Fluorescence Microscopy: A Biological Perspective

Transcription

1 Fluorescence Microscopy: A Biological Perspective

2 From nanometre to metre: the scale of life Instrumentation and accessible scale limits the questions that can be addressed in biology Why are there limits?

3 Resolution and microscopy Resolution: ability to distinguish two adjacent objects. Depends on the wavelength (λ) and numerical aperture (NA) of the objective. Limit: R = 0.61λ/NA so at 488 nm R ~ 0.2 µm. Small structures in cells can have a size close to or below this limit, may be imaged but as depicted in the image will appear larger than the real structure. Co-localizations:things may appear to co localize but are separate. NA = n x sin α where n is the refractive index

4 Five Dimensional Imaging: Goal of Fluorescence Microscopy in Biomedical Sciences 3 spatial dimensions, X, Y and Z Ability to follow multiple probes/targets Following processess in real time

5 Typical wide field fluorescence microscope Note key elements Objectives delivers exciting light and collects emmission signal Dichroic mirror; reflects excitation wavelength light but transmits emission wavelength. Filters: Filtering of different wavelengths. Different cuts off wavelenths and band passess Dichroic mirror Reflects the green light in excitation light but transmits the emitted red light from the fluorescent probe in sample Excitation light

6 Light Sources for fluorescent microscopy Mercury lamps ( W) & Excite HbO lamps variable intensities. LEDs long lasting, bright, use multiple wavelengths, variable power. Lasers (gas/solid state) high-intensity monochromatic light sources that are coherent and highly collimated to form a tight beam with a very low rate of expansion. Limited number of wavelengths available

7 Detector systems: CCD cameras Axiocam HRc, colour camera resolution 1.4 megapixels (1388 x 1040) Size: 6.45 µm x 6.45 µm. Frame rate: 5/sec (20 ms exposure) at full resolution. DP71: 1.45 million pixel CCD with a Bayer color filter. This CCD is coupled to pixel-shifting technology; resulting in images with an ultrahigh resolution of 4080 x 3072 pixels. (high resolution) monochrome FVII: 1376 x 1032 x 6.45µm square pixel CCD monochrome imaging sensor. High frame rate, up to 22 fps in binning mode. colour High speed up 30 FPS with binning.

8 Electron multiplying CCD (EMCCD) technology EMCCDs operate by amplifying weak signal events (down to single photons) to a signal level that is well clear of the read noise floor of the camera, at any readout speed. cooling ~ -90 C in deep vacuum. ixon EM +885 Camera: high resolution with high QE. 32 FPS at full resolution. ixon EM +897 Camera: ultimate in sensitivity back illuminated EMCCD has single photon detection capability without an image intensifier, combined with greater than 90% QE of a back-illuminated sensor. Containing a 512 x 512 Frame. 16 µm square. Combines high sensitivity with high speed readout. EMCCD technology has revolutionized high speed imaging of live cells Solves problem of low light intensities and high speed acquisition

9 The Zeiss Axiovert 100TV Microscope

10 Visualization of multiple targets: Multi-channel imaging

11 Commonly used synthetic fluorophores Key concepts Selection: avoid spectral overlap. Try to separate emmission maxia. Sensitivity: high quantum yield: strong signals Stable: bleeching or quenching of signal A wide range of Alexa fluorophores available from Molecular Probes that meet the three requirements

12 Immunolocalization experiments in cells Fixation of cells (paraformaldehyde) Attachment of cells to slide Permeabilization & Blocking Probe with primary antibody. Probe with secondary antibodies with fluorophores (dyes) attached.. Washing. Viewing Y Y Y Fluor label Secondary ab Primary ab

13 Example of immunolocalization of a flagellar associated protein in fixed Trypanosomes MERGE of all fluorescence channels MERGE of all channels Multichannel imaging shows DAPI (blue)staining dsdna in nucleus/kinetoplast Alexa 488/staining target protein using antibody Phase merge on wide field

14 Quantitative microscopy Analysis of cell populations reveals a dynamic aspect to distribution of a myosin motor in trypanosomes Discrete spots per cell 1-6

15 Morphology: molecular analysis HeLa Cells stained with anti-tubulin (green) and PI (red). Control Drug treated

16 Olympus IX81 with long focal length objectives Major advantage: direct viewing of cells in culture flasks Limitation: longer focal length objectives have lower NA values Long focal length objectives Dual camera system DP 71 and FVII Dual condenser system Excite HbO system Phase and DIC

17 Video imaging for analysis of cell motility Flagellar mutants

18 trackit software analysis gives quantitative data Detached flagellum cell Object ID Distance Length Extent Direction Speed Start X Start Y End X End Y Lost in fra µm µm µm µm/s µm µm µm µm Count Minimum Maximum Range Mean Std.-Dev Normal cell

19 Organelle Membrane-Specific Probes: DiOC6(3), NBD ceramide, Rhodamine These dyes stain specifically the organelle membranes, such as the endoplasmic reticulum membrane, Golgi membrane, and mitochondrial membrane. Organelle specific Fluorescent Probes for use in cells Organelle specific probes based on features of the comparment: Other probes accumulate specifically into organelles such lysosomes (weak bases) and mitochondria (weak acids or positively changed dyes). The nucleus can be stained with a variety of dyes that bind to dsdna e.g. DAPI, Hoest stain etc Membrane Potential-Sensible Probes: WW 781, RH-155, and Di-8-ANEPPS - These dyes are incorporated into the cell membrane. Their absorption or fluorescence intensity varies depending on the membrane potential. Fast-type dyes with response rates on the order of milliseconds should be used. Live cells usually incubated with the probe and then viewed directly fixed for viewing in combination with other probes

20 Visualization of single mitochondrion in live procyclic form trypanosomes

21 Fluorescent proteins: A vital tool in imaging in live cells Osamu Shimomura and Frank Johnson, a protein that lacked was able to produce green fluorescence when illuminated with ultraviolet light. The protein was eventually christened with the unceremonious name of green fluorescent protein (GFP). The GFP gene has been isolated and improved to obtain different color variants. These proteins are used to construct fluorescent chimeric proteins that can be expressed in living cells, tissues, and entire organisms, after transfection with the engineered vectors. The fluorescent protein technique avoids the problem of purifying, tagging, and introducing labeled proteins into cells or the task of producing specific antibodies for surface or internal antigens. Used widely in live cell imaging

22 Fluorescent proteins in cells, tissues and animals

23 Can also be used to make novelty pets

24 Problems with use of Fluorescent proteins 1. In the natural state most are oligomeric: can cause problems in vivo 2. Quantum yield of some variants is low. 3. Red proteins. Restricted choice in the red far red region. 4. Because of size (~20-30 kda) insertion of tag may affect location/function of tagged target protein.

25 The Z dimension: Confocal Microscopy

26 Why use Confocal Microscopy? High resolution images of your cells or sample, beyond that of a normal fluorescent microscope Localisation of a particle of interest within a cell Co-localisation of different particles We can take 2-D sections through 3-D cells & tissues and reconstruct an extended-focus series Use of 3 or 4 different fluorescent probes simultaneously We can combine fluorescence contrast with phase contrast and differential interference contrast (DIC) We can control the laser to do dynamic experiments

27 1. Single beam laser scanning confocal microscope Uses point illumination and point detection Uses a pinhole Both of these specifications restrict image information to the plane of focus Laser beam scans the specimen from left to right and is rapidly transported back to the start point in a process termed flyback

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29 From Spot to image

30 Scanning Speed Field of View 512x 512 pixels = 262,144 points Build up entire field of view in 1 second Laser must dwell on each point for 3.8 µsec Need a PMT to read each sampling pixel

31 Advantages of Confocal Ability to serially produce thin ( µm) optical sections through fluorescent specimens up to 100 µm thick, non-invasively Contrast & definition dramatically improved cf widefield microscopy Reduction of out-of-focus fluorescence due to the presence of a pinhole Better lateral and axial resolution than wide-field Improved signal to noise ratio (SNR) Can un-mix two spectrally close fluors and image simultaneously without cross-talk Magnification zoom can be adjusted electronically As well as imaging in 3D (XYZ) one can image in 4D (XYZT)-live cell imaging with optical sectioning

32 Olympus FV1000 Point-Scanning Confocal microscope

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34 Cells & Tissues are 3-dimensional3

35 Z-stacks

36 Multi colour labelling, Z-stack

37 Multi-colour labelling, single slice

38 Wide field imaging of a myosin motor TbMyo I reveal localization with various elements of the endocytic pathway TbMyo I Flagellar pocket/ Early endosomes TbMyo I Late endosomes TbMyo I Lysosome TbMyo I does not locate specifically with any individual compartment

39 Imaris Image analysis software Iso-surface Model showing the nucleus, kinetoplast (blue), lysosome (red) and Myosin B (green) in T. brucei

40 Iso-surface model of fluorescently stained trypanosome Bloodstream forms of Trypanosoma brucei stained with antimyosin antibody(green) and DNA stain-dapi (blue)

41 Filter cube sets for DAPI, FITC & rhodamine chromophores Laser System contains: Argon laser, multi-line 458/ 488/ 515 nm, Green Helium-Neon laser 543 nm, Red Helium-Neon laser 633 nm, Near-Violet Laser diode 405 nm 10x, 20x & 40x dry and 40x & 60x oil objectives all of standard focal length 2 Stages available, one for standard slides and the other a heated stage for 35 mm or 50 mm glass bottom petri dishes Incubator for live cell experiments FluoView software has 3D & 4D capabilities spectral unmixing ; detection, spectral separation and visualization of multiple fluorescent labels with overlapping emission spectra such as CFP, GFP and YFP, which cannot be separated using conventional methods. The SIM scanning system can synchronize laser light stimulation and imaging to capture rapid changes in living cells immediately following fluorophore excitation e.g. photoactivation & FRAP

42 Live cell analysis and use of Multi-beam laser-scanning confocal microscope Uses point illumination and point detection Uses many pinholes in a Nipkow disc Very Fast

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44 Basics of Spinning Disk Confocal

45 Andor Revolution XD Spinning Disc Confocal Microscope

46 Live Cell Microscopy 35/50 mm glass bottom petri dishes from Willco/MatTek LabTek chamber coverglass slides from Nunc

47 FEATURES CoolLED light source with wavelength peaks at 445nm, 490nm & 565nm. Filter cube sets for CFP, GFP, rhodamine & a triple filter cube to view all three flours simultaneously. Laser combiner comprises four solid state lasers, 445 nm, 488 nm, 514 nm, 561 nm The FRAPPA unit allows user defined ROI laser scanning for FRAP and Photo-activation. Yokogawa CSU-X1 Spinning Disk Unit, motorised version with brightfield bypass option. 5000rpm disk spin speed giving scan rate of 1000 scans per second. 2 interchangeable high-res cameras available for acquisition of images, using ixon +Ultra sensitive EMCCD technology

48 Advantages of Spinning Disk Confocal Can use CCD camera ( instant optical sectioning) rather than a PMT Parallel beam goes much faster-higher frame rate-limited only by the CCD camera read out rate Uses lower intensity laser illumination than single point scanning

49 Uptake of alum particles by dendritic cells

50 Cytoplasmic GFP-protein in live bloodstream forms of T. brucei, Spinning disk Confocal image

51 Specialized applications of Confocal Microscopy FRAP - photo-bleaching studies FRET Florescence Resonance Energy Transfer FLIM-Fluorescence Lifetime Imaging FLAP-Fluorescence localisation after photobleaching

52 FRET: useful for protein interaction studies

53 Using lasers on region of interest Looking for fluorescent tag to move back into area that is bleached. Used as a measure of mobility of tagged protein or organelle.

54 FRAP in bloodstream forms of T. brucei Bleeching of a protein in the flagellar pocket and recovery of signal due to trafficking of new protein to surface.

55 For multidimensional analysis What to use? Use Confocal for thick samples: up to 100 µm Use wide-field for thin samples: less than 2µm Use multi-beam confocal for moving or photo sensitive samples All can be used for multichannel imaging

56 Useful Links: Good Java Tutorials: Microscopy Primer website: Info on Fluorescent excitation & emission spectra