Article pubs.acs.org/ac
Superlocalization Spectral Imaging Microscopy of a Multicolor Quantum Dot Complex Xingbo Shi,† Zhongqiu Xie,† Yuehong Song,† Yongjun Tan,† Edward S. Yeung,† and Hongwei Gai*,‡ †
School of Biology, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan, China 410082 ‡ School of Chemistry and Chemical Engineering, Xuzhou Normal University, Xuzhou, Jiangsu, China 221116 S Supporting Information *
ABSTRACT: The key factor of realizing super-resolution optical microscopy at the single-molecule level is to separately position two adjacent molecules. An opportunity to independently localize target molecules is provided by the intermittency (blinking) in fluorescence of a quantum dot (QD) under the condition that the blinking of each emitter can be recorded and identified. Herein we develop a spectral imaging based color nanoscopy which is capable of determining which QD is blinking in the multicolor QD complex through tracking the first-order spectrum, and thus, the distance at tens of nanometers between two QDs is measured. Three complementary oligonucleotides with lengths of 15, 30, and 45 bp are constructed as calibration rulers. QD585 and QD655 are each linked at one end. The measured average distances are in good agreement with the calculated lengths with a precision of 6 nm, and the intracellular dual-color QDs within a diffractionlimited spot are distinguished.
M
blinking.20,23 For example, in a QD pair, the intensity decreasing to half is a sign that one QD is “off”. Here, there is a prerequisite that the fluorescence intensity of each QD is nearly equal and constant during measurement. Otherwise we cannot exclude the possibility that the variations in fluorescence intensity resulted from other fluctuations in a QD pair such as orientation and energy transfer rather than blinking. Moreover, one cannot tell from which QD the blinking originates, leading to ambiguity in the PSF simulation of each image. Super-resolution optical fluctuation imaging (SOFI)21 is a simple microscopy that correlates the temporal fluctuation in each pixel and identifies the origination of photons by eliminating the low-order correlation contributors to acquire the high-order SOFI image in a series of images. The down side is that the pixels used for image acquisition need to be smaller than the diffraction limit and the positions of the emitters have to remain unchanged during the whole acquisition process. The second requirement calls for high stability of the optics. Very recently, Hell et al.22 were able to recognize the blinking QD markers within a diffraction-limited spot by using asynchronous spectral bluing and realized super-resolution/superlocalization. Unfortunately, this method also failed to uniquely determine which QD was blinking. Furthermore, all of the methods mentioned above can only deal with single-color QD labels. The superior approach of multicolor QD superlocalization imaging is an ongoing project for nanoscopy researchers.
ultiple powerful approaches have demonstrated the possibility of breaking the diffraction limit in optical microscopy, including the ensemble methods of 4Pi microscopy,1 reversible optical linear fluorescent transition,2 the superlens,3 and stimulated emission depletion microscopy,4,5 plus the single-molecule methods of photoactivated localization microscopy (PALM),6 stochastic optical reconstruction microscopy (STORM),7 and photoswitchable nanoparticles.8 The common principle of super-resolution/superlocalization measurements at the single-molecule level is that only one pointlike light source is active at any time, so that the active single molecule or single particle can be localized by fitting its fluorescence intensity profile with a two-dimensional Gaussian function or point-spread function (PSF).9−14 Betzig15 first proposed the concept of superlocalization in 1995. According to this concept, Schmidt et al.16 localized spatial positions of individual pentacene molecules embedded in a p-terphenyl host crystal within a diffraction-limited volume. Quantum dots (QDs) are a class of fluorescent materials that show some interesting optical phenomena such as photobleaching, bluing, and blinking.17 These properties are thought to be detrimental in the study of biological imaging, and attempts have been made to minimize them.18,19 However, the blinking effect of quantum dots offers an opportunity to distinguish the signal from the individual quantum dots in a pair to achieve super-resolution/superlocalization imaging.20−25 The interference between adjacent QDs in fluorescent fluctuation makes it difficult to separately record and identify two neighboring QDs, but recording the changes in the fluorescence intensity of a QD pair is a way to recognize © 2012 American Chemical Society
Received: October 20, 2011 Accepted: January 10, 2012 Published: January 24, 2012 1504
dx.doi.org/10.1021/ac202784h | Anal. Chem. 2012, 84, 1504−1509
Analytical Chemistry
Article
Heinlein et al.26 measured the distance below the diffraction limit between two fluorophores (Bodipy 630/650 and Cy5.5) by their different fluorescence lifetimes and fluorescence emission maxima. In practice, however, the complicated equipment, small spectral window, and difficulties in choosing suitable fluorophore pairs restricted this method's further application. Nie et al.27 proposed a less complex scheme by using a color charge-coupled device (CCD) to split a yellow spot originating from a fluorescent bead pair into green and red and then fitting separate PSFs to localize each. However, the color CCD detector was not suitable for analyzing singlequantum-dot pairs or single-molecule pairs in general due to its low sensitivity. At the same time, the lower spectral resolution of the color CCD resulted in a narrow multiplexed spectral window compared with that resulting from the use a grating. In this paper, we report a spectral imaging nanoscopy approach to measure the distance between two single QDs. Spectral imaging of single molecules or single QDs utilizing a transmission grating was demonstrated in our previous work.18,28,29 A simple transmission grating inserted between the detector and the microscope objective disperses the light into zeroth-order and first-order images. Dual-color or multicolor QD complexes exhibit one overlapped zerothorder spot and two or multiple (depending on the number of colors) distinct first-order streaks. When one of the QDs in the pair is in the off state, its corresponding first-order streak disappears. The remaining zeroth-order spot (in the case of dual color) can be unambiguously identified and thus can be positioned by PSF. This method clearly recognizes which QD is off and makes it possible for multiple images to be accumulated to improve the localization precision.
the carboxyl of the QDs to form an amine-reactive Oacylisourea intermediate which may react with an amine on the oligonucleotide probes, yielding a conjugate of the two parts joined by a stable amide bond. The reaction was continued for 2 h in a sonicator at room temperature. Oligonucleotide-modified QDs were then purified with S-400 microspin columns (AmershamBioscience) at 700g. Hybridization was performed in pH 9.0, 0.1 mM sodium borate buffer. The reactions were carried out by mixing QD585−ssDNAwith QD655−ssDNA at 45 °C for 4 h (the molecular ratio of QD585−ssDNA to QD655−ssDNA was kept at 1:1). After being cooled to room temperature, the dual-color hybridized product (QD585−dsDNA−QD655) was subjected to singlemolecule detection. The three-color hybridized product (QD525−dsDNA−QD585−dsDNA−QD655) was formed from oligo-C, oligo-A3, and oligo-B1. Cell Culture. Human embryo kidney cells (293A cells) were grown on a 25 × 76 mm single concave slide at 37 °C in a 5% CO2 incubator in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). After 4 h of culture, two kinds of QDs (QD585 and QD655) were added into the medium, and the final concentrations were kept at 0.25 nM. After 24 h of incubation, the cells were washed with PBS buffer (pH 7.4) three times. The concave slide was taken out and was ready for imaging. Single-Molecule Fluorescence/Spectral Imaging. An aliquot (4−8 μL) of the dual-color QD complex solution was deposited on the glass slide and immediately covered with a coverslip. To observe a single dual-color QD complex, an epifluorescence microscope (Olympus BX51) with a 100× oil immersion objective (numerical aperture (NA) of 1.45, UPLSAPO, Olympus, Tokyo, Japan) and an electron-multiplied charge-coupled device (EMCCD; DV887, Andor Technology, North Ireland) were used. The gain was set at 200, and the camera was maintained at −60 °C. The exposure time was set at 0.1−0.5 s. A mercury arc lamp was used to excite the dual-color QD complex at different wavelengths using different filter sets. All filter sets were purchased from Semrock (Rochester, NY). A transmission grating with 70 lines/mm (Edmund Scientific, Barrington, NJ) was placed in front of the EMCCD camera to disperse the fluorescence of each QD. A long-pass filter (Figure 1E) was chosen to record the dual-color emission of the QD complexes. Data Analysis. The center of the QD was located by a twodimensional Gaussian fit:9,13,32
■
EXPERIMENTAL SECTION Materials. QDs (carboxyl Qdots 525, 585, and 655 ITK) were purchased from Invitrogen/Molecular Probes (Eugene, OR). 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was obtained from Bio Basic Inc. (Canada). Seven oligonucleotide probes were designed in-house and purchased from Shanghai Sangon Biological Engineer Technology & Service. (Sangon, China). They are oligo-A1 (5′NH2-C12-TCAGG CTATA ATGGC-3′), oligo-B1 (3′-AGTCC GATAT TACCG-C12-NH2-5′), oligo-A2 (5′-NH2-C12-CATAA AAGAG CTCCA TATCC AACCT GCACG-3′), oligo-B2 (5′NH 2 -C 12 -CGTGC AGGTT GGATA TGGAG CTCTT TTATG-3′), oligo-A3: (5′-NH 2 -C 12 -CATAA AAGAG CTCCA TATCC AACCT GCACG TCAGG CTATA ATGGC-3′), oligo-B3 (5′-NH 2 -C 12 -GCCAT TATAG CCTGA CGTGC AGGTT GGATA TGGAG CTCTT TTATG-3′), and oligo-C (5′-NH2-C12-ATATG GAGCT CT3′). All oligonucleotides were purified by high-performance liquid chromatography. Microscope coverslips (0.17 mm thick, 22 × 22 mm) were purchased from Fisher Scientific (Fisherbrand), and glass microscope slides were obtained from Shitai Experiment Equipment (Shitai, China). Ultrapure water was prepared using a Milli-Q purification system (Millipore). Human embryo kidney cells (293A) were purchased from Invitrogen. Other chemical reagents were of analytical grade and purchased from local reagent suppliers. Sample Preparation. Oligonucleotides were covalently conjugated to quantum dots using the carboxyl-linked amino method.30,31 QDs (8 μM, 4 μL) were mixed with 3.2 μL of DNA (100 μM), and then carbodiimide cross-linking agent EDC (58.6 μg) was added into the mixture. EDC reacts with
⎡ ⎛ y − y ⎞2 ⎤ ⎛ x − x 0 ⎞2 ⎢ 0⎟ ⎥ Ixy = B + I00 exp⎢ − 0.5⎜ ⎟ − 0.5⎜⎜ ⎟ ⎥ σ σ ⎝ x ⎠ ⎝ y ⎠ ⎦ ⎣ where (x0, y0) is the centroid of the point source, Ixy and I00 are the intensities at pixels (x, y) and (x0, y0) on the CCD detector, σ is the width of the point spread function in the x and y directions, and B is the background. The surface fitting tool in MATLAB.R2009a was used to localize the centroids of the dual-color QD pair. After the positions of the differently colored QD centroids were obtained, the distances between the QDs were calculated.
■
RESULTS AND DISCUSSION Since double-stranded DNA segments shorter than the persistent length (
