Ultrahigh-resolution multicolor colocalization of single fluorescent nanocrystals

Abstract

A new method for in vitro and possibly in vivo ultrahigh-resolution colocalization and distance measurement between biomolecules is described, based on semiconductor nanocrystal probes. This ruler bridges the gap between FRET and far-field (or near-field scanning optical microscope) imaging and has a dynamic range from few nanometers to tens of micrometers. The ruler is based on a stage-scanning confocal microscope that allows the simultaneous excitation and localization of the excitation point-spread-function (PSF) of various colors nanocrystals while maintaining perfect registry between the channels. Fit of the observed diffraction and photophysics-limited images of the PSFs with a two-dimensional Gaussian allows one to determine their position with nanometer accuracy. This new high-resolution tool opens new windows in various molecular, cell biology and biotechnology applications.

Keywords: Superresolution; confocal; diffraction limit; fluorescence; microscopy; quantum dot; semiconductor nanocrystal; single molecule.

Figure 1

Setup used for ultrahigh-resolution colocalization of multicolor fluorescent probes. The microscope is a homemade stage-scanning confocal microscope using a nanometer-resolution closed-loop piezo-stage scanner (PS). A single laser line is brought via a fiber and a beam expander (BE) to the back focal plane of the objective (Ob) after reflection on a dichroic mirror (DC). Emission of the fluorescent probes is collected by the same objective, and either sent to a dual-channel recording arm using avalanche photodiodes (APD), or to a full-spectrum recording arm using a prism and an intensidifed CCD camera (ICCD). Sh: Shutter, TL: Tube lens, PH: Pinhole, TM: Tiltable Mirror, L: lens, LP: Longpass Filter, BP: Bandpass Filter.

Figure 2

Normalized bulk emission spectra of 4 different NC samples (large dots) obtained with a standard spectrofluorimeter, superimposed with individual nanocrystals spectra (thin curves) as obtained with our multicolor imaging setup using the integrated data of 11×11 pixels and an integration time of 100 ms per pixel (200 nW incident excitation power). FWHM of bulk spectra are of the order of 30 nm: 34, 37, 34 and 32 nm, respectively for the 540, 575, 588 and 620 nm emissions. Peak ± FWHM of individual spectra from left to right (in nm): 537 ± 16, 565 ± 24, 578 ± 15, 598 ± 18, 605 ± 20, 627 ± 24.

Figure 3

Multicolor imaging of a mixture of 4 NC batches (bulk spectra as indicated in Fig. 2). The area corresponds to a 3 × 3 μm² scan (bar indicates 1 μm) using 128 steps. Incident laser power: 200 nW, integration time: 100 ms per pixel. 4 spectral bands were selected as discussed in the text and false-colored (green: 540 ± 6, red: 571 ± 7, blue: 594 ± 8, orange: 643 ± 9). Some overlapping NCs are clearly visible in this contrast-enhanced image (case a, spectrum: 586 ± 6 nm), as well as cases of nanocrystals appearing simultaneously in the blue and red false-color channels (violet, case b, spectra: 546 ± 10 and 602 ± 10 nm).

Figure 4

Spectra of two different 11×11 pixels area of Fig. 3. Recorded spectra are displayed as scatter plots, with the best Lorentzian fits indicated by continuous curves. Boundaries of spectral bands used to build the false-color image planes of Fig. 3 are indicated by dashed vertical lines. Case a correspond to two nearby NCs whose PSFs overlap over most of their physical extension, but with clearly separated spectra (and different emission intensity). Case b corresponds to a single NC whose spectrum overlaps the two chosen bands used to build the red and blue image plane.

Figure 5

Repeated dual-color imaging of the same 2×2 μm² area of a mixture of green and red NCs, excited at 488 nm (incident excitation power: 200 nW, integration time: 50 ms, pixel size: 50 nm). The overall scan duration is about 80 s and the next scan is started almost immediately. Bar indicates 500 nm. The measured distance in each case is indicated with its corresponding 95 % confidence limit error bar as obtained with 1000 bootstrap simulations. Scan d gives a single stripe of signal for the red nanocrystal, so that no position can be determined for this NC. In subsequent scans, this NC gives more complete PSF images.