RAPID COMMUNICATION |
Application of Quantum Dots as Probes for Correlative Fluorescence, Conventional, and Energy-filtered Transmission Electron Microscopy
Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
Correspondence to: David P. BazettJones, 555 University Avenue, Toronto, ON M5G 1X8, Canada. E-mail: dbjones{at}sickkids.ca
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(J Histochem Cystochem 52:1318, 2004)
Key Words: quantum dots energy-filtered transmission electron microscopy electron spectroscopic imaging multiple labeling protein co-localization cell structure immunogold
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To illustrate the value of QDs as probes for EM studies of cell components, the nuclear promyelocytic leukemia (PML) protein was chosen as the target biomolecule. The localization of PML protein in discrete subnuclear bodies has been well characterized with both fluorescence and electron microscopy (Boisvert et al. 2000,2001
).
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Growth, Fixation, and Embedding
HEp-2 PML I (GFP-PML I/IV stable cell line; a kind gift from Dr. J. Taylor) cells were cultured per ATCC recommendations. Cells were seeded onto coverslips, cultured overnight in Dulbecco's modified Eagle's medium (DMEM) (GIBCO; Carlsbad, CA) with 500 µg/ml G418 sulfate (Calbiochem; San Diego, CA) and 10% FBS (Invitrogen; Carlsbad, CA), then fixed in a 1% paraformaldehyde and 2% glutaraldehyde mix in PBS (pH 7.5) at ambient temperature for 10 min and rinsed three times with PBS. Then the cells were dehydrated with a graded ethanol series, consisting of 30%, 50%, and 70% ethanol at 10 min each. Cells were then incubated in a 2:1 mix of 70% ethanol:LR White (Electron Microscopy Sciences; Fort Washington, PA) for 10 min and then rinsed twice with LR White. The coverslip was placed cell side down onto a mold filled with LR White. The assembly was inserted into a vacuum chamber and placed in a 60C oven for 24 hr.
Thin Sectioning
The block of resin attached to the coverslip was removed from the mold, placed in liquid nitrogen briefly, and then peeled off the coverslip. A selected area of the block was excised, glued on a bullet of resin, and mounted in the ultramicrotome for sectioning. Sections 6070-nm thick were picked up on 400-mesh nickel grids.
Post-section Labeling
For all incubations, drops of solution were placed on a sheet of Parafilm and grids were floated on the drops, section side down. All antibody incubations were performed for 1 hr, and all blocking and rinsing steps were performed for 10 min. The sections were blocked twice with a solution of 0.5% BSA, 0.15% glycine, then three times with 2% BSA in PBS (incubation buffer). The sections were then incubated with a rabbit polyclonal anti-PML antibody (Chemicon; Temecula, CA) diluted in incubation buffer, then rinsed three times with the incubation buffer. For immunogold labeling, a goat anti-rabbit IgG10-nm gold antibody (Electron Microscopy Sciences) was used for the secondary antibody incubation. After rinsing three times with the blocking buffer, goat anti-rabbitCy3 was employed to fluorescently label the remaining sites on the primary antibody. The sections to be labeled with QDs were incubated with a biotin-SP-conjugated donkey anti-rabbit biotinylated antibody (Jackson ImmunoResearch; West Grove, PA) and rinsed as before. The Qdot 605 conjugate was incubated at 10 nM for 30 min with the Qdot Incubation Buffer (Quantum Dot). The section was then placed on a drop of this solution. After the immunogold or QD incubation, the samples were rinsed once with PBS for 10 min. Grids were then placed on drops of distilled water (Invitrogen; Grand Island, NY) four times for 10 min each and finally air-dried. For the double-labeling experiment, the rabbit anti-NH2-terminal CBP antibody (Santa Cruz Biotechnology; Santa Cruz, CA) was incubated concurrently with a mouse monoclonal anti-PML antibody 5E10 (a kind gift from R. van Driel). Immunogold was targeted against PML and QD against CBP.
Staining
Staining was performed by placing grids on drops of 1% uranyl acetate (Electron Microscopy Sciences) solution for 5 min. The grids were then rinsed three times on drops of distilled water.
Fluorescence Microscopy
Thin sections supported on grids were placed on glass slides and covered with coverslips for examination at high magnification (x63) with a Leica DMRA2 epifluorescence microscope (Leica Microsystems; Wetzlar, Germany). Digital images were collected with a 14-bit CCD camera. Images were captured and processed with Open Lab 3.0.7 software (Improvision; Boston, MA). The fluorescence filter set used for Cy3 was also well suited for imaging the Qdot 605 tag.
Electron Microscopy
Before ESI analysis the sections were coated with a carbon film approximately 3 nm thick to stabilize the section from physical distortions caused by the electron beam (Ren et al. 2003). The regions of interest were imaged with a Tecnai 20 (FEI; Eindhoven, The Netherlands) transmission electron microscope equipped with an electron imaging spectrometer (Gatan; Pleasanton, CA) and operated at 200 kV. Elemental maps were generated by dividing the element-enhanced post-edge image by the pre-edge image after alignment by cross-correlation. Net ratio elemental maps were produced from pre- and post-edge images recorded at 120 and 155 eV (LII,III edge) for phosphorus, edges recorded at 385 and 415 eV (K edge) for nitrogen (BazettJones and Hendzel 1999
), and edges collected at 415 and 510 eV (MIV,V edge) for cadmium. The recording times required to obtain the pre-edge and post-edge images are in the range of 1030 sec. The images were collected with a cooled CCD camera. Because the images are captured at a resolution of 1024 x 1024 pixels, they have a pixelated appearance when inspected closely or zoomed. The images were processed using Digital Micrograph software (Gatan).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The electron dose for detection via ESI is on the order of 300 electrons/Å2 for the pre- or post-edge images. This is approximately a 100-fold increase from the dose used for brightfield imaging. Terminal mass loss occurs within the first second of imaging and no further loss occurs during image collection. Nevertheless, damage of structural detail at the level that is required is not a major problem. An order of magnitude greater dose exposure would be required before physical damage from the beam would become apparent. The integrity of the plastic sections is enhanced with a carbon coating 23 nm thick.
We observed that colloidal gold used for immunodetection can be distinguished from the QDs on the basis of mass density and shape. This provides an opportunity for multiple labeling in EM studies. As an example of this application, we investigated the localization of the transcriptional co-activator CREB binding protein (CBP) with respect to the PML body, using a double-labeling technique. We have observed with ESI and in situ nanogold labeling that the CBP protein localizes primarily to the periphery of PML bodies and not to their core (our unpublished data). Here we labeled PML protein with an immunogold probe and CBP with a QDstreptavidin probe directed against a biotinylated secondary antibody (Figure 3) . With sufficient magnification, the gold probes can be easily distinguished from QDs on the basis of electron density and shape. In the overlay of the high contrast brightfield image, which clearly shows the QD and gold distributions on the nitrogen elemental map (Figure 3C), we observe that a domain on the periphery of the ring-shaped PML body is enriched in CBP. The PML protein, as indicated by the colloidal gold spheres, is distributed throughout the ring structure.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The full potential of this class of probes is yet to be realized, because the ability to use chemical signatures with ESI to determine the co-localization of subcellular components hinges on the development of QDs of various chemical compositions. The core/shell configuration offers the choice of several atomic constituents, although the larger number of atoms in the core of nanocrystals results in an inherent advantage of the core vs the shell component. For the CdSe/ZnS material, cadmium provides the highest ESI sensitivity. The determinant criteria for an element suitable for energy-filtered mapping are the energy and the profile of its ionization edge. Ionization edges far from the zero loss peak are less favored than edges in the low-energy loss region of the spectrum. This is due to higher beam exposures required for edges that are farther out in the spectrum. Therefore, elemental maps of Cd are easier to obtain than those for Se. Moreover, the ionization edge should not overlap with the edge of another element of interest within the sample. Nanocrystals composed of CdTe (Wuister et al. 2003), GaSb (MullerKirsch et al. 2003
), InGaAs/GaAs (Guffarth et al. 2003
), and Fe2O3 (Lu et al. 2003
) have been synthesized, and Te, Sb, In, As, and Fe are all elements that have appropriate ionization edges for obtaining ESI maps. Furthermore, doping nanocrystals with transition metal ions suitable for ESI, to achieve materials such as Co2+-doped ZnAl2O4 (Duan et al. 2003
) and Mn2+-doped CdS/ZnS (Yang and Holloway 2003
), is another method of creating novel probes. Doping techniques can be adjusted to produce up to 37 atom % of the dopant in a nanocrystal (Zhuang et al. 2003
). For a nanocrystal consisting of 1500 atoms, 2% of the dopant would result in up to 30 atoms, which is theoretically within the limit of detection of ESI, for an element of high ESI sensitivity (BazettJones and Hendzel 1999
).
As conditions for nanocrystal growth are optimized for a variety of elemental components and bioconjugation of these materials becomes routine, QDs will attain prominence in the arsenal of probes for high-resolution mapping of biochemicals in macromolecular complexes visualized in vitro and in situ by ESI.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BazettJones DP, Hendzel MJ (1999) Electron spectroscopic imaging of chromatin. Methods 17:188200[Medline]
BazettJones DP, Hendzel MJ, Kruhlak MJ (1999) Stoichiometric analysis of protein- and nucleic acid-based structures in the cell nucleus. Micron 30:151157[Medline]
Boisvert FM, Hendzel MJ, BazettJones DP (2000) Promyelocytic leukemia (PML) nuclear bodies are protein structures that do not accumulate RNA. J Cell Biol 148:283292
Boisvert FM, Kruhlak MJ, Box AK, Hendzel MJ, Bazett-Jones DP (2001) The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body. J Cell Biol 152:10991106
Chan CW, Nie S (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281:20162018
Dabbousi BO, RodriguezViejo J, Mikulec FV, Heine JR, Mattoussi H, Ober R, Jensen KF, et al. (1997) (CdSe)ZnS Core-shell quantum dots: synthesis and characterization of a size series of highly luminescent nanocrystallites. J Phys Chem 101:94639475
Duan XL, Yuan DR, Cheng XF, Sun ZH, Sun HQ, Xu D, Lu MK (2003) Spectroscopic properties of Co2+: ZnAl2O4 nanocrystals in sol-gel derived glass ceramics. J Phys Chem Solids 64:10211025
Goldman ER, Anderson GP, Tran PT, Mattoussi H, Charles PT, Mauro JM (2002) Conjugation of luminescent quantum dots with antibodies using an engineered adaptor protein to provide new reagents for fluoroimmunoassays. Anal Chem 74:841847[Medline]
Guffarth F, Heitz R, Geller M, Kepteyn C, Born H, Sellin R, Hoffmann A, et al. (2003) Radiation hardness of InGaAs/GaAs quantum dots. Appl Phys Lett 82:19411943
Hines MA, GuyotSionnest P (1996) Synthesis and characterization of strongly luminescing ZnS-capped CdSe nanocrystals. J Phys Chem 100:468471
Hendzel MJ, BazettJones DP (1996) Probing nuclear ultrastructure by electron spectroscopic imaging. J Microsc 182:114[Medline]
Jensen HL, Norrild B (1999) Easy and reliable double-immunogold labeling of Herpes simplex virus type-1 infected cells using primary monoclonal antibodies and studied by cryosection electron microscopy. Histochem J 31:525533[Medline]
Lu J, Fan JD, Xu RS, Roy SJ, Ali N, Gao Y (2003) Synthesis of alkyl sulfonate/alcohol-protected gamma-Fe2O3 nanocrystals with narrow size distributions. J Colloid Interface Sci 258:427431[Medline]
MullerKirsch L, Ledentsov NN, Sellin R, Pohl UW, Bimberg D, Hausler I, Kirmse H, et al. (2003) GaSb quantum dot growth using InAs quantum dot stressors. J Crystal Growth 248:333338
Ren Y, Kruhlak MJ, BazettJones DP (2003) Same serial section correlative light and energy-filtered transmission electron microscopy. J Histochem Cytochem 51:605612
Ueda J, WentzHunter KK, Cheng EL, Fukuchi T, Abe H, Yue BYJT (2000) Ultrastructural localization of myocilin in human trabecular meshwork cells and tissues. J Histochem Cytochem 48:13211329
Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson JP, Ge N, et al. (2002) Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nature Biotechnol 21:4146
Wuister SF, van Driel F, Meijerink A (2003) Luminescence and growth of CdTe quantum dots and clusters. Phys Chem Chem Phys 5:12531258
Yang H, Holloway PH (2003) Enhanced photoluminescence from CdS: Mn/ZnS core/shell quantum dots. Appl Phys Lett 82:19651967
Zhuang J, Zhang X, Wang G, Li D, Yang W, Li T (2003) Synthesis of water-soluble ZnS:Mn2+ nanocrystals by using mercaptopropionic acid as a stabilizer. J Mater Chem 13:18531857