Use of quantum dots for live cell imaging

Quantum dots
Monitoring interactions within and among cells as they grow and differentiate is a key to understanding
organismal development. Fluorescence microscopy is among the most widely used approaches for highresolutiA photograph and representative spectrum of ph...Image via Wikipediaon, noninvasive imaging of live organisms1,2, and organic fluorophores are the most commonly used tags for fluorescence-based imaging3. Despite their considerable advantages in live cell imaging,
organic fluorophores are subject to certain limitations. Fluorescent quantum dots (QDs) are inorganic
fluorescent nanocrystals that overcome many of these limitations and provide a useful alternative for studies that require long-term and multicolor imaging of cellular and molecular interactions4,5.

      For labeling specific cellular proteins, QDs must be conjugated to biomolecules that provide binding
specificity. Bioconjugation approaches vary with the surface properties of the hydrophilic QD used. The
mixed surface self-assembly approach is recommended for conjugating biomolecules to QDs capped
with negatively charged dihydroxylipoic acid (DHLA) (Fig. 1). In this approach DHLA-capped QDs are
conjugated to proteins using positively charged adaptors6–8, for example, a naturally charged protein
(e.g., avidin9), a protein fused to positively charged leucine zipper peptide (zb)8 or a protein fused to
pentahistidine peptide (5× His)10. The use of avidin permits stable conjugation of the QDs to ligands,
antibodies or other molecules that can be biotinylated, whereas the use of proteins fused to a positively
charged peptide or oligohistidine peptide obviates the need for biotinylating the target molecule. This
procedure describes the bioconjugation of QDs and specific labeling of both intracellular and cell-surface
proteins4,5. For generalized cellular labeling, QDs not conjugated to a specific biomolecule may be used;
various strategies are presented in Box 1, Generalized Labeling of Live Cells.

 MATERIALS REAGENTSQuantum dots (QDs; e.g., Quantum Dot Corporation or Evident Technology)
Cells or tissue for labeling, prepared appropriately depending on the application (QDs can be used to tag live cells, label cell-surface
proteins, or label fixed cells or tissue sections)
Amylose resin (New England Biolabs)
Antibodies of interest
Avidin (Sigma Chemicals)
Bovine serum albumin (BSA), 1% in PBS
Lipid-based transfection reagent (e.g., Lipofectamine 2000 (Invitrogen) or Fugene 6 (Roche))
Maltose (Sigma Chemicals)
Maltose-binding protein fused to the basic leucine zipper domain (MBP-zb) and protein G fused to the basic leucine zipper domain
(PG-zb) expressed and purified from bacteria as described elsewhere7.
Phosphate-buffered saline (PBS; Sigma Chemicals)
Sodium tetraborate (Sigma Chemicals)
Sulfo-NHS-SS biotin (Pierce Biotechnology)
Tris-buffered saline (TBS; Sigma Chemicals)
Hand-held UV lamp
Fluorescence microscope (for details, see the section Imaging the Labeled Cells)

Bioconjugation of the QDs

Figure 1 | The mixed surface approach for
conjugating biomolecules to DHLA-capped QDs
using avidin as the linker molecule. The positive
charge on MBP-zb and avidin allow self-assembly
of these molecules on the negatively charged
surface of DHLA-capped QDs. Use of MBP-zb in
molar excess to the avidin makes it possible to
regulate the number of avidin molecules present
on each QD. This, in turn, permits regulating the
number of biomolecules (cholera toxin B subunit
in this case) on each QD.

1| Prepare the QD mix: combine 200 pmol avidin and 600 pmol MBP-zb; to this mixture add 100 pmol QD and bring the final volume to 200 μl with 10 mM sodium tetraborate buffer (pH 9.0). QDs are often synthesized and conjugated to specific biomolecules in the investigator’s laboratory. We use CdSe-ZnS QDs, which are rendered water soluble by capping with DHLA as described elsewhere7. Quantum Dot Corporation provides QDs conjugated to avidin for use with biotinylated proteins and antibodies. Evident
Technology offers biotin-conjugated QDs and QDs that can be conjugated to the N or C terminus of a protein. Both suppliers provide QDs conjugated to specific antibodies as well as protocols for conjugating proteins to their QDs.

2| Allow the mixture to stand at 20–25 °C for 15 min.

3| Add an additional 150 pmol MBP-zb to the mixture and let it react at 20–25 °C for a further 15 min.

4| Set up a 500 μl amylose column, equilibrated with 10 mM sodium tetraborate buffer.

5| Load the entire preparation of the QD bioconjugate mix (from step 3) onto the column and wash the column twice with sodium tetraborate buffer.

6| Add 200 pmol of the biotinylated molecule of interest (in the case of avidin-conjugated QDs) or specific antibody (in the case of specific antigen-conjugated QDs) to the column and let it react at 20–25 °C for 30–60 min.

7| Wash the column twice with sodium tetraborate buffer and elute using 10 mM maltose (in PBS or sodium tetraborate buffer) until all the QDs are eluted from the column (QD elution can be easily monitored by placing the column in UV light and monitoring the QD fluorescence of the eluant)7. This approach provides a pure population of conjugated QDs, free of unbound QDs and of the unbound biomolecules.

8| To label live cells, follow option A, Labeling of cell-surface proteins in live cells; for labeling fixed cells, proceed to option B, Labeling of proteins in fixed cells. Bioconjugated QDs are used for the specific labeling of both intracellular and cell-surface proteins4,5. In live cells, however, these approaches permit labeling only of the cell-surface proteins4.

Option A. Labeling of cellsurface proteins in live cells
1| Wash the cells with fresh growth medium.
2| Incubate the cells for 30 min, at either 37 °C or 4 °C, in growth medium containing the appropriate
amount of QDs bioconjugated to biotinylated ligand or antibody (from step 7 above). Incubating the cells at 4 °C will help to minimize endocytic uptake of ligands, antibodies and QD bioconjugates.
3| Remove excess QD bioconjugates by washing the cells two or three times with growth medium or
PBS. When using biotinylated ligand or antibodies, continue with step 4.
 4| Incubate the cells for 10–15 min (at 37 °C or 4 °C) with avidin-conjugated QDs.
5| Remove the excess unbound QDs by washing the cells two or three times with growth medium or
These cells can be monitored live or subsequently fixed using chemical fixatives such as 4%
paraformaldehyde without affecting the QD fluorescence11.
6| To visualize results, proceed to the section Imaging the Labeled Cells.

Option B. Labeling of proteins in fixed cells
1| Fix the cells using appropriate chemical fixative and wash the cells two or three times with PBS.
2| Incubate the cells for 30 min at 20–25 °C in 1% BSA in PBS.
3| Replace the 1% BSA solution with an appropriate amount of QD bioconjugates or biotinylated antibody or ligand prepared in 1% BSA solution and incubate at 20–25 °C for 30–45 min.
4| Wash the cells two or three times with PBS. When using biotinylated ligand or antibodies continue with step 5.
5| Incubate the cells for 10–15 min at 20–25 °C with QD avidin, and then wash two or three times with PBS to remove excess unbound QD avidin.
6| To visualize results, proceed to the section Imaging the Labeled Cells

Imaging the labeled cells
 QDs can be imaged using any type of fluorescence microscope, including epifluorescence, confocal
and multiphoton. However, unlike with conventional fluorophores, a single wavelength of light can be
used to excite several different color QDs. Because most commercially available QDs emit in the green
to red region of the visible spectrum, a microscope capable of providing an excitation beam (from
lamp or laser) in the UV to blue region of the spectrum and capable of resolving multiple emission
wavelengths could be used. As QDs are better excited by UV light, fixed cells can be imaged using
a UV light source. To minimize UV-induced photodamage, live cells should be imaged using a blue
(wavelength >400 nm) excitation light. For two-photon imaging, excitation at 800 nm is optimal, but
any wavelength of light between 700 and 1,000 nm could be used12. The choice of emission filter will
depend on the emission spectrum of the QD in use.

Bioconjugation of QDs, Step 1. To regulate the number of linker molecules (e.g., avidin) on each QD, the molar ratio of MBP-zb to the linker molecule should be altered. Because of their net positive charge, these proteins compete with each other to bind the negatively charged DHLA coat on the surface of the QD. Altering the ratio of these proteins in the mixture facilitates regulating their relative numbers on each QD. Because these proteins bind to QDs in a competitive manner, it is critical that both proteins be mixed thoroughly before addition of QDs. Nonhomogeneous mixing could result in greater variation in the ratio of the two molecules on each QD. The ratio presented here results in an average of one avidin molecule present for three MBP-zb molecules bound to each QD. To optimize the number of linker molecules, a series of QD bioconjugates should be prepared with ratio of linker to MBP-zb above and below the suggested ratio of 1:3. These ratios should be individually tested for specificity and affinity of binding before deciding upon the best QD bioconjugate for cellular labeling.
Imaging the labeled cells. During the course of imaging for all live cell studies, it is recommended that the cells be maintained at 37 °C and not at room temperature (15–25 °C). Although QDs are highly photostable, long exposures to an excitation light source or exposure to UV light can lead to photodamage to the labeled cells. Thus, during long-term imaging, attempts should be made to minimize the length of exposure to excitation light and avoid the use of UV excitation. For multicolor imaging, instead of taking sequential images for each color QD, the emission from each color QD should be acquired simultaneously (if possible) using devices, such as dual view, quad view (Optical Insights), META detector (Zeiss) or AOBS (Leica), that allow simultaneous resolution of different-color QDs.

The approaches described here have been found to be nontoxic both for DHLA-capped QDs and for some commercially available QDs; nevertheless, it is advisable to assess each new system or new QD formulation being used for live cell labeling. Despite the several advantages of QDs, such as their enhanced brightness12 and resistance to metabolic degradation and photodamage, there are a few impediments to their successful use. Two of these are the tendency of QDs to aggregate in the cytosol and the tendency of single QDs to bind
multiple molecules13. As these features limit the application of QDs for imaging molecules in the cytosol of live cells, there is great interest in overcoming them. Other difficulties arising from the physical and chemical properties of currently available QDs are also the focus of ongoing research aimed at developing QDs as routine tools for bioimaging13.

Example of Application
GM1 gangliosides are a group of galactose-containing cerebrosides found in the plasma membranes of neurons and other cells. Cholera toxin B (CTxB) binds these gangliosides, which exist in lipid microdomains called rafts. Fluorescently labeled cholera toxin is thus frequently used to visualize these membrane microdomains. To specifically label the GM1 ganglioside, we used biotinylated CTxB in conjunction with
QD-avidin conjugates9, prepared as described in Bioconjugation of the QDs. Live HeLa cells were labeled by a first incubation with biotinylated CTxB followed by incubation with QD-avidin and Hoechst 3342. Figure 2 shows an image of the lateral membrane staining for GM1 using QDs (in red) and nuclear staining using Hoechst (in blue). Punctuate labeling of the cell surface by QD bioconjugate is typical for molecules such as GM1 that are present in membrane rafts.
Figure 2 | Distribution of
GM1 gangliosides found in
the plasma membrane. Live
HeLa cells growing on a glass
coverslip were incubated for 15
min with biotinylated CTxB and
then for 10 min with QD avidin
(610 nm) and Hoechst 3342.
The fluorescence image through
the middle of a cell shows
lateral membrane staining for
GM1 using QDs (in red) and
nuclear staining using Hoechst
(in blue). The cells were imaged
sequentially using 370/30-nm
excitation and 420/40-nm emission filters for Hoechst and 480/40-nm
excitation and 570-nm long-pass emission filters for QDs.

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