1. Application of Fluorescent Nanodiamonds to Biological Imaging Joseph Magliocco Physics H190 Spring 2011

2. The Basics: Excitation and Emission Jablonski Diagram Image from www.icecube.berkeley.edu

3. The Basics: Excitation and Emission Image from www.currentprotocols.com Spectra -“Mirror image” rule about ZPL -apply filters for specific excitation and emission wavelengths

4. The Basics: Phosphorescence and Photobleaching -Slow emission rates for phosphorescence since the transitions are “not allowed” -Phosphorescence is almost never seen in solution, only in solid state -When a fluorophore is bleached, it is essentially unusable Image from Phys177 Lecture by Carlos Bustamante, Spring 2010

5. The Basics: Quantum Yield Quantum Yield = number of photons emitted via fluorescence vs. number of photons absorbed kfkf ΣkiΣki Φ = k i could be due to fluorescence, internal conversions (phonons), quenching, intersystem crossing (phosphorescence), or any other decay process from the excited state

6. Two types of applicable FNDs From Nanodiamonds, First Applications in Biology and Nanoscale Medicine by Dean Ho; Chapter 6 – Development and Use of Fluorescent Nanodiamonds as Cellular Markers by Huan-Cheng Chang

7. Two types of applicable FNDs Red FNDs (rFNDs) due to NV centers –140nm rFNDs are mainly NV -, 35nm rFNDs show spectral features of both NV 0 and NV -

8. Two types of applicable FNDs Green FNDs (gFNDs) due to H3 centers –Both 70nm particles and 140nm particles are predominantly H3, but 140nm shows signs of NV - sideband

9. Comparison to other fluorophores: Bleaching -rFNDs (red) vs. fluorescent polystyrene nanospheres (blue) -gFNDs (red) vs. fluorescent polystyrene nanospheres (blue) rFNDs exhibited no photobleaching for up to 8 hours while gFNDs exhibited no photobleaching for up to 5 hours.

10. Comparison to Alexa Fluorophores: Quantum Yields N-V-N (λ em = 531):.95 NV - (λ em = 685):.99 Alexa 488 (λ em = 519):.92 Alexa 660 (λ em = 690):.37 Note that Alexa dyes are named after absorption wavelengths Conclusion: FNDs are more efficient and therefore brighter than common Alexa dyes FNDsAlexa Fluor

11. Comparison to Alexa Fluorophores: Photostability

12. Other useful properties of FNDs Cells can take in FNDs (only shown in HeLa cells) Can conjugate FNDs to proteins, nucleic acids, and carbohydrates Nontoxic (as opposed to Quantum Dots and other dyes) Chemically and thermally stable

13. One potential application: single- particle tracking Motor proteins? DNA translocases?

14. Future Directions Cost! Single vacancy per nanodiamond

15. References and Acknowledgements Nanodiamonds, First Applications in Biology and Nanoscale Medicine by Dean Ho; Chapter 6 – Development and Use of Fluorescent Nanodiamonds as Cellular Markers by Huan- Cheng Chang Physics 177 (Biophysics), Spring 2010, Carlos Bustamante Alexa Fluorophore website (www.invitrogen.com)www.invitrogen.com Images cited on slides