QDot Nanocrystals Insights

Transcription

1 QDot Nanocrystals Insights Invitrogen Corporation Eugene, OR. HeLa cell labeled with three Qdot conjugates for nucleus, Golgi, and tubulin

2 What are quantum dots? Highly fluorescent, nanometer-size, crystals of semiconductor materials Size of the nanocrystal determines the color Size is tunable from ~2-10 nm (± 3%) Size distribution determines emission band width

3 Anatomy of a Qdot Nanocrystal

4 Quantum Dot Benefits Feature Bright, photostable Large Stokes Shift Same excitation range (UV- Violet, 405 nm laser) Inert and Biologically stable Emissions across the spectrum, 525, 545, 565, 585, 605, 625, 655, 705, 800 nm Benefit Ultrasensitive, even single molecule detection possible. Avoid autofluorescence and can be used with other fluorescence markers. Multiplexing off single excitation source. Long term in vivo tracking. Small animal in vivo imaging. Many choices for multiplexing and multiparameter labeling.

5 Spectral Properties Organic dye (FITC) Qdot Conjugate (525) Small Stokes shift Large Stokes shift Multiple source excitation req d. Single-source excitation Broad emission Narrow emission

6 Excitation Spectra of Qdot Conjugates High extinction >> High brightness All colors can be excited at the same wavelength, 425DF45

7 Emission spectra of Qdot Conjugates Minimal (<5%) cross-talk using 20nm bandpass filters Simplified signal un-mixing >> simplified multiplex labeling Narrow Symmetrical Emission Spectra

8 Photostability of Qdot Conjugates Top panel (a-e): Nucleus labeled with Qdot conjugates and microtubules labeled with Alexa Fluor 488 Bottom panel (f-j): Nucleus labeled with Alexa Fluor 488 and microtubules labeled with Qdot conjugates Left: quantitative data showing effect of antifade medium Wu, X., et al. Nature Biotechnology, 21(1) 2003.

9 Photostability of Qdot Nanocrystals Qdot Nanocrystals vs. Organic Dyes -- Comparative Photobleach Rates % % Cyto QD525 Avg Percent Orig Intensity % % 80.00% 60.00% 40.00% Cyto AF555 Cyto QD % 0.00% Cyto AF488 00: : : : :52.8 Time (min)

10 Challenges of Using Dots Dots are relatively large discrete objects Steric issues Penetration issues? Valency Blinking Mounting of fixed specimens

11 Size of Qdot Nanocrystals

12 Size of Qdot Nanocrystals (continued) Images provided by Mark Ellisman, National Center for Microscopy and Imaging Research, UCSD, San Diego, CA

13 Size of Qdot Nanocrystals (continued)

14 Degree of Labeling Valency Typical Fluorescent Dye Conjugate: 145,000 D IgG antibody Each Fluorophore is between D Usually 3-5 dyes per IgG Depending on protocol may have several IgGs per dot

15 Blinking of Qdot Nanocrystals

16 Applications of Quantum Dot Nanocrystal technology In vivo Imaging Live cell tracking Imaging Flow Cytometry Western Dots

17 Antibody Conjugation Kit: Hinge Chemistry Quantum dot activation with SMCC Antibody reduction with DTT Removal of excess SMCC Removal of excess DTT React Quench Size exclusion

18 Kit Components in order of use

19 Qdot Nanocrystals for Cell and Tissue Imaging Image of cerebellum labeled for glial fibrillary acidic protein using Qdot 655 (red), inositol triphosphate receptor using Qdot 525 (green), and nuclear labeling using DAPI. Image provided by Mark Ellisman, UCSD.

20 Comparable Label Quality to Organic Dyes Alexa Fluor 488 Goat Anti-Rabbit Qdot 525 Goat Anti-Rabbit

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22 Multiplex Labeling Using Qdot Conjugates Tubulin labeled with Qdot 525 streptavidin Golgi bodies labeled with Qdot 585 goat anti-rabbit Nuclei labeled with Qdot 655 goat anti-mouse Tubulin labeled with Qdot 525 streptavidin Plasma membrane labeled with Qdot 655 wheat germ agglutinin conjugate Existing Demo Slide

23 Qdot 655 Wheat Germ Agglutinin for Plasma Membrane Labeling WGA AF488 WGA QD655 Combo Live HeLa cells labeled with 5ug/ml of Alexa Fluor 488 WGA and 20nM Qdot 655 WGA, then imaged with confocal microscopy.

24 Spectral Unmixing with QDot Nanocrystals If you can unmix 3 QDot conjugates, why not six (or more)? 1) (Tubulin) QDot 525 Streptavidin / GRat Biotin / Rat anti-a-tubulin 2) (Fibronectin) QDot 565 Goat anti-chicken (whole) / chicken anti-fibronectin 3) (Golgi) QDot 585 Goat anti-rabbit / rabbit anti-giantin 4) (Nuclei) QDot 605 Goat anti-mouse / mouse anti-nucleosome 5) (Plasma Membrane) QDot 655 Wheat germ agglutinin 6) (Mitochondria) QDot 800 Streptavidin / endogenous biotin

25 Spectral Unmixing with QDot Nanocrystals Tubulin Golgi Fibronectin Nuclei Plasma Membrane Combo

26 Her-2 in Breast Cancer

27 Correlative light and electron microscopy

28 Correlative light and electron microscopy

29 Qtracker Cell Labeling Kits Live HeLa cells with Qtracker 525 cell labeling reagent (green, endosomes) and wheat germ agglutinin Qdot 655 (red, plasma membrane and endosomes); confocal microscopy image. Live U2OS cells with Qtracker 655 cell labeling reagent (red, endosomes); Emerald GFP on both mitochondria and nuclei (pseudocolored blue); YFP on plasma membrane (pseudocolored greenyellow); inverted widefield microscopy image.

30 Three color Qtracker cell labeling 10x 3T3 (green), HeLa (red), and U188 (white) cells labeled with Qtracker 565, 655, and 705 reagents, respectively Co-cultured in 8-well chambers for 24 hours without cross-over Images captured with a Leica confocal microscope (488 nm excitation)

31 Qdot Nanocrystals for In Vivo Imaging Qtracker non-targeted quantum dots 800 labeling mouse vasculature in vivo. Image was obtained using the CRi Maestro imaging system.

32 Finding Fluorescent Needles in the Cardiac Haystack: Tracking Human Mesenchymal Stem Cells Labeled with Quantum Dots for Quantitative in Vivo 3-D Fluorescence Analysis. Rosen et al., 2007, Stem Cells, online.

33 In Vivo Tumor Imaging Anti-CEA-AlexaFluor 680 Composite Qtracker 800 non-targeted quantum dots Combining Blood Flow with Targeting

34 Qdot Nanocrystals for Western Blot Imaging

35 Chemiluminescence vs WesternDot Workflow ECL on Film Transfer (1 hr) Block (1 hr) Primary (1 hr) Rinse (0.25 hr) Secondary (1 hr) Rinse (0.25 hr) Substrate, Film, Develop (1 hr) 5.5 Hours WesternDot 625 Transfer (1 hr) Block (1 hr) Primary (1 hr) Rinse (0.25 hr) Secondary (1 hr) Rinse (0.25 hr) Qdot 625 streptavidin (0.5 hr) Rinse (0.25 hr) 5.25 Hours So How Much Is Your Time Worth?

36 WesternDot 625 vs. ECL plus Invitrogen WesternDot 625 with UV epi-illumination, 620/40 bandpass emission filter and 10 second exposure Target protein biotin PVDF western blot hν ox substrate H 2 O 2 substrate GE-Amersham ECL Plus and 15 minute exposure HRP Target protein biotin Biotinylated MW marker dilution series PVDF western blot

37 Increased Sensitivity of Chemiluminescence Detection of GAPDH. 2-fold dilution series of a Jurkat cell extract. 11 lane series. Lane 1: 2.5 ug of extract. Lane 11: ~ 2.5 ng of extract WesternDot 625 ECL-Plus 254 nm epi-illumination 5 seconds PVDF membrane 10 minutes PVDF membrane Increased sensitivity with longer exposures, but loss of signal ( burnout ) at high protein concentration

38 Detecting Western Blots Westernbreeze Chromogenic Detection Instrument Eyesight Film Gel imager UV-illumination Color WesternDot 625 Fluorescence* ** NOVEX ECL Chemiluminescence Westernbreeze Chemiluminescent Chemiluminescence * Can be efficiently excited from ~254 nm 488 nm and detected with ethidium bromide filters ** May not be able to detect lowest protein levels with eyesight alone.

39 Qdot Nanocrystals for Flow Cytometry

40 Qdot Nanocrystals in Flow Cytometry Fluors for 488 nm and 633 nm excitation Ability to add 1-2 fluors for each laser 405 nm excitation Easy addition of 2-8 fluors Qdot conjugates and their properties Comparable intensity to best organic dyes Narrow emission spectra of the Qdot nanocrystals facilitate use of more fluors Selected Qdot reagents exhibit minimal spectral overlap within laser; must compensate across lasers Can be used with common sample preparation reagents Easily multiplexed for multicolor analysis in single- and multi-laser systems, including substitution for tandem dyes

41 Qdot Direct Conjugates for Flow Cytometry Gating: lymphocytes Gating: lymphocytes Gating: leucocytes Unstained Lymphocytes Unstained Lymphocytes Lymphocytes Monocytes Human PBL stained with conjugates at nm nanocrystals

42 Resolving Difficult Populations hpbl: biotin-cd19 + Qdot 655-streptavidin RPE-Cy7-CD4 biotin-cd152 + Qdot 605-streptavidin PerCP-Cy5.5-CD3 Courtesy of Ian Dimmick, Northeast England Stem Cell Institute CD19-biotin + Qdot 655-streptavidin

43 Qdot Nanocrystals versus Tandem Dyes CD3 CD4 CD8 Qdot 655 Antibody Conjugate PE-Cy5 Antibody Conjugate

44 Multi-Color Use: 6 Color Stain (655/20) 1.4% (655/20) 22.1% (605/20) 41.6% 2.5% 3.5% 5.4% (605/20) (670/14) (620/10) (655/20) 4.6% (655/20) 0.2% (620/10) 33.2% 3.1% 0.3% 11.8% (620/10) (575/26) (575/26) Human PBL, gated on lymphocytes by CD45 / SSC

45 Qdot Conjugate Portfolio Human Mouse Isotype Streptavidin

46 General Summary Quantum dot conjugates offer new possibilities for fluorescent applications due to brightness, spectral properties, and photostability. Qtrackers cell labeling reagents offer an enhanced ability to track cells. There is a wide range of conjugates and colors available (primary antibodies for both microscopy and flow cytometry, secondary antibodies, streptavidins, wheat germ agglutinin) as well as ITK kits for making your own modifications. Conjugation kits are available for those needing the flexibility to conjugate their own antibodies. Our Qdot nanocrystals have been shown to have a wide range of utility, including microscopy of cells and tissues, cell tracking, flow cytometry, Western blot detection, and in vivo imaging.

47 THANK YOU! Questions? A four-qdot combination of antibodies on HeLa cells, labeling nuclei, Golgi, tubulin, Invitrogen and Proprietary mitochondria & Confidential