cell and tissue imaging by fluorescence microscopy

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1 cell and tissue imaging by fluorescence microscopy Steven NEDELLEC Plateforme Micropicell SFR Santé François Bonamy Nantes 1

2 A matter of size Limit of resolution 0.15mm aims: building the image of an object using photons (or electrons) enlarge the image size thanks to lens 2

Fluorescence and fluorochromes Jablonski diagram Fluorochrome (also termed fluorophore), is a fluorescent chemical compound that can re-emit light upon light excitation.

3 Fluorescence and fluorochromes Jablonski diagram Fluorochrome (also termed fluorophore), is a fluorescent chemical compound that can re-emit light upon light excitation. The emission of light through the fluorescence process is nearly simultaneous with the absorption of the excitation light due to a relatively short time delay between photon absorption and emission, ranging usually less than a microsecond in duration 3

4 What does a fluorescence microscope look like? 4

Wide-field fluorescence microscopy Observation tube, camera Emission filter Dichroic beamsplitter Excitation light Excitation filter Lamp

5 Wide-field fluorescence microscopy Observation tube, camera Emission filter Dichroic beamsplitter Excitation light Excitation filter Lamp (mercury, xenon ) Acquisition of in-focus well defined images (solid line) but also out-of-focus blurry (dotted line) images objective Focal plane 5

Optical slicing Confocal microscopy Photomultiplier detector Emission filter The pinhole aperture eliminates out-of-focus interfering events, allowing detection of

6 Optical slicing Confocal microscopy Photomultiplier detector Emission filter The pinhole aperture eliminates out-of-focus interfering events, allowing detection of the only infocus objects Dichroic beamsplitter Confocal imaging approach provides a significant improvement in both axial and lateral resolution objective Focal plane 6

Hypothetical specimen under a confocal microscope The initial scan collects an optical slice of the image and then stores that image in the computer Below is a

The lower right image is a projection image made from the series The stage is moved in the Z direction and another optical section is taken.

7 Hypothetical specimen under a confocal microscope The initial scan collects an optical slice of the image and then stores that image in the computer Below is a collection of individual slices called a mosaic. The series of slices were taken of a pollen grain. Each slice shows only a portion of the entire sample. The lower right image is a projection image made from the series The stage is moved in the Z direction and another optical section is taken. The digital image is again stored in the computer The final optical slice is scanned and stored in the computer From the image stack an image is produced called a projection Source: Paperproject.org 7

8 Wide-field vs Confocal imaging Wide field confocal A wide-field fluorescent image (left) vs a laser scanning confocal image (right) of a mouse intestine (from Carl Zeiss). 8

9 Applications of fluorescence microscopy to cellular and tissular biology Most molecules constituting living cells display very low fluorescence levels that can t be quantified Fluorescent staining of sub-cellular compartments needs specific tools 9

10 Fluorescent staining: Probing Cell Structure and functions - High absorption - High fluorescent quantum yield - Water solubility - Cell permeant - Affinity to a particular part of the cell - Chemical and photostability - Stability in cell conditions - Low cytotoxicity Cell structure Nucleus Cytoskeletal proteins Plasma membrane and lipids Organelles (mitochondria, reticulum ) Cell functions Viability, proliferation Endocytosis Ions channels Signal transduction Intracellular ions indicators Intracellular ph Membrane potential 10

11 Probing Cell Structure: Nuclear stains DAPI Λabs=358nm λem=461nm Hoechst Λabs=350nm Λem=461nm HeLa cells Molecular probes handbook 11

Λabs=488nm λem=518nm Gustatory mouse neuron Molecular probes handbook Fibroblast

12 Probing Cell Structure: Plasma membrane stains Cytoskeleton stains Alexa 488 phalloidin DiL (lipophilic probe) Λabs=540nm λem=580nm Phalloidin alexa 488 Λabs=488nm λem=518nm Gustatory mouse neuron Molecular probes handbook Fibroblast labeled with probes for actin (green-fluorescent Alexa488 phalloidin) and the nucleus (TO-PRO-3) 12

13 Probing Cell Structure: mitochondria MitoTracker Red CMXRos λabs=578nm λem=599nm MitoTracker Green FM λabs=490nm λem=516nm Green: actin, blue: nucleus, red, mitochondria Molecular probes handbook 13

14 Probing Cell Function: Calcium indicators Fura-2 λabs=340/380nm λem=510nm Fluo-4 λabs=480nm λem=520nm Hi Ca 2+ level Lo Ca 2+ level Source: Dr Michele Sweeney, Newcastle University (Andor technology) 14

15 Probing Cell Function: Calcium indicators Fura2 loaded T cells Hi Ca 2+ level Non fluorescent infected target cell (dendritic cell) In vitro time lapse imaging with a wide-field fluorescence microscope: images were recorded every 15 seconds Lo Ca 2+ level 15

Hi Ca 2+ level In vitro time lapse imaging with a wide-field fluorescence microscope: images were recorded every 15

16 Probing Cell Function: Calcium indicators Tumoral target B cells are co-cultured with fura-2 probed T lymphocytes. On the right, target cells are pre-treated with a blocking antibody, preventing recognition by effector cells. Hi Ca 2+ level In vitro time lapse imaging with a wide-field fluorescence microscope: images were recorded every 15 seconds Dilek N. et al, 2011 Lo Ca 2+ level Such probe is very useful in order to quantify the activation kinetic of an effector cell 16

17 Probing Cell Function: Cytoplasm stains In vitro time lapse imaging with a wide-field fluorescence microscope: images were recorded every 15 seconds Tumoral target cells loaded with calcein-green probe Non fluorescent T cells Loss of target cell fluorescence is linked to cell death 17

18 Probing Cell Function: mitosis Mitosis in pig kidney epithelium Green: tubulin,red: DNA MicroscopyU.com 18

Immuno staining: fluorescent antibodies Immunostaining refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to

19 Immuno staining: fluorescent antibodies Immunostaining refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. It takes its name from the roots "immuno," in reference to antibodies used in the procedure. Specific molecular markers are characteristic of particular cellular events (such as proliferation, cell death, physiologic activity etc ). Fluorescent antibodies are designed to specifically stain those markers and can be used to detect surface molecules on living cells or intracellular molecules on fixed cells. By using this technique, it s possible to identify accurately protein location. Various antibodies tagged with different fluorochromes can be used simultaneously. 19

nuclear probe (DAPI), FITC conjugated phalloidin (green) and TRITC

20 Immuno staining: fluorescent antibodies HeLa cells labeled with nuclear probe and fitc conjugated anti-keratin antibody HeLa cells labeled with nuclear probe (DAPI), FITC conjugated phalloidin (green) and TRITC conjugated anti-vinculin antibody Chatin B et al, 2012 Chatin B et al,

Immuno staining: colocalization A B CD5 GM1 overlay CD5 IgM overlay Colocalization experiment of angiotensinconverting-enzyme (ACE) with beta-2 adrenergic

ACE is stained with an alexa 568 (red) conjugated antibody anti ACE, B2 receptors with an alexa 488 (green) conjugated antibody anti B2.

et al, 2006 A) Colocalization experiment of membrane receptor CD5 (anti-cd5 FITC staining) and lipid rafts (stained in red with a probe specific of M1

21 Immuno staining: colocalization A B CD5 GM1 overlay CD5 IgM overlay Colocalization experiment of angiotensinconverting-enzyme (ACE) with beta-2 adrenergic receptors in Chinese hamster ovary (CHO) cells. ACE is stained with an alexa 568 (red) conjugated antibody anti ACE, B2 receptors with an alexa 488 (green) conjugated antibody anti B2. Overlay of both channels (red and green) displays a strong colocalization viewable in yellow. Chen z. et al, 2006 A) Colocalization experiment of membrane receptor CD5 (anti-cd5 FITC staining) and lipid rafts (stained in red with a probe specific of M1 ganglioside). Overlay of GM1 and CD5 (red and green) displays a strong colocalization viewable in yellow, whereas CD5 and IgM don t colocalize. B) Colocalization experiment of membrane receptor CD5 (anti-cd5 FITC staining) and the B Cell Recptor BCR (stained in red with an anti-igm alexa 568). Overlay of both channels reveals an absence of colocalization between these receptors. 21

22 Fluorescent fusion proteins, a tool for live cell imaging Step 1: Fusion X gene GFP gene X protein Plasmid insertion Fusion gene Fusion protein GFP gene GFP: Green Fluorescent Protein Step 2: Transfection 22

This method allows to generate living cells with intracellular fluorescent

23 Fluorescent fusion proteins Mitochondria GFP nucleus Generation of a cell line expressing a mitochondrial fusion protein F1-ATP-synthase GFP. This method allows to generate living cells with intracellular fluorescent proteins. Immunofluorence can reveal intracellular elements only after cell fixation. 23

Fixed tissue labelling: in vitro granuloma

microscopy time series showing leukocytes from a healthy

Representative movie from confocal microscopy showing

24 Fixed tissue labelling: in vitro granuloma characterization pathogen Representative movie from microscopy time series showing leukocytes from a healthy donor infected with GFP-tagged C. albicans cells. Representative movie from confocal microscopy showing mature macrophages (red) interacting with 24 C. albicans hyphae (green) inside granuloma.

25 Living tissues labelling: human lung carcinoma biopsy Salmon H et al., 2011 Preactivated T cells (Hoechst; green) were added to a human lung tumor slice that was subsequently stained for fibronectin and EpCAM to respectively identify stromal (red) and tumor epithelial cell (blue) regions 25

Fluorescence Recovering After Photobleaching (FRAP) The mobility of a fluorescent protein fusion can be assessed using a technique known as fluorescence recovery after photobleaching (FRAP).

non-bleached fluorescent molecules into the photobleached region using low laser power. This technique is used for mobility measurement of membrane or intracellular molecules.

26 Fluorescence Recovering After Photobleaching (FRAP) The mobility of a fluorescent protein fusion can be assessed using a technique known as fluorescence recovery after photobleaching (FRAP). In practice, the fluorescently labeled molecules in a small region of the cell are irreversibly photobleached using high laser power, followed by monitoring the subsequent movement of the surrounding non-bleached fluorescent molecules into the photobleached region using low laser power. This technique is used for mobility measurement of membrane or intracellular molecules. control Treated cell HEK cells were transduced to express a GFP tagged of the butyrophilin molecule Control and treated cells display different patterns of BT3 membrane mobility. This differential behaviour is linked to the ability of T lymphocytes to recognize and eliminate cells. 26

FLIM/FRET FLIM (Fluorescence Lifetime Imaging Microscopy) is a powerful technique to measure protein-protein interactions, and is based on the FRET (Forster Resonant Energy Transfert) principle, as

27 FLIM/FRET FLIM (Fluorescence Lifetime Imaging Microscopy) is a powerful technique to measure protein-protein interactions, and is based on the FRET (Forster Resonant Energy Transfert) principle, as shown below. X Y The fundamental mechanism of FRET involves a donor fluorophore in an excited electronic state, which may transfer its excitation energy to a nearby acceptor fluorophore. FRET occurs between two appropriately positioned fluorophores only when the distance separating them is 8 to 10 nanometers or less. 27

Two photons microscopy Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a very high depth, that is up to about one millimeter.

28 Two photons microscopy Two-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a very high depth, that is up to about one millimeter. The concept of two-photon excitation is based on the idea that two photons of comparably lower energy than needed for one photon excitation can also excite a fluorophore in one quantum event 28

Two photons microscopy Radially expanding transglial calcium waves in the intact cerebellum Spontaneous radial expanding calcium wave in the cerebellar molecular layer (xz).

. Overlaid color map shows relative fluorescence changes with time (playback is 4 times original speed, diameter of the field of view is 195 m). Hoogland et al.

29 Two photons microscopy Radially expanding transglial calcium waves in the intact cerebellum Spontaneous radial expanding calcium wave in the cerebellar molecular layer (xz). A radial expanding calcium wave.. Overlaid color map shows relative fluorescence changes with time (playback is 4 times original speed, diameter of the field of view is 195 m). Hoogland et al., 2009 ATP-triggered calcium wave in the cerebellar molecular layer (xy). Green channel displays raw fluorescence reported by fluo-5f; red channels shows Alexa 594 in pipette and during ATP injection as well as SR101 staining of the molecular layer (playback is 4 times original speed, diameter of the field of view is 286 m). 29

Scanner Light Sheet Microscopy Light sheet microscopy is a rapidly emerging technology that combines optical sectioning with multiple-view imaging to observe tissues and living organisms with

30 Scanner Light Sheet Microscopy Light sheet microscopy is a rapidly emerging technology that combines optical sectioning with multiple-view imaging to observe tissues and living organisms with impressive resolution. the technique can image virtually any plane with multiple views obtained from different angles. Thus, light sheet microscopy is ideal for examining of both large (animals) and small (cells) specimens labeled with fluorescent proteins and other fluorophores. long-term imaging of zebrafish development for 24 h. Keller et al.,

31 Use of molecular fluorescence imaging to guide surgery Source: USCD medical center 31

Fluorescence Light Microscopy for Cell Biology

Fluorescence Light Microscopy for Cell Biology Why use light microscopy? Traditional questions that light microscopy has addressed: Structure within a cell Locations of specific molecules within a cell