B; Roper Scieintific)a dual imager for simultaneous imaging the channels (Optical Insights)
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100x 1.46 NA objective 100x (Carl Zeiss, Inc.)488- and 568-nm laser lines (Laser Physics USA)rear-illuminated CCD camera (Cascade 512B; Roper Scieintific)a dual imager for simultaneous imaging the channels (Optical Insights)
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http://www.olympusfluoview.com/applications/opticalhighlighters.html
STANDARD
PHOTOACTIVABLE
PHOTOCONVERTIBLE
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≈1
𝑁
𝜆
2𝑛𝑠𝑖𝑛𝛼
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University of Szeged, Department of Optics and Quantum Electronics
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