time correlated single photon counting (tcspc) tf · time correlated single photon counting (tcspc)...
TRANSCRIPT
![Page 1: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/1.jpg)
Time Correlated Single Photon
Counting (TCSPC) (tF)
![Page 2: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/2.jpg)
TCSPC(Fluotime 300):
Capabilities
• Steady state emission (detection in the UV-Vis/
IR)
• Excitation (Xe lamp: 200 nm – 900 nm)
• Time resolved emission ( UV-Vis/ IR)
• Phosphorescence measurements (singlet
oxygen)
• Fluorescence upconversion
• Anisotropy
• TRES
![Page 3: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/3.jpg)
S0
S1
Sn
Tn
T1
Absorbance
10-15 s
Fluorescence
10-9 - 10-7 s
Internal Conversion
10-14 - 10-11 s
Inter System Crossing
10-8 - 10-3 s
Phosphorescence
10-4 - 10-1 s
Vibrational Relaxation
10-14 - 10-11 s
![Page 4: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/4.jpg)
Fluorescence lifetime
Exponential curve fitting
can be used to calculate
the fluorescence lifetime
(tF), which is the time it
takes for the intensity to
fall to 1/e of its initial
value
![Page 5: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/5.jpg)
Why are fluorescence lifetimes
important?
Lifetime measurements can give us
information about:
• Fluorophore structure
• Fluorophore/ environmental interactions
i.e. effects of temperature, polarity,
presence of fluorescence quenchers
• Imaging - fluorescence lifetime microscopy
![Page 6: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/6.jpg)
Phosphorescence
measurements (singlet oxygen)
• Sepia PDL820 and TimeHarp (Software
updates).
• This then allows use of burst mode
• Enables EasyTau to go to IR region of
1000 -1400 nm
![Page 7: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/7.jpg)
Fig. 1: Sepia PDL820 and Time
Harp software
![Page 8: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/8.jpg)
Sample prep..
• Absorbance (0.2 to 0.3)
• Dimethylformamide (Organic solvent)
• Deuterated water
![Page 9: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/9.jpg)
Steps to follow..
![Page 10: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/10.jpg)
Conti…
tF 𝜱𝜟
![Page 11: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/11.jpg)
Emission icon
![Page 12: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/12.jpg)
Near-Infrared phosphorescence
spectrum
Zn Standard Sample
phosphorescence spectrum was plotted as a function of the fractionof absorbed light (1 − 10−A)
![Page 13: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/13.jpg)
Calculation
• The slope of these plots (rR and rS, respectively) are proportional to the corresponding ΦΔ
R and ΦΔS
• The absorption needs to be taken into concentration as the samples and standard were both excited at 670 nm. The Equation was multiplied by the absorption factor: Aabs
R/ AabsS
•
![Page 14: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/14.jpg)
Results
Previous calculated value is 0.61The calculated value using TCSPC is 0.58
![Page 15: Time Correlated Single Photon Counting (TCSPC) tF · Time Correlated Single Photon Counting (TCSPC) (t F) TCSPC(Fluotime 300): Capabilities •Steady state emission (detection in](https://reader033.vdocuments.us/reader033/viewer/2022052800/5f0f92527e708231d444d2c6/html5/thumbnails/15.jpg)