Confocal fluorescence microscopy: characterization of

Confocal characterization and imaging calibration, SIPcharts.

Confocal fluorescence microscopy: characterization of imaging and calibration using thin uniform fluorescence layers, SIPcharts.

G. J. Brakenhoff Swammerdam Institute for Life Sciences, University of

Amsterdam, Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands

E-mail: [email protected]

Varenna 12 Juli 2010 International School of Physics Enrico Fermi

Advanced methods in optical fluorescence microscopy towards nanoscopy

objectintensity

lmageintensity

Imaging by a lens

focalpoint

focalpoint

Shading or non-uniform intensity imaging:

focalpoint

focalpoint

objectintensity

lmageintensity

Vignettting:partial block of light rays

Image of uniform layer:

Lower intensity at the edges!

Problems in microscopy affecting quantitative imaging

Shading effects

Imaging conditions difficult to characterize

Imaging conditions difficult to reproduce

�� Non-quantitative intensity imaging

Present both Present both in 2D - regular microscopy

3D - confocal microscopy

Propose correction/calibration procedures based on

Thin uniform fluorescent layers

Sectioned Imaging Property or SIPcharts analysis INTENSITY CORRECTION AND NORMALIZATION INFLUORESCENCE CONFOCAL MICROSCOPY WITH

SECTIONED IMAGING PROPERTY CHARTS OR SIPCHARTS

J. M. Zwier #, G.W.H. Wurpel #, K. Jalink +, L. Oomen+, L. Brocks + & G. J. Brakenhoff #

#Swammerdam Institute for Life Sciences, Section of Molecular Cytology and Center ofAdvanced Microscopy, University of Amsterdam,, The Netherlands,

+ Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, TheNetherlands

0.01% fluorescing compound in polyvinylalcohol spin-coated on microscope slide.

Protected and sealed with a microscope cover glass

Calibration for 2D and 3D Fluorescence Microscopy

Basis :Spatially uniform fluorescing reference layer

Thickness 100 to 200 nm.Thickness uniform within a few %

Reference layers have to be to a high degree

spatially uniform and reproducible.

• various info

NN

O

O

O

O

concentration of

N,N'-bis(2,5-di-tert-butylphenyl)-3,4,9,10-N,N'-bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenebis(dicaboximide) in polystyrene/toluene

0.01 w% in polystyrene

concentration measured with absorption spectroscopy (ε(490nm) = 86000 l•mol•cm-1 ) -> A= 0.09 -> 3.0•106 mol/l -> in a layer of 100 nm thickness this leads to

1.8 •1010 molecules/cm2

Average distance between molecules about 100 nm.

No quenching effects!

• Absorption spectrum

• emission spectrum

350

300

250

200

150

100

50

x10

3

550500450400350

wavelength (nm)

abso

rptio

n c

oef

f. (a

.u)

160

120

80

40

0

x10

3

700650600550500

wavelength (nm)

inte

nsi

ty (

cou

nts

)

Fluorescence specimen image:

Ps (x,y) = I(x,y) . D(x,y) . Fs (x,y)

illumination detection specimen fluoresence

Reference layer image: taken under same conditions !

Pr(x,y) = I(x,y) D(x,y) Fr

Reference layer fluorescence : uniform

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.



Arb. Intensity

Arb. Intensity

Ps (x,y)

P (x,y)Calibrated image Pc(x,y)

Ps(x,y F(x,y)Pc(x,y) = ------ = ------- ( F L U ’ S ) ------------ =

Pr(x,y) Fr

expressed in Fluorescence (of the reference) Layer Units

: ” F L U ’s ”

Actual imaging conditions as described by I(x,y) D(x,y) have dropped out !2D

(regular microscopy)



FLU’s Intensity





Specimen: Lipid DPPCtwo phase monolayer segregation

Pr(x,y)

QuickTime™ and aTIFF (PackBits) decompressorare needed to see this picture.

Microscope20x image

Microscope 40 x image

Images of object

Images of

Shading correction


Images with different magnifications can be well correllated!

Images of reference layer

Calibrated images

2D(regular microscopy)

500

400

300

4000

3000

A 500

400

3001.21.0

B

before shading correction after shading correction

Application:Shading correction Specimen:

Lipid DPPC monolayer,two phases with low (LC) and high (LE) NPB fluorescencer concentration

300

200

100

0

6004002000

3000

2000

1000

x-pixel

y-pi

xel 300

200

100

0

6004002000

1.00.80.60.40.2

x-pixely-

pixe

l

1200

1000

800

600

400

200

0

40003000200010000 fluorescence intensity

c) 1200

1000

800

600

400

200

0

1.20.80.40.0relative fluorescence intensity

d)

Are the reference layers really uniform?

Image Reference image

Two images of the reference layer taken at two different locations are used one as image to be calibrated and the other as reference image.

Calibrated image

drawing

Referenceimage

Image

Proves that our fluorescent reference layers for the calibration that are to a high degree

spatially uniform

reference layer

2D(regular microscopy)

2DIm age calibra tion in fluore scen ce microscopyJ. M. Zwier, G . J. VAN R oo ij, J . W , Ho fst raa t & G . J. Brak enhof fJ. of Micr., 216 , 15 -24 , 2004

3D

Confocal fluorescence microscopy sectioned images

(Anaphase, Allium Cepa (onion), DNA stain: mitrmycine)

Integrated or Total Confocal Image



Method: Making a z-stack (through focus) of the uniform polymer fluorescence reference layer (ca. 100 nm)

Sectioned image characterization with thin uniform fluorescent layers

Through focus stack

coverslip

reference layer

reference image

slide


1

1 2

4

3 5

z

Through focus stack

x

z

z1

1 24

3 5

Axial Point Spread Function

Axial PSF

Through-focus stack of “ thin”uniform fluorescence reference layer

LaterallyIntegrated Intensity

z

yx


0.00E+00

1.00E+02

2.00E+02

3.00E+02

4.00E+02

5.00E+02

6.00E+02

1 6

11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96

Series1

Series2

0.00E+00

1.00E+02

2.00E+02

3.00E+02

4.00E+02

5.00E+02

6.00E+02

7.00E+02

1 6

11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96

Series1

Series2

0.00E+00

1.00E+02

2.00E+02

3.00E+02

4.00E+02

5.00E+02

6.00E+02

1 6

11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96

Series1

Series2

0.00E+00

1.00E+02

2.00E+02

3.00E+02

4.00E+02

5.00E+02

6.00E+02

7.00E+02

1 6

11

16

21

26

31

36

41

46

51

56

61

66

71

76

81

86

91

96

Series1

Series2

0.00E+00

1.00E+02

2.00E+02

3.00E+02

4.00E+02

5.00E+02

6.00E+02

7.00E+02

Series1

Series2

1

43

5

2

I intensity

0.00E+00

1.00E+02

2.00E+02

3.00E+02

4.00E+02

5.00E+02

6.00E+02

7.00E+02

Series1

Series2

Series3

Series4

Series5

Confocal trough-focus response: local off axis responses

Super position plot axial PSF responses over image field

a)

b) c)

Axial PSF

zconfocal PSF 700nm and up

z



Layer thickness thin with respect to axial confocal dimensions.Essential !

fluorescer layer thickness 100nm

m max intensitys =a/b skew

a b

1

1 2

4

3 5 z


How to characterize the spatial imaging conditions in confocal and (later) multi-photon microscopy?

Proposed parameters for charcterization:

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

0 20 40 60 80 100 120

Series1

zm axial position of maximum

fwhm resolution

a b

integrated intensity

40000000

45000000


a b

Analysis of confocal imaging conditionsResolution variation over image

50 1.15

bin_fwhmAvg = 1.05 µm

SD = 0.048 µm

1

1 2

4

3 5

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

0 20 40 60 80 100 120

Series1


fwhm resolution

exp 230603 100nm layer pinhole 1 Airy

40

30

20

10

0

y

40200x

1.10

1.05

1.00

0.95µm

Beam scanning:

Confocal imaging determined by overlap of

illumination and detection distributions

50

40

30

20

10

0

y

40200x

8.8

8.4

8.0

7.6

x106

bin_i Avg = 8.20187e+06SD = 306548

x,y confocal intensity distribution


1

1 2

4

3 5

m max intensitys =a/b skewBeam scanning:no aberration off-axis chromatic aberration

xxInt.

Int.

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

0 20 40 60 80 100 120

Series1



fwhm resolution

a b

How to standardize the characterization of the spatial imaging conditions in confocal and (later) multi-photon microscopy?

Propose:Sectioned Imaging Property charts


Sectioned Imaging Property charts or

SIPcharts

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ItotalAvg = 90.6395SD = 4.68024

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ImaxAvg = 79.9347SD = 10.0326

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

6150

6100

6050

6000

5950

5900

5850

zmaxAvg = 6012.89SD = 75.4975

60

50

40

1400

1300

fwhm Avg = 1026.68SD = 92.209

30000000

35000000

40000000

45000000

Imax max intensity

a b Axial PSF

skew : s =a + b

a − bskew = a − b

a + b Sectioned Imaging

Property chart

SIPchart


60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

-30

-20-10

0

1020

30

x10-3

skew Avg = -0.0488328SD = 0.0299356

40

30

20

10

0

y (

pix)

6040200

x (pix)

1200

1100

1000

900

12

10

8

6

4

2

0

Flu

ores

cenc

e In

t. (1

03 )

121086420

z (knm)

I z(16,16)

I z(16,48)

I z(32,32)

I z(48,16)

I z(48,48)

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

0 20 40 60 80 100 120

Series1

Zmax axial position of maximum

Imax max intensity

fwhm resolution

a b

Itot integrated intensityunder axial PSF

AxialPSF

skew : s =a + b

a − b

�SIP Chart SectionedImagingPropertyChart

Confocal System 1 Obj. 63x, oil, 1,4 NA pinhole 1 Airy NKI 12-08-04 Bin 8 from 512*512

0

5000000

10000000

15000000

20000000

25000000

0 20 40 60 80 100 120

Series1


fwhm resolution


PSF SIPchart

Analysis 3D image formation with SIPcharts


60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ItotalAvg = 90.6395SD = 4.68024

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ImaxAvg = 79.9347SD = 10.0326

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

6150

6100

6050

6000

5950

5900

5850

zmaxAvg = 6012.89SD = 75.4975

60

501400

fwhm Avg = 1026.68SD = 92.209

4000 SIP Chart SectionedImagingPropertyChart

Confocal System 1

Comparing confocal systems, system1


60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

-30

-20-10

0

1020

30

x10-3

skew Avg = -0.0488328SD = 0.0299356

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

1300

1200

1100

1000

900

12

10

8

6

4

2

0

Flu

ores

cenc

e In

t. (1

03 )

121086420

z (knm)

I z(16,16)

I z(16,48)

I z(32,32)

I z(48,16)

I z(48,48)

Point params bin_avg bin_sd

0 I_max 79.9347 10.0326

1 z_max 6012.89 75.4975

2 fwhm 1026.68 92.209

3 skew -0.0488328 0.0299356

4 I_total 90.6395 4.68024

3000

2000

1000

0

fwhm

(nm

)

Confocal System 1Obj . 63x, oil, 1,4 NA pinhole 1 AiryNKI 12-08-04 Bin 8 from 512*512

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

0 20 40 60 80 100 120

Series1


Imax max intensity

fwhm resolution

a b


Axial PSF

skew : s = a + b

a − b

Comparing confocal systems, system 2

60

40

20

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ItotalAvg = 81.7096SD = 9.1452

60 1350

fwhm Avg = 1194.62SD = 66.1558

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ImaxAvg = 83.4493SD = 8.27537

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

6000

5500

5000

4500

4000

3500

zmaxAvg = 4656.19SD = 666.415

4000 SIP Chart SectionedImagingPropertyChart

Confocal System 2


60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

1350

1300

1250

1200

1150

1100

105030

0020

0010

000

fwhm

(nm

)

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

skew Avg = 0.0526943SD = 0.0480111 200

150

100

50

0

Flu

ores

cenc

e In

t. (1

03 )

80006000400020000

z (nm)

I z(16,16)

I z(16,48)

I z(32,32)

I z(48,16)

I z(48,48)


0 I_max 83.4492 8.27537

1 z_max 4656.19 666.415

2 fwhm 1194.62 66.1558

3 skew 0.0526943 0.0480111

4 I_total 81.7096 9.1452

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

0 20 40 60 80 100 120

Series1


Imax max intensity

fwhm resolution

a b


Axial PSF

skew: s =a + b

a − b

Confocal System 2Obj . 63x, oil, 1,4 NA pinhole 1 AiryCLSM 04-05-04 no4 Bin 8 from 512*512

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ItotalAvg = 90.6395SD = 4.68024

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ImaxAvg = 79.9347SD = 10.0326

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

6150

6100

6050

6000

5950

5900

5850

z maxAvg = 6012.89SD = 75.4975

60

50

40

y (

pix) 10

20

30

x10

skew Avg = -0.0488328SD = 0.0299356

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

1400

1300

1200

1100

1000

900

fwhm Avg = 1026.68SD = 92.209

12

10

8

Flu

ores

cenc

e In

t. (1

03 )

I z (16,16)

I z (16,48)

I z (32,32)

I (48,16)


0 I_max 79.9347 10.0326

1 z_max 6012.89 75.4975

2 fwhm 1026.68 92.209

3 skew -0.0488328 0.0299356

4 I_total 90.6395 4.68024

4000

3000

2000

1000

0

fwhm

(nm

)

SIP Chart SectionedImagingPropertyChart


30000000

35000000

40000000

45000000

Imax max intensity

a b

Axial

skew : s = a + b

a − b

60

40

20

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ItotalAvg = 81.7096SD = 9.1452

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

1350

1300

1250

1200

11501100

1050

fwhm Avg = 1194.62SD = 66.1558

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ImaxAvg = 83.4493SD = 8.27537

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

6000

5500

5000

4500

4000

3500

zmaxAvg = 4656.19SD = 666.415

4000

3000

2000

1000

0

fwhm

(nm

)

60

50

40

y (

pix)

0.00

0.05

0.10

0.15

skew Avg = 0.0526943SD = 0.0480111 200

150

100

Flu

ores

cenc

e In

t. (1

03 )

I z(16,16)

I z(16,48)

I z(32,32)

I z(48,16)

I (48,48)


0 I_max 83.4492 8.27537

1 z_max 4656.19 666.415

2 fwhm 1194.62 66.1558

3 skew 0.0526943 0.0480111

4 I_total 81.7096 9.1452

20000000

25000000

30000000

35000000

40000000

45000000

Series1

Imax max intensity

a b

Axial PSF

skew: s = a + b

a − b



Comparing confocal systems


60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

1400

1300

1200

1100

1000

900

fwhm Avg = 1026.68SD = 92.209

60

50

40

30

20

10

0y

(pi

x)

6040200

x (pix)

1350

1300

1250

1200

1150

1100

1050

fwhm Avg = 1194.62SD = 66.1558

30

20

10

0

y (

pix)

6040200

x (pix)

-30

-20-10

0

x10-3

6

4

2

0

Flu

ores

cenc

e In

t. (1

0

121086420

z (knm)

I z (48,16)

I z (48,48)0

5000000

10000000

15000000

20000000

25000000

0 20 40 60 80 100 120

Series1


fwhm resolution


PSF 30

20

10

0

y (

pix)

6040200

x (pix)

-0.15

-0.10

-0.05

0.00

50

0

Flu

ores

cenc

e In

t. (1

0

80006000400020000

z (nm)

I z(48,48)

0

5000000

10000000

15000000

0 20 40 60 80 100 120


fwhm resolution


Comparison resolution

4000

3000

2000

1000

0

fwhm

(nm

)

4000

3000

2000

1000

0

fwhm

(nm

)

System 1 System 2

Comparison of lenses:Overview from 4 SIPcharts each taken with a different 63x objective


Conclusion: easy to spot differences between lenses: quality control!

160

140

120

100

80

60

40

20

0

x10

3

806040200

z1616 z1648 z3232 z4816 z4848

150

100

50

0

x10

3

806040200

z1616 z1648 z3232 z4816 z4848

60

50

40

30

20

10

0

y

6040200

x

12.0

11.0

10.0

bin_fwhm Avg = 10.5374SD = 0.551831

60

50

40

30

20

10

0y

6040200

x

22

20

18

16

14

12

bin_fwhm Avg = 16.844SD = 2.11699

objective 2 phase contrast63x oil immersion170204 fred012

objective 163x oil immersion170204 fred010

4 Objectives: resolution at pinhole 1 Airy equiv.

160

140

120

100

80

60

40

20

0

x10

3

806040200

z1616 z1648 z3232 z4816 z4848

140

120

100

80

60

40

20

0

x10

3

806040200

z1616 z1648 z3232 z4816 z4848

60

50

40

30

20

10

0

y

6040200

x

11.0

10.0

9.0

8.0

bin_fwhm Avg = 9.27375SD = 0.640423

60

50

40

30

20

10

0

y

6040200

x

11

10

9

8

7

bin_fwhm Avg = 7.90227SD = 0.738263

x

objective 363x oil immersion170204 fred013

objective 4 100 x oil immersion170204 fred014

Confocal course with partial beam block

Leica TCSNA= 1.463Xwith partial beam block

lmageintensity

with partial block

Confocal course without partial beam block

Leica TCSNA= 1.463XWithout partial beam block

wiih partialblock removed

lmageintensity

Confocal course function NA



Leica SP2-2 NA= 1.2

Confocal course function NA

Leica SP2-4 NA= 1.4100X

Sipchart_before_cleaning objective

Sipchart_after_cleaning objective

Spinning Disk -1.1.pdf

Dependence of imaging on proper immersion oil.

At temperatures for live cell imaging –37 C-regular oils provide not optimal imaging.

Sipchart representations show:Total photon yield is about 23% higher in the DF2 oil for 37 C then for the regular DF23 and Leica oils.then for the regular DF23 and Leica oils.

With proper immersion oil: resolution is improved together with imaging field uniformity

Dependence of imaging on proper immersion oil.Imaging at 37C with standard immersion oil.

Dependence of imaging on proper immersion oil.Imaging at 37C with immersion oil for 37 C.

60

40

20

0

y (

pix)

6040200

x (pix)

6100

6000

5900

zmaxAvg = 5985.81SD = 87.651

60

40

20

0

y (

pix)

6040200

x (pix)

5900

5800

5700

5600

zmax Avg = 5767.09SD = 79.6776

Imaging wavelengths: Imaging wavelengths :

Axial image plane dependence on wavelength settings


Imaging wavelengths: ext: λ 488, detection: λ bp 505-530

Imaging wavelengths : exitation: λ 543, detection: λ 560-615

Shift axial image plane with wavelength settings

60

40

20

0

pix

6040200

pix

z-max(b-a)

240

200

160

Avg= 218.723SD= 18.7466

nm

Important for co-localizationand FRET etc. studies!

Multi-excitation, multi-detector confocal microscope

variablepinhole

detectionchannels

(PMT) dichroics

computer

display

lasers

scanmirror

dichroic

pinhole

objective

pinhole

BRAKENHOFF, G. J., WURPEL, G. W. H., JALINK, K., OOMEN, L., BROCKS, L. &ZWIER, J. M. (2005)Characterization of sectioning fluorescence microscopy with thin uniformfluorescent layers: Sectioned Imaging Property or SIPcharts.Journal of Microscopy 219 (3), 122-132.

3D characterization reference:


Next:

Sectioning microscopy image Sectioning microscopy image calibrationcalibration

• SIPchart based relative and absolute image calibration

– Relative image calibration• uniform sensitivity of the lateral image field (= shading compensation)• images can be related by expressing their intensities in

fluorescence layer units (FLU’s)

Calibrated image Pc(x,y)

– Examples:• fluorescent beads• shading correction BPAE cells

Bovine pulmonary artery endothelial cells.

• comparison of images taken with different magnifications

Ps(x,y) F(x,y) . PSF(x,y) F(x,y)Pc(x,y) = ------ = --------------------- = --------- ( F L U ’s )

Pr(x,y) Fr . PSF(x,y) Fr

Shading correction, beads

I total SIPchartbased

• SIPchart based relative and absolute image calibration– Using the SIPchart data to do correction of the total intensities of image

stacks• proof of principle on fluorospheres

15

12

3.0

2.5

coun

ts)

1 3

befo

re s

hadi

ng c

orre

ctio

n

afte

r s

hadi

ng c

orre

ctio

n

12

9

6

3

0

15129630I (total) field 2 ( Irel)

I (to

tal)

field

1 (

Ire

l)2.5

2.0

1.5

1.0

0.5

0.0

3.02.52.01.51.00.50.0

I(to

tal)

field

1 (

x10

6 co

unts

)

I(total) field 2 (x106

counts)

0 1

2

3

4

56

78

befo

re s

hadi

ng c

orre

ctio

n

afte

r s

hadi

ng c

orre

ctio

n

Calibration imaging with different magnifications / NA

Reference layer image Pr(x,y)

from I total SIPchart!

I total SIPchart’s

Before calibration

from I total SIPchart!

NA 1.2540x

Planapo

NA 1.3263x

Planapo

SIPchart’s

after calibrationin FLU’s

Calibration between two confocal microscopes: • SP2 Leica system• NT Leica system

– 63x objectives (1.32 NA) (exc 488 nm)– Only detection filters were somewhat different: NT - lp530 nm

SP2 - +530 nm

500

4001.0

nt-microscope (63x, 1.32 NA)500

4001.0

sp2-microscope (63x, 1.32 NA)

Calibration imaging with between different microscopes

NT SP2300

200

100

0

5004003002001000

0.8

0.6

0.4

0.2

0.0

FLU's

300

200

100

0

5004003002001000

0.8

0.6

0.4

0.2

0.0

FLU's

• After calibration (FLU intensity scales are comparable)

• Intensities within 30%! > Small differences in spectral filtering conditions

– object image:

– reference layer image:

Fobj (x,y ) = QE obj * σref .Nobj (x,y ).Peff (x,y )

fluorescence quantum yield

absorption cross section

number of molecules

eff,PSF

500

400

300

200

100

0

5004003002001000

imagestack-bg(counts)

160x103

120

80

40

0

500

400

I total (counts)

140x103

SIPchart based absolute image calibrationSIPchart based absolute image calibration

– reference layer image:

– ratio image:

Fref (x ,y ) = QE ref * σref .N ref .Peff (x ,y )

Fobj(x,y)

Fref (x,y)= QEobj

QEref.σobj

σref.Nobj(x ,y)

Nref.

300

200

100

0

5004003002001000

140x101301201101009080

Nobj(x,y) = 1.7∗104.Fobj(x,y)

Fref (x,y)mol /µm2

SIPchart based absoluteimage calibration

–ratio image:

then:

Fobj(x,y)

Fref (x,y)= QEobj

QEref.σobj

σref.Nobj(x ,y)

Nref.


Used parameter values:QE (BodipyFL) = 0.8 QE (dtbpd ref layer) = 0.95 dtbpd (488 nm) : ε = 84000 l.mol-1cm-1=> σ = 3.2 * 10-16 cm 2

bodipyFL (488 nm) : ε = 60000 l.mol-1cm-1 => σ = 2.3 * 10-16 cm (σ = ε * ln 10/ NA = 3.825 * 10-21 * ε)

Nref = 1.0 *104 molecules / µm2

• SIPchart based relative and absolute image calibration

500

400

molecules bodipyFL

(micrometer-2

)

Nobj(x,y) = 1.7∗104.Fobj(x,y)

Fref (x,y)mol /µm2


400

300

200

100

0

5004003002001000

(micrometer-2

)

2.0x104

1.5

1.0

0.5

0.0

Total number of BodipyFl

fluorophores in the cell:

4.7 * 107

100µm

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ItotalAvg = 90.6395SD = 4.68024

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ImaxAvg = 79.9347SD = 10.0326

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

6150

6100

6050

6000

5950

5900

5850

z maxAvg = 6012.89SD = 75.4975

60

50

40

y (

pix) 10

20

30

x10

skew Avg = -0.0488328SD = 0.0299356

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

1400

1300

1200

1100

1000

900

fwhm Avg = 1026.68SD = 92.209

12

10

8

Flu

ores

cenc

e In

t. (1

03 )

I z (16,16)

I z (16,48)

I z (32,32)

I (48,16)


0 I_max 79.9347 10.0326

1 z_max 6012.89 75.4975

2 fwhm 1026.68 92.209

3 skew -0.0488328 0.0299356

4 I_total 90.6395 4.68024

4000

3000

2000

1000

0

fwhm

(nm

)



30000000

35000000

40000000

45000000

Imax max intensity

a b

Axial

skew : s = a + b

a − b

60

40

20

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ItotalAvg = 81.7096SD = 9.1452

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

1350

1300

1250

1200

11501100

1050

fwhm Avg = 1194.62SD = 66.1558

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

100

90

80

70

60

50

40

ImaxAvg = 83.4493SD = 8.27537

60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

6000

5500

5000

4500

4000

3500

zmaxAvg = 4656.19SD = 666.415

4000

3000

2000

1000

0

fwhm

(nm

)

60

50

40

y (

pix)

0.00

0.05

0.10

0.15

skew Avg = 0.0526943SD = 0.0480111 200

150

100

Flu

ores

cenc

e In

t. (1

03 )

I z(16,16)

I z(16,48)

I z(32,32)

I z(48,16)

I (48,48)


0 I_max 83.4492 8.27537

1 z_max 4656.19 666.415

2 fwhm 1194.62 66.1558

3 skew 0.0526943 0.0480111

4 I_total 81.7096 9.1452

20000000

25000000

30000000

35000000

40000000

45000000

Series1

Imax max intensity

a b

Axial PSF

skew: s = a + b

a − b



Comparing confocal systems


60

50

40

30

20

10

0

y (

pix)

6040200

x (pix)

1400

1300

1200

1100

1000

900

fwhm Avg = 1026.68SD = 92.209

60

50

40

30

20

10

0y

(pi

x)

6040200

x (pix)

1350

1300

1250

1200

1150

1100

1050

fwhm Avg = 1194.62SD = 66.1558

30

20

10

0

y (

pix)

6040200

x (pix)

-30

-20-10

0

x10-3

6

4

2

0

Flu

ores

cenc

e In

t. (1

0

121086420

z (knm)

I z (48,16)

I z (48,48)0

5000000

10000000

15000000

20000000

25000000

0 20 40 60 80 100 120

Series1


fwhm resolution


PSF 30

20

10

0

y (

pix)

6040200

x (pix)

-0.15

-0.10

-0.05

0.00

50

0

Flu

ores

cenc

e In

t. (1

0

80006000400020000

z (nm)

I z(48,48)

0

5000000

10000000

15000000

0 20 40 60 80 100 120


fwhm resolution


Comparison resolution

4000

3000

2000

1000

0

fwhm

(nm

)

4000

3000

2000

1000

0

fwhm

(nm

)

System 1 System 2

How to make a SIPchart?

• Web-application– Accepts large 3D data sets– Automatic generation of SIPcharts– SIPchart delivered to user via

website:

www.SIPchart.org

www.SIPchart.com

Sipchart creation

Get a calibration layer.Collect 3D data set from thiscalibration layerGo to:

Click restricted area:

• We-application:– uplboad page:

• Webapplication– Return email:

• Webapplication:– SIPchart

Sipchart creationYou need a uniform fluorescence calibration layer

Send me an email at: [email protected] can then send you a layer. For free or else?

The “layer project” has ended succesfully.No real grounds for a continuation grant.

If the layers are usefull for maintaining and calibrating microscope performancethey should be of some value to people.

I want to keep the project going by inviting people to buy a few layers.comes with unlimited website access to produce SIPcharts.Euro 175 each?

Money goes to the University, to pay people etc.

20071113-14094813553

Sipchart Double Layer.pdf

20071113-141826-13644Sipchartbollen.pdf

Application:Generation of loacl 3D PSF’sCorrection of datasets acquired at different wavelengths for chromatic axial and lateral image shifts

Applications SIPcharts :

Testing and Evaluation , Optimization and alignmentMaintenance of confocal systems

Co-localization studiesidentify axial image plane shifts with spectral settings

Suitable basis for more detailed analysis

of sectioned imaging systems

Suitable basis for more detailed analysis

Correction of sectioned imaging first order correction fluorescence intensity variations

optimization of de-convolution algorithms

Basis for: absolute calibration of sectioned imaging

Email : [email protected]

Close

Documents

Confocal fluorescence microscopy: characterization of