Fluorescent Imaging in Cell Biology. Invitrogen Cellular Analysis
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1 Fluorescent Imaging in Cell Biology Invitrogen Cellular Analysis
2 Topics Fluorescent Dyes and Dots Cellular Structure. Multicolor Immuno-Labeling. rganelle Stains Cellular Physiology Intracellular Ions Viability Assays/Vital Stains Cellular processes Endocytosis Division Migration
3 The Power of Fluorescence High Sensitivity Single molecule to macro scale detection Multiplexing Multiple targets in single sample. Live Cell Compatible Main detection system for live cell studies.
4 Classic Dyes Cascade Blue (399/421) Dansyl (335/515) Aminomethylcoumarin (345/445) Lucifer Yellow (428/544) BDIPY FL (503/512) DiIC (550/565) 18 Sulforhodamine 101 (595/615) Fluorescein (490/520) Tetramethylrhodamine (555/580) Larger molecule Longer wavelength
5 Green Fluorescent Protein (GFP)
6 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
7 Alexa Fluor Dye Conjugates
8 The Alexa Fluor family of dyes First and foremost- decent primary antibody. 19 structurally homologous dyes. High molar absorption. (ε max ). High Quantum yields. Low self quenching. High photostability. Insensitive to ph. Excitation maximums tailored to common excitation sources.
9 Alexa Fluor Family members
10 SpectraViewer
11 Just a note : Antifade Reagents ProLong Gold and SlowFade Gold Antifade Reagents ptimized formulation for widest range of dyes. Superior antifade with little or no impact on initial fluorescence intensity. Ready to use - dropper bottle. With Prolong Gold Without Prolong Gold
12 Prolong Gold: More than just bleach protection. Prolong Gold does not affect the initial fluorescence intensity.
13 Just a Note: Image-iT FX Signal Enhancer No Block Blocked with Image-IT FX Blocking Solution 16um Mouse Hippocampus Section, No primary, AF488 GAM Image-IT FX Signal Enhancer suppresses non-specific background
14 Cellular Structure Where it s at? Principal method: Fixed Cell Immunofluorescence Use primary antibody to detect target Use conjugated secondary antibody to detect primary. Fluorescent dyes with intrinsic binding properties (both live and fixed cells). Nucleic acid stains rganelle stains Membrane stains ther fluorescent conjugates Lectins Toxins Receptor Ligands Target Fluorescent Protein Expression systems. rganelle Lights.
15 Antibody Labeling Anti-paxcillin Alexa Fluor 488, Alexa Fluor 594 Phalloidin, DAPI
16 rganelle Antibodies Anti-Calreticulin detected with Alexa Fluor 488 secondary, Mitotracker Red and DAPI
17 Cell Surface Receptors Alexa Fluor 488 Transferrin, Receptor detected with Alexa Fluor 555 secondary and Hoechst
18 Plasma Membrane Stains (Above) Live 293 MSR cells labeled for 1 min in complete media, washed, and imaged Label maintained in PM for minutes before internalization Formaldehyde-fixable (not shown in this slide) but not detergent resistance Near 100% labeling of cells after 1-10 min labeling
19 Lectin Conjugates: Plasma Membrane Labeling Wheat Germ Agglutinin Alexa Fluor 594
20 Multicolor Detection
21 Live Cell rganelle Labeling Live BPAE cells incubated simultaneously with MitoTracker Deep Red FM, LysoTracker Green DND-26, and Hoechst 33342
22 Live Cell Nucleic Acid Labeling SYT 13 Live-Cell Nucleic Acid Stain
23 rganelle Lights How they really work Baculovirus with only one gene that mammalian cell can transcribe Following cellular entry and transport to nucleus, GFP-construct is expressed Depending on targeting tag, the fusion proteins traffics to the specified organelle or sub-cellular structure
24 rganelle Lights rganelle Lights Cytoplasm Endoplasmic Reticulum Endosomes CFP GFP YFP FP RFP (440/480) (488/510) (514/528) (548/565) (555/584) Golgi Lysosomes Mitochondria Nuclear Envelope Nucleus Peroxisome Plasma Membrane Synaptophysin Null virus (control) C10080 C10130
25 rganelle Lights rganelle Markers Mito-GFP ER-FP BacMam virus delivered expression of organelle targeted FPs
26 Cellular Lights Cellular Lights CFP GFP YFP FP RFP (440/480) (488/510) (514/528) (548/565) (555/584) Actin C10126 C10127 CFS1r Exo1 C10078 C10079 Histones C10128 C10129 MAP4 C10105 Tubulin C10106 C10112 Null virus (control) C10130
27 Cell Physiology Intracellular Ions Calcium Hydrogen Ions (ph) Sodium Heavy metals Viability/Vitality LIVE/DEAD Assays Metabolic indicators.
28 How ion Indicators Work Ion-dependent spectral shift Ion-dependent fluorescence increase, no spectral shift Examples Fura-2 Indo-1 Premo Cameleon Dye Dye Examples Fluo-3/Fluo-4 Rhod-2 Calcium Green regon Green 488 BAPTA Ca 2+ Ca 2+
29 What is an Indicator? Cl C Cl CCH2 DYE NH Magnesium Green C N(CH 2 C ) 2 CHELATR DYE= dichlorofluorescein DYE NH CH 3 Calcium Green -1 (X=H) Calcium Green -5N (X=N 2 ) DYE NH X CH 3 N Sodium Green ( CCH 2 ) 2 N CH 2 CH 2 N(CH 2 C ) 2 CH 3 N NH CH 3 DYE
30 What s a Biosensor? A biological element (protein) that has been rendered sensitive to an external signal Naturally occurring (e.g. light-sensitive pigments in the eye) Constructed (e.g. blood glucose biosensor) Can be targeted to cell type or organelle Based on naturally occurring fluorescent signal proteins (e.g. GFP) and detector proteins (e.g. calmodulin) P36207 Premo Cameleon Calcium Sensor
31 Biosensors: Premo Cameleon calcium indicators Adult stem cell from left atrial appendage of pig heart stimulated with 20 um ATP Image obtained in collaboration with Drs. Michael Rutten and Kenton Gregory, regon Medical Laser Center Bioimaging Suite, Providence St. Vincent Hospital, Portland, regon
32 Tools for Cell Health Fluorescence-based cell viability assessment methods use various criteria including Mitochondrial potential detection, Membrane integrity, Intracellular enzyme activity. To control for method dependence, two assays with different assessment criteria can be run in parallel using wavelength-multiplexed detection Bacteria, yeast and mammalian cells require different detection methods
33 Viability/Cytotoxicity Assays (CH CCH CCH ) N N(CH CCH CCH ) CH CH 2 2 CH C CCH 3 3 calcein AM nonpolar nonfluorescent esterase ( CCH ) NH NH(CH C - ) CH CH C - calcein polar green fluorescent H 2 N NH 2 H 2 N NH 2 N+ + N + + (CH ) NH CH CH NH (CH ) ethidium homodimer-1(ethd-1) membrane impermeant red fluorescence enhanced on DNA binding
34 Viability assays using two colors 3-D rendition of rat lung stained with calcein AM (live cells) and EthD-1 (dead cells). Lance Rodenkirch, University of Wisconsin
35 Cell Vitality using Mitochondrial Stains. Total dye concentration in all three images is the same + 1 µm FCCP Self-quenching of rhodamine 123 (R302) in rat cortical astrocytes
36 Cellular Processes Cell Division and Proliferation Click-iT EdU for cell division Phagocytosis and Endocytosis phrodo particles FM dyes Cell Migration and movement QTrackers CellTrackers
37 Nucleoside analogue structures H Click-analogue Ethynyl du Bromo du
38 A better way to detect new DNA synthesis
39 Click-iT EdU vs. BrdU Workflow Incubate with EdU or BrdU, fix & permeabilize sample Click-iT EdU Protocol Click-iT detection reaction Wash 2X Nuclear counterstain Wash 3X Image With Click-iT EdU 30 minutes 10 minutes 15 minutes 15 minutes TTAL TIME 70 minutes Measure proliferation in cells or tissue Time to complete: <2 hours Detect by fluorescence microscopy, flow cytometry or high-throughput imaging (HCS) BrdU Protocol Wash 3X HCl Denaturation Neutralize Wash 3X Block Anti-BrdU incubation Wash 3X Secondary antibody incubation Wash 3X Nuclear counterstain Wash 3X Image 15 minutes 40 minutes 12 minutes 15 minutes 60 minutes 1-16 hours 15 minutes 120 minutes 15 minutes 15 minutes 15 minutes TTAL TIME 6-21 hours
40 Phagocytosis Assays using phrodo Particles.
41 phrodo particles for phagocytosis
42 Cell migration using Qtracker Cell Labeling 10x 3T3(green), HeLa(red), and U188(white) cells labeled with Qtracker 565, 655, and 705 respectively. Co-cultured in 8-well chambers for 24 hrs. Images captured with a Leica Confocal microscope (ex. = 488nm).
43 CellTracker Dyes
44 Summary Fluorescence imaging is a powerful technique for probing cellular structure and activity. Multiple markers can be used in a single specimen to extract more data. Fluorescence probes are key tools for live cell imaging applications.
45 THANK YU! Questions?
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