Application of Fluorescent Nanodiamonds to Biological Imaging

Joseph Magliocco

Physics H190

Spring 2011

The Basics: Excitation and EmissionJablonski Diagram

Image from www.icecube.berkeley.edu

The Basics: Excitation and Emission

Image from www.currentprotocols.com

Spectra

-“Mirror image” rule about ZPL-apply filters for specific excitation and emission wavelengths

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

The Basics: Quantum Yield

Quantum Yield = number of photons emitted via fluorescence vs. number of photons absorbed

kf

ΣkiΦ =

ki could be due to fluorescence, internal conversions (phonons), quenching, intersystem crossing (phosphorescence), or any other decay process from the excited state

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

Two types of applicable FNDs

• Red FNDs (rFNDs) due to NV centers– 140nm rFNDs are mainly NV-, 35nm rFNDs

show spectral features of both NV0 and NV-

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

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.

Comparison to Alexa Fluorophores: Quantum Yields

• N-V-N (λem = 531): .95

• NV- (λem = 685): .99

• Alexa 488 (λem = 519): .92

• Alexa 660 (λem = 690): .37Note that Alexa dyes are named after absorption wavelengths

Conclusion: FNDs are more efficient and therefore brighter than common Alexa dyes

FNDs Alexa Fluor

Comparison to Alexa Fluorophores:Photostability

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

One potential application: single-particle tracking

Motor proteins? DNA translocases?

Future Directions

• Cost!

• Single vacancy per nanodiamond

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)

• Images cited on slides