(𝑑𝑥 , 𝑑𝑦) = ∆= 0.61𝜆

𝑁𝐴~200 𝑛𝑚

𝑑𝑧 = 2𝑛𝜆

𝑁𝐴 2~800 𝑛𝑚

𝑁𝐴 = 𝑛𝑠𝑖𝑛𝛼

http://www.nature.com/nature/journal/v178/n4543/abs/1781194a0.html

𝛼1 = 𝛼𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙

𝑛1

𝑛2𝛼2 = 900

𝛼1 < 𝛼𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙

𝛼1 > 𝛼𝑐𝑟𝑖𝑡𝑖𝑐𝑎𝑙

αcritical ≡ α1 𝛼2 = 900

𝒏𝟏𝒏𝟐

𝒔𝒊𝒏𝜶𝒄𝒓𝒊𝒕𝒊𝒄𝒂𝒍 = 𝟏

𝑛1𝑠𝑖𝑛𝛼1 = 𝑛2𝑠𝑖𝑛𝛼2

𝒏𝟏 > 𝒏𝟐

𝛼1 < 𝛼2

α1

α2

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

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)

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

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 → ∞

B16/F1 melanoma cell

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

𝑟 = 𝑛𝑓𝑠𝑖𝑛𝛼

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 ↓

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 → ∞

Inner life of the cell

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

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

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

m

Prokop A. et al. Journal of Cell Science 2013

Barkó Sz. Bugyi B. et al. Journal of Biological Chemistry 2010Goncalves-Pimentel et al. PLOS One 2011

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

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

Vig A. Bugyi B. et al. manuscript in preparation 2016

Vig A. Bugyi B. unpublished

Vig A. Bugyi B. unpublished

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).

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)

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)

http://www.olympusfluoview.com/applications/opticalhighlighters.html

STANDARD

PHOTOACTIVABLE

PHOTOCONVERTIBLE

≈1

𝑁

𝜆

2𝑛𝑠𝑖𝑛𝛼

University of Szeged, Department of Optics and Quantum Electronics