Quantum dot imaging platform for single-cell molecular profiling

Abstract

Study of normal cell physiology and disease pathogenesis heavily relies on untangling the complexity of intracellular molecular mechanisms and pathways. To achieve this goal, comprehensive molecular profiling of individual cells within the context of microenvironment is required. Here we report the development of a multicolour multicycle in situ imaging technology capable of creating detailed quantitative molecular profiles for individual cells at the resolution of optical imaging. A library of stoichiometric fluorescent probes is prepared by linking target-specific antibodies to a universal quantum dot-based platform via protein A in a quick and simple procedure. Surprisingly, despite the potential for multivalent binding between protein A and antibody and the intermediate affinity of this non-covalent bond, fully assembled probes do not aggregate or exchange antibodies, facilitating highly multiplexed parallel staining. This single-cell molecular profiling technology is expected to open new opportunities in systems biology, gene expression studies, signalling pathway analysis and molecular diagnostics.

Figure 1. Schematic illustration of the M3P technology.

(a) A universal QD-SpA platform is used for single-step purification-free assembly of functional QD-SpA-Ab probes via capture of free antibodies from solution by SpA. (b) Once bound, Abs cannot be exchanged between QD-SpA probes, thus enabling mixing of multicolour probes within a single cocktail. (c) The QD-SpA-Ab cocktail is used for single-step parallel multiplexed staining. (d) Spectral imaging is performed for unmixing individual QD colours, quantitative analysis of target expression and depiction of relative target distribution within the specimen. (e) Complete de-staining restores specimen for another staining cycle. (f) Sequential repetition of N-colour parallel staining for M cycles enables analysis of N × M molecular targets.

Figure 2. Characterization of QD-Ab probe specificity.

(a) Five model targets (Ki-67, HSP90, Lamin A, Cox-4 and β-tubulin, from left to right) are labelled with QD585-SpA-Ab probes in a single-step procedure, producing staining patterns consistent with those obtained with either QD565-labelled (b) or Alexa Fluor 568-labelled (c) 2′Abs in a conventional two-step staining. Scale bar, 50 μm.

Figure 3. Characterization of QD-SpA-Ab probe stability.

(a) As QD-SpA can capture free Abs from solution, when anti-Lamin A IgG, QD545-SpA and QD585-SpA are mixed simultaneously and immediately applied to cells, both probes successfully capture Ab and produce Lamin A staining with ~50% contribution from each (a,d). At the same time, regardless of whether QD545 (b) or QD585 (c) is pre-assembled with anti-Lamin A antibody, only the pre-assembled probe shows specific nuclear envelope staining, solely contributing to all fluorescence signal registered (d). Furthermore, presence of a competitor QD-SpA probe does not interfere with target staining by the pre-assembled QD-SpA-Ab probe, as observed signal intensity in a two-probe mixture is comparable to that obtained with single-colour staining experiments in the absence of a competitor probe (e). HSI is used to unmix true-colour images (left panels) into individual QD545 (middle panels) and QD585 (right panels) channels, remove background and perform quantitative analysis of staining intensity. Brightness of individual QD channels is normalized to aid in direct comparison of staining intensity. Intensity of the QD545 channel is scaled up by a factor of 2 relative to QD585 channel to compensate for differential brightness of QD probes. Error bars represent s.d. of the average staining intensity between three different fields of view imaged on the same specimen. FL intensity denotes fluorescence QD signal intensity measured by HSI. Scale bar, 50 μm.

Figure 4. Parallel multiplexed staining with five pre-assembled QD-SpA-Ab probes.

Ki-67, HSP90, Lamin A, Cox-4 and β-tubulin are simultaneously stained with QD-SpA-Ab probes emitting at 525, 545, 565, 585 and 605 nm, respectively (a). HSI and spectral unmixing are used to extract individual QD channels (b), perform quantitative analysis of staining intensity (d) and reconstruct a false-colour composite image (a) depicting relative distribution and colocalization of different targets. Staining patterns obtained in this manner (b) are consistent with single-colour staining performed with colour-matched QD-SpA-Ab probes (c). Furthermore, average staining intensities for five model targets measured with multiplexed staining (five-colour bars in d) are consistent with quantitative analysis performed with singleplexed staining using either colour-matched QD-SpA-Ab probes (single-colour bars in d) or reference singleplexed staining performed with same QD585-SpA probe for all targets (reference bars in d). Individual channels (b) are false-coloured and fluorescence intensity of individual channels is automatically adjusted by HSI software to achieve clear target representation in a composite false-colour image (a). For quantitative analysis (d), fluorescence intensity of individual QDs is normalized to reference QD585 using appropriate correction factor (Supplementary Fig. S3). Error bars represent s.d. of the average staining intensity between three different fields of view imaged on the same specimen. FL intensity denotes fluorescence QD signal intensity measured by HSI. Scale bar, 50 μm.

Figure 5. Specimen regeneration and target re-staining with multicycle staining procedure.

(a) Characteristic nuclear envelope staining is obtained with QD545-SpA probes pre-assembled with anti-Lamin A antibody. Brief incubation with regeneration buffer removes QD-SpA-Ab probes, achieving specimen restoration to pre-staining condition and enabling nearly complete target re-staining (b,d). At the same time, incubation of regenerated specimen with ‘blank’ QD545-SpA probes (lacking target-specific Abs) fails to produce any residual staining (c,d), confirming complete removal of 1'Abs during de-staining. Same subset of cells is imaged, normalized by intensity and false-coloured with a heat map for this study. (e–l) Further application of the de-staining/re-staining procedure to dual-colour cell labelling enables complete exchange of QD reporters between two different molecular targets. Specifically parallel staining of Lamin A with QD545 and HSP90 with QD585 is achieved (e–h). HSI reveals distinct Lamin A staining pattern in QD545 channel (f) and HSP90 pattern in QD585 channel (g). Following de-staining, during the second cycle the same targets are stained with the counterpart QD probes (i–l), yielding clear HSP90 pattern in QD545 channel (j) and Lamin A pattern in QD585 channel (k). (e) and (i) are true-colour images and (h) and (l) are false-colour composite images. Fluorescence intensity of individual channels is adjusted to achieve clear target representation in composite false-colour images. Same subpopulation of cells is imaged after each step to aid in direct comparison of staining patterns. To quantitatively assess effect of repeated regeneration procedure on specimen stability and staining intensity, up to 10 ‘degradation’ cycles are performed on separate specimens before cells are stained for Lamin A (m) or HSP90 (n). For both targets, measured average fluorescence staining intensity does not vary significantly regardless of the number of ‘degradation’ cycles performed, demonstrating sufficient preservation of specimen antigenicity required for cyclic staining. Error bars represent s.d. of the average staining intensity between four different fields of view imaged on the same specimen. FL intensity denotes fluorescence QD signal intensity measured by HSI. Scale bar, 50 μm.

Figure 6. Evaluation of the robustness of sequential staining procedure.

Single-colour staining of five molecular targets (Lamin A, HSP90, Ki-67, Cox-4 and β-tubulin) on the same cell subpopulation is performed using QD545-SpA-Ab probes in sequential manner in the order from highly condensed to diffusely distributed targets (Lamin A to β-tubulin, a–c) and in reverse (β-tubulin to Lamin A, d–f). Quantitative analysis of target staining intensity for both sequences in comparison with reference single-cycle single-colour staining performed with QD585-SpA probes is shown in (g). Independent of the order, all targets are reliably stained, showing correct staining pattern and relative staining intensity. No carry-over fluorescence, build-up of background fluorescence or cross-staining between cycles can be observed. Original images obtained with HSI (a,d) are false-coloured, aligned and cropped (b,e) for clear representation of each target in composite false-colour images (c,f). Error bars represent s.d. of the average staining intensity between three different fields of view imaged on the same specimen. FL intensity denotes fluorescence QD signal intensity measured by HSI. Scale bar, 50 μm.

Figure 7. Validation of M3P technology with 25-target staining.

Utility of M3P technology for highly multiplexed molecular profiling is demonstrated by re-staining the same set of five model molecular targets (Ki-67, HSP90, Lamin A, Cox-4 and β-tubulin labelled with QD-SpA-Ab probes emitting at 525, 545, 565, 585 and 605 nm, respectively) through five cycles (a–e) and performing quantitative analysis of average staining intensity for each target at every cycle (f). Qualitative evaluation of individual QD channels (columns 1–5 in a–e) as well as composite images (column 6 in a–e) of the same cell subpopulation imaged after every cycle shows robust re-staining of each target with precisely preserved subcellular morphology, whereas quantitative analysis (f) demonstrates consistency of target staining profiles throughout all cycles. Note that complete specimen regeneration is achieved after each cycle, leaving no detectable fluorescence signal (Supplementary Fig. S7), thus ensuring that observed staining is generated by incubation of cells with a new QD-SpA-Ab cocktail during each cycle. Error bars represent s.d. of the average staining intensity between three different fields of view imaged on the same specimen. FL intensity denotes fluorescence QD signal intensity measured by HSI. Scale bar, 50 μm.