Fluorescence Microscopy: A Biological Perspective · 2012-04-30 · Detector systems: CCD cameras...

56
Fluorescence Microscopy: A Biological Perspective

Transcript of Fluorescence Microscopy: A Biological Perspective · 2012-04-30 · Detector systems: CCD cameras...

Fluorescence Microscopy:A Biological Perspective

From nanometre to metre: the scale of lifeInstrumentation and accessible scale limits the questions that can

be addressed in biology

Why are there limits?

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

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

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

Excitation light

Dichroic mirror

Reflects the green light in excitation light but transmits the emitted red

light from the fluorescent probe in sample

Light Sources for fluorescent microscopy

Mercury lamps (50 -100 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

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 ultra-high 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. colourHigh speed up 30 FPS with binning.

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.

iXonEM +885 Camera: high resolution with high QE. 32 FPS at full resolution.

iXonEM +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.

Electron multiplying CCD (EMCCD) technology

EMCCD technology has revolutionized high speed imaging of live cells

Solves problem of low light intensities and high speed acquisition

The Zeiss Axiovert 100TV Microscope

Visualization of multiple targets:Multi-channel imaging

Commonly used synthetic fluorophoresKey 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

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

Fluorlabel

Y

Y

Y

Primary ab

Secondary ab

Example of immunolocalization of a flagellar associated protein in fixed Trypanosomes

DAPI (blue)staining dsDNA in nucleus/kinetoplastAlexa 488/staining target protein using antibodyPhase merge on wide field

Multichannel imaging shows

MERGE of all channels

MERGE of all

fluorescence

channels

Quantitative microscopy

Analysis of cell populations reveals a dynamic aspect to

distribution of a myosin motor in trypanosomes

Discrete spots per cell 1-6

Morphology: molecular analysis

HeLa Cells stained with anti-tubulin (green) and PI (red).

Drug treatedControl

Olympus IX81 with long focal length objectives

Long focal length objectivesDual camera system DP 71 and FVIIDual condenser systemExcite HbO systemPhase and DIC

Major advantage:direct viewing of cells in culture flasks

Limitation:longer focal length objectives have lower NA values

Video imaging for analysis of cell

motility Flagellar mutants

Object ID Distance Length Extent Direction Speed Start X Start Y End X End Y Lost in frame No.

µm µm µm ° µm/s µm µm µm µm

0 36.13915 149.9734 40.3578 205.9402 4.065604 163.4328 3.260833 147.6244 35.75899 17

1 3.433133 201.9237 13.38392 229.1044 0.386223 9.021012 56.0965 6.425893 58.34411 17

2 70.1888 243.0118 85.53769 260.2697 7.896141 86.38691 65.57176 17.20784 77.43444

3 9.816114 24.51143 10.01864 264.4338 7.656875 103.0572 73.79773 93.28735 74.74984 17

4 11.62538 198.0033 20.88619 285.0797 1.307839 122.1842 75.90844 110.9592 72.88395

5 18.12689 198.4273 27.29209 223.3044 2.03925 201.4052 89.28699 188.9724 102.4783

6 13.23022 185.2609 19.82163 80.06522 1.488382 124.8998 94.33535 137.9317 92.05278

7 45.10288 149.9547 53.14376 268.1638 5.074011 49.62214 99.18964 4.54242 100.6348 4

8 18.46314 275.1163 27.40254 206.3204 2.077077 10.87646 128.311 2.690081 144.8601

9 36.1553 184.1578 50.14509 248.7898 4.06742 194.1883 136.9865 160.4822 150.0671

10 35.04096 129.7525 43.24707 256.6404 3.942058 109.1934 139.9408 75.10069 148.0375

11 64.66975 201.4379 132.1987 320.1399 7.275256 116.109 148.4101 74.66113 98.7689

12 2.892181 11.8245 5.260734 233.4559 8.954122 206.643 150.8284 204.3194 152.5506 4

13 21.51279 44.45225 22.21497 270.4689 2.420159 150.6569 163.951 129.1448 163.775

Count 14 14 14 14 14 14 14 14 14 5

Minimum 2.892181 11.8245 5.260734 80.06522 0.386223 9.021012 3.260833 2.690081 35.75899

Maximum 70.1888 275.1163 132.1987 320.1399 8.954122 206.643 163.951 204.3194 163.775

Range 67.29662 263.2918 126.938 240.0747 8.567899 197.622 160.6902 201.6294 128.016

Mean 27.59976 156.9863 39.35077 250.2646 4.189315 117.6912 101.8482 96.66781 105.1712

Std.-Dev. 20.49135 76.72476 32.81751 35.58685 2.687812 61.61184 43.60499 66.80291 38.81878

trackIT software analysis gives quantitative data

Detached flagellum cell

Normal cell

Organelle specific Fluorescent Probes for

use in cells

• Organelle Membrane-Specific Probes: DiOC6(3), NBD ceramide, Rhodamine 123 - These dyes stain specifically the organelle membranes, such as the endoplasmic reticulum membrane, Golgi membrane, and mitochondrial membrane.

• 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

Visualization of single mitochondrion in live procyclic form trypanosomes

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

Fluorescent proteins in cells, tissues and animals

Can also be used to make novelty pets

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.

The Z dimension: Confocal Microscopy

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

• 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’

1. Single beam laser scanning confocal microscope

From Spot to image

• 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

Scanning Speed

• Ability to serially produce thin (0.5 – 1.5 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

Advantages of Confocal

Olympus FV1000 Point-Scanning Confocal microscope

Cells & Tissues are 3Cells & Tissues are 3--dimensionaldimensional

Z-stacks

Multi colour labelling, Z-stack

Multi-colour labelling, single slice

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

Imaris Image analysis software – Iso-surface Model showing the nucleus, kinetoplast (blue), lysosome

(red) and Myosin B (green) in T. brucei

Iso-surface model of fluorescently stained trypanosome

Bloodstream forms of Trypanosoma brucei stained with anti-myosin antibody(green) and DNA stain-DAPI (blue)

• 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

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

Basics of Spinning Disk Confocal

Andor Revolution XD Spinning Disc Confocal Microscope

Live Cell Microscopy

35/50 mm glass bottom petri dishes from Willco/MatTek

LabTek™ chamber coverglass slides from Nunc

• 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 by-pass 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

FEATURES

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

Uptake of alum particles by dendritic cells

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

Specialized applications of Confocal Microscopy

• FRAP - photo-bleaching studies

• FRET – Florescence Resonance Energy Transfer

• FLIM-Fluorescence Lifetime Imaging

• FLAP-Fluorescence localisation after photo-bleaching

FRET: useful for protein interaction studies

Looking for fluorescent tag to move back into area thatis bleached. Used as a measure of mobility of tagged

protein or organelle.

Using lasers on region of interest

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.

For multidimensional analysisWhat 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

Good Java Tutorials: http://www.olympusfluoview.com/

Microscopy Primer website: http://microscopy.fsu.edu/primer/

Info on Fluorescent excitation & emission spectra

http://www.iss.com/resources/reference/data_tables/index.html

http://www.mcb.arizona.edu/IPC/spectra_page.htm

http://www.olympusfluoview.com/applications/index.html

Useful Links: