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Week 2 Excitation, fluorescence, optical systems, resolution BME 695Y / BMS 634 Confocal Microscopy: Techniques and Application Module Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine & Department of Biomedical Engineering, Schools of Engineering J.Paul Robinson, Ph.D. Professor of Immunopharmacology & Biomedical Engineering Director, Purdue University Cytometry Laboratories J These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not be used for any commercial purpose. One useful text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of the ideas and figures in these lecture notes are taken from this text. UPDATED February 2004
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Overview of lecture 2 1. Excitation Sources 2. Fluorescence
3. Raman & Raleigh Scatter 4. Photobleaching 5. CCD cameras for fluorescence 6. Fluorescent probes for biological material 7. The structure of a confocal microscope 8. Optical properties of confocal systems 9. Confocal principals 11.Resolution, gray scales and image structure 12 Sampling theory and electronic zoom 13.Reflection Imaging.
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Excitation Sources Excitation Sources Lamps Xenon Xenon/Mercury Lasers
Argon Ion (Ar) Krypton (Kr) Helium Neon (He-Ne) Helium Cadmium (He-Cd) Krypton-Argon (Kr-Ar)
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Fluorescence Chromophores are components of molecules which absorb light They are generally aromatic rings
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Fluorescence Jablonski Diagram S2 T2 S1 T1 S0 ENERGY Singlet States
Triplet States S2 Vibrational energy levels Rotational energy levels Electronic energy levels T2 S1 IsC ENERGY T1 ABS FL I.C. PH IsC S0 [Vibrational sublevels] ABS - Absorbance S Singlet Electronic Energy Levels FL - Fluorescence T 1, Corresponding Triplet States I.C.- Nonradiative Internal Conversion IsC Intersystem Crossing PH - Phosphorescence
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Simplified Jablonski Diagram
Energy S1 hvex hvem
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Fluorescence Intensity Wavelength
The longer the wavelength the lower the energy The shorter the wavelength the higher the energy eg. UV light from sun causes the sunburn not the red visible light Intensity related to the probability of the event Wavelength the energy of the light absorbed or emitted
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Fluorescence Stokes Shift
is the energy difference between the lowest energy peak of absorbence and the highest energy of emission Stokes Shift is 25 nm Fluorescein molecule 495 nm 520 nm Fluorescnece Intensity Wavelength
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PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid
Common Laser Lines 600 nm 300 nm 500 nm 700 nm 400 nm 457 350 514 610 632 488 PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid
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Parameters Extinction Coefficient Quantum Yield
refers to a single wavelength (usually the absorption maximum) Quantum Yield Qf is a measure of the integrated photon emission over the fluorophore spectral band At sub-saturation excitation rates, fluorescence intensity is proportional to the product of and Qf
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Excitation Saturation
The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime f) Optical saturation occurs when the rate of excitation exceeds the reciprocal of f In a scanned image of 512 x 768 pixels (400,000 pixels) if scanned in 1 second requires a dwell time per pixel of 2 x 10-6 sec. Molecules that remain in the excitation beam for extended periods have higher probability of interstate crossings and thus phosphorescence Usually, increasing dye concentration can be the most effective means of increasing signal when energy is not the limiting factor (ie laser based confocal systems)
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How many Photons? Consider 1 mW of power at 488 nm focused to a Gaussian spot whose radius at 1/e2 intensity is 0.25m via a 1.25 NA objective The peak intensity at the center will be 10-3W [.(0.25 x 10-4 cm)2]= 5.1 x 105 W/cm2 or x 1024 photons/(cm2 sec-1) At this power, FITC would have 63% of its molecules in an excited state and 37% in ground state at any one time
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Raman Scatter A molecule may undergo a vibrational transition (not an electronic shift) at exactly the same time as scattering occurs This results in a photon emission of a photon differing in energy from the energy of the incident photon by the amount of the above energy - this is Raman scattering. The dominant effect in flow cytometry is the stretch of the O-H bonds of water. At 488 nm excitation this would give emission at nm
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Rayleigh Scatter Molecules and very small particles do not absorb, but scatter light in the visible region (same freq as excitation) Rayleigh scattering is directly proportional to the electric dipole and inversely proportional to the 4th power of the wavelength of the incident light the sky looks blue because the gas molecules scatter more light at shorter (blue) rather than longer wavelengths (red)
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Photobleaching Defined as the irreversible destruction of an excited fluorophore (discussed in later lecture) Methods for countering photobleaching Scan for shorter times Use high magnification, high NA objective Use wide emission filters Reduce excitation intensity Use “antifade” reagents (not compatible with viable cells)
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Photobleaching example
FITC - at 4.4 x 1023 photons cm-2 sec-1 FITC bleaches with a quantum efficiency Qb of 3 x 10-5 Therefore FITC would be bleaching with a rate constant of 4.2 x 103 sec-1 so 37% of the molecules would remain after 240 sec of irradiation. In a single plane, 16 scans would cause 6-50% bleaching
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Antifade Agents Many quenchers act by reducing oxygen concentration to prevent formation of singlet oxygen Satisfactory for fixed samples but not live cells! Antioxidents such as propyl gallate, hydroquinone, p-phenylenediamine are used Reduce O2 concentration or use singlet oxygen quenchers such as carotenoids (50 mM crocetin or etretinate in cell cultures); ascorbate, imidazole, histidine, cysteamine, reduced glutathione, uric acid, trolox (vitamin E analogue)
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Excitation - Emission Peaks
% Max Excitation at nm Fluorophore EXpeak EM peak FITC Bodipy Tetra-M-Rho L-Rhodamine Texas Red CY Note: You will not be able to see CY5 fluorescence under the regular fluorescent microscope because the wavelength is too high.
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Fluorescent Microscope
Arc Lamp EPI-Illumination Excitation Diaphragm Excitation Filter Ocular Dichroic Filter Objective Emission Filter
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Fluorescence Microscope with Color Video (CCD) 35 mm Camera
Camera viewer ocular filters objectives stage condensor
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Cameras and emission filters
Cooled color CCD camera Camera goes here Color CCD camera does not need optical filters to collect all wavelengths but if you want to collect each emission wavelength optimally, you need a monochrome camera with separate emission filters shown on the right (camera is not in position in this photo).
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Types of Probes Proteins Nucleic Acids DNA Ions
pH Sensitive Indicators Oxidation States Specific Organelles
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Probes for Proteins Probe Excitation Emission FITC 488 525 PE 488 575
APC PerCP™ Cascade Blue Coumerin-phalloidin Texas Red™ Tetramethylrhodamine-amines CY3 (indotrimethinecyanines) CY5 (indopentamethinecyanines)
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Probes for Ions INDO-1 Ex350 Em405/480 QUIN-2 Ex350 Em490
Fluo-3 Ex488 Em525 Fura -2 Ex330/360 Em510
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pH Sensitive Indicators
Probe Excitation Emission SNARF BCECF /620 440/ [2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein]
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Probes for Oxidation States
Probe Oxidant Excitation Emission DCFH-DA (H2O2) HE (O2-) DHR 123 (H2O2) DCFH-DA - dichlorofluorescin diacetate HE - hydroethidine DHR dihydrorhodamine 123
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Specific Organelle Probes
Probe Site Excitation Emission BODIPY Golgi NBD Golgi DPH Lipid TMA-DPH Lipid Rhodamine 123 Mitochondria DiO Lipid diI-Cn-(5) Lipid diO-Cn-(3) Lipid BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium
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DNA Probes AO AT/GC binding dyes Metachromatic dye
concentration dependent emission double stranded NA - Green single stranded NA - Red AT/GC binding dyes AT rich: DAPI, Hoechst, quinacrine GC rich: antibiotics bleomycin, chromamycin A3, mithramycin, olivomycin, rhodamine 800
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Multiple Emissions Many possibilities for using multiple probes with a single excitation Multiple excitation lines are possible Combination of multiple excitation lines or probes that have same excitation and quite different emissions e.g. Calcein AM and Ethidium (ex 488) emissions 530 nm and 617 nm
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Energy Transfer Effective between 10-100 Å only
Emission and excitation spectrum must significantly overlap Donor transfers non-radiatively to the acceptor PE-Texas Red™ Carboxyfluorescein-Sulforhodamine B
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Fluorescence Resonance Energy Transfer Intensity Wavelength Molecule 1
ACCEPTOR DONOR Intensity Absorbance Absorbance Wavelength
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Benefits of Confocal Microscopy
Reduced blurring of the image from light scattering Increased effective resolution Improved signal to noise ratio Clear examination of thick specimens Z-axis scanning Depth perception in Z-sectioned images Magnification can be adjusted electronically
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Fluorescent Microscope
Confocal Microscope Arc Lamp Laser Excitation Diaphragm Excitation Pinhole Excitation Filter Excitation Filter Ocular PMT Objective Objective Emission Filter Emission Filter Emission Pinhole
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MRC 1024 System UV Laser Optical Mixer Kr-Ar Laser Scanhead Microscope
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Bio-Rad MRC 1024
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MRC 1024 System Light Path PMT
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Optical Mixer - MRC 1024 UV Fast Shutter Argon Laser 353,361 nm
Visible Filter Wheels UV Correction Optics Argon- Krypton Laser 488, 514 nm 488,568,647 nm Beam Expander To Scanhead
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MRC 1024 Scanhead 2 3 Emission Filter Wheel From Laser PMT 1
Galvanometers To and from Scope
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From Scanhead To Scanhead
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Scanning Galvanometers
Point Scanning x y Laser out To Microscope Laser in
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The Scan Path of the Laser Beam
767, 1023, 1279 Start Specimen 511, 1023 Frames/Sec # Lines
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How a Confocal Image is Formed
Condenser Lens Pinhole 1 Pinhole 2 Objective Specimen Detector Modified from: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989
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Fundamental Limitations of Confocal Microscopy
From Source To Detector . x,y,z 2 n2 photons 2 1 n1 photons 1 z y x PIXEL VOXEL From: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989
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Optical Resolution: Gray Level & Pixelation
Analogous to intensity range For computer images each pixel is assigned a value. If the image is 8 bit, there are 28 or 256 levels of intensity If the image is 10 bit there are 1024 levels, 12 bit 4096 levels etc. The intensity analogue of a pixel is its grey level which shows up as brightness. The display will determine the possible resolution since on a TV screen, the image can only be displayed based upon the number of elements in the display. Of course, it is not possible to increase the resolution of an image by attributing more “pixels” to it than were collected in the original collection!
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T Pixels Pixels & image structure
Hardcopy usually compromises pixel representation. With 20/20 vision you can distinguish dots 1 arc second apart (300 m at 1 m) so 300 DPS on a page is fine. So at 100 m, you could use dots 300 mm in size and get the same effect! Thus an image need only be parsimonius, i.e., it only needs to show what is necessary to provide the expected image. T
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320x240 x 24 Zoom x 4 The final image appears to be very “boxy” this is known as “pixilation”. Zoom x 2 Magnifying with inadequate information. This is known as “empty magnification” because there are insufficient data points. Zoom x 8
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Socrates?….well perhaps not...
180x200x8 (288,000) 1X 361x400x8 (1,155,200) 2x Magnifying with adequate information. Here, the original image was collected with many more pixels - so the magnified image looks better! 541x600x8 (2,596,800) 1.5x)
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320x240 x 24 Originals collected at high resolution - compared to a low resolution image magnified 1500x1125x24
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Sampling Theory The Nyquist Theorem
Nyquest theory describes the sampling frequency (f) required to represent the true identity of the sample. i.e., how many times must you sample an image to know that your sample truly represents the image? In other words to capture the periodic components of frequency f in a signal we need to sample at least 2f times Nyquist claimed that the rate was 2f. It has been determined that in reality the rate is 2.3f - in essence you must sample at least 2 times the highest frequency. For example in audio, to capture the 22 kHz in the digitized signal, we need to sample at least 44.1 kHz
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Digital Zoom Note that we have reduced the field of view of the sample
1 x 1024 points 4 x 1024 points 2 x 1024 points Note that we have reduced the field of view of the sample
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Reflection Imaging Backscattered light imaging
Same wavelength as excitation Advantages: no photobleaching since not using a photo-probe (note: does not mean no possible damage to specimen) Problems: optical reflections from components of microscope CD-ROM pits Increasing mag Collagen
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Issues for good confocal imaging
Axial Resolution Must determine the FWHM (full width half maximum) intensity values of a vertical section of beads Field Flatness Must be able to collect a flat field image over a specimen - or z-axis information will be inaccurate Chromatic Aberration must test across an entire field that emission is constant and not collecting radial or tangential artifacts due to chromatic aberration in objectives Z-drive precision and accuracy must be able to reproducibily measure distance through a specimen - tenths of microns will make a big difference over 50 microns
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Conclusions Fluorescence is the primary energy source for confocal microscopes Dye molecules must be close to, but below saturation levels for optimum emission Fluorescence emission is longer than the exciting wavelength The energy of the light increases with reduction of wavelength Fluorescence probes must be appropriate for the excitation source and the sample of interest Correct optical filters must be used for multiple color fluorescence emission Sampling rate must be appropriate for specimen(Nyquist Theorem)
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