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Download Lecture 5. Confocal microscopy and instrumentation I
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Stimulated Emission Spontaneous Emission Excited States are metastable and must decay Excited States have lifetimes ranging from milliseconds (10-3 s) to nanoseconds (10-9 s) Stimulated Emission: through collisions emitted photon causes other excited atoms to decay in phase • Faster emission than spontaneous • Emitted Photons are indistinguishable LASER: Light Amplification by Stimulated Emission Radiation Stimulated Emission Rate: Absorption Rate: -σ12FN1 Absorption Cross-Section Units → cm2 Number of atoms or molecules in lower energy level (Unit: per cm3) Photon Flux Units → #/cm2sec -σ21FN2 Stimulated emission Cross-Section Units → cm2 (typical value ~ 10-19 to 10-18 cm2) Number of atoms or molecules in lower energy level (Unit: per cm3) Photon Flux Units → #/cm2sec Stimulated Emission Rate: Absorption Rate: -σ12FN1 Absorption Cross-Section Units → cm2 Number of atoms or molecules in lower energy level (Unit: per cm3) Photon Flux Units → #/cm2sec -σ21FN2 Stimulated emission Cross-Section Units → cm2 (typical value ~ 10-19 to 10-18 cm2) Einstein showed: σ12 = σ21 Number of atoms or molecules in lower energy level (Unit: per cm3) Photon Flux Units → #/cm2sec Population Inversion: is the condition for light amplification through stimulated emission. Population inversion is not achievable through direct excitation in a two-level system. http://www.olympusconfocal.com/java/stimulatedemission/index.html Lasing begins as fluorescence Need at least a three-level system R1 and R2 are the external “pump” rates. 1/20 is spontaneous emission rate 2→0 1/21 is spontaneous emission rate 2→1 1/1 is spontaneous emission rate 1→0 1/2= 1/ 21 + 1/20 2→(anything) dN 2 R2 (1/ 2 ) N2 F ( N2 N1 ) dt dN 2 R2 (1/ 2 ) N2 F ( N2 N1 ) = 0 dt dN1 R2 (1 / 21 ) N 2 F ( N 2 N1 ) (1 / 1 ) N1 = 0 dt R2 2 (1 1 21 ) R1 1 N 2 N1 1 ( 1 2 1 2 21 ) F Four Level System is Most Common for Lasing E3-E2 radiationless decay 10-12 E2-E1 spontaneous lifetime of 10-6 E2-E1 stimulated emission of 10-9 E1-E0 radiationless decay 10-12 Log(Iout/Iin) Gain Pump Gain region No absorption Below energy gap Loss region 0 Photon Energy Absorption So What Is LASER? cavity output Pump Population inversion in lasing medium Laser cavity to create the resonance amplification Gain from the medium > loss in the optics of the cavity Laser Cavity Laser Cavity is also a Fabry-Perot optical resonator Not too different from the soap bubble FSR=/ = / 2nL or = c / 2nL : frequency c: speed of light n: refractive index L: cavity length For an optical cavity of 20 cm emitting visible laser light at 500nm (blue) the number of integer wavelengths between the two mirrors would be Lines adjacent to the 500nm line are very close: 500.0013 nm 499.9987 nm Each line allowed by the cavity is a longitude mode. Propagate Cross-section ? Transverse Modes Low-order axisymmetric resonator modes Low-order Hermite-Gaussian resonator modes A single mode (such as TEM00) maintain beam cross-section shape during propagation Gaussian Beam Propagation Near Beam Field waist Far Field • Light is a wave and diffract. It is therefore impossible to have a perfectly collimated beam. • If a Gaussian TEM00 laser-beam wavefront were made perfectly flat at some plane, it would quickly acquire curvature and begin spreading. R -> z if z >> 0 At which point Gaussian beam looks like a point source. w = w0 if z << w02/ zR If = 500nm w0 = 2mm zR = 25.12 m zR also called Raleigh range R: wavefront curvature z: propagation distance w: Beam width defined as the width at 1/e (13.5%) of the peak intensity. w0: Beam width at beam waist. Beam Quality For a theoretical single transverse mode Gaussian beam, the value of the waist radius–divergence product is: For any real laser beam: M2 is a dimensionless parameter to describe how “clean” is the mode of the laser beam, i.e., how close is it to a true Gaussian beam. Very good quality laser beam from low power He-Ne laser can have a M2 ~1.05. Most lasers does not have such ideal beam. Embedded Gaussian A mixed-mode beam: 1. Has a waist M (not M2) times larger than the embedded Gaussian. 2. Will propagate with a divergence M times greater than the embedded Gaussian 3. Has the same curvature and Raleigh range. Mode Control • Larger (therefore higher order) modes are easier to get into lasing condition, because it goes through more active medium. • Aperture is commonly used to increase the loss for larger modes, so that only TEM00 mode is allowed to survive. • In many lasers, the limiting aperture is provided by the geometry of the laser itself. Some Common Lasers To build a laser, you need 1. Two Mirrors 2. Gain Medium 3. Pump Nd:YAG 1. Four level laser 2. Host solid is single crystal YAG: yttrium aluminum garnet Y3Al5O12 3. Optically active atoms Nd. Only <1% of the Medium. 4. Useful for cw operation, becauseYAG has high thermal conductivity and can handle a lot of heat. 5. Also useful for pulsed operation Ar Ion Laser 1. One electron gets pulled off of one atom. 2. In the large field, the electron gets accelerated and impacts other atoms, knocking off other electrons. 3. A current of electrons is now flowing; the positive ions also cause a current. 4. Multiple electron collisions pump the Ar+, which emit light 400-500 nm 5. Very inefficient. 0.03% Semiconductor Laser Mobile electrons Mobile holes A better band picture • Many-state system • Optical transition reserves k • Population inversion easily achievable k P-N Junction P type N type Emission Electron Injection N type + + + + - Hole Injection P type • Small size • Very small cavity, large mode spacing. • Very efficient: ~50% efficiency • Most band gap is small, so emit IR light (shortest wavelength at ~760nm) • Can easily form arrays to increase total power output. • Mostly used as pump laser in microscopy applications. DPSS Laser Common Continuous Wave (CW) Lasers for Microscopy Argon UV (cw) Argon Vis Argon-Krypton He-Ne DPSS laser 364 nm 488, 514 nm (458, 477 nm) 488, 568, 647 nm 633 nm (laser pointer) 532 nm, 565 nm Not tunable Dye lasers are tunable and covers broad spectrum, but very difficult to operate. None appropriate for 2-p absorption, wrong colors, low power Wide Field vs Confocal Fluorescence Imaging Confocal Greatly reduces Out of focus blur Wide-field Brighter but No sectioning More examples widefield confocal medulla muscle pollen Epi-illumination widefield is form of Kohler Illumination: Objective is also condenser Lamp or laser lens detector Detect at 90 degrees Split with dichroic mirror Confocal detection with 3 dimensional scanning Image one plane, Move focus Confocal Aperture •Decreasing the pinhole size rejects more out of focus light, therefore improving contrast and effective z resolution. •Decreasing the pinhole will increase x,y resolution (1.3x widefield) •Decreasing pinhole size decreases the amount of the Airy disk that reaches the detector. This results in less light from each point being collected •Generally, collecting the diameter of 1 Airy disk is considered optimal. This collects about 85% of light from a subresolution point. Limits: Open pinhole: nearly widefield resolution (still some confocality) Closed: no image Signal, S/N (out of focus) opposite trends Closed: better axial sectioning, but no photons for contrast Open: no sectioning, lots of photons Confocal Aperture ALIGNMENT OF APERTURES IS CRITICAL •X, Y alignment : Different wavelengths focus at different lateral position. Lateral color aberrations can be important for multi-color imaging (multiple dyes with multiple lasers) •Z alignment: Different wavelengths focus at different depths in image plane. Chromatic aberrations can be important. Need wellcorrected lenses Intermediate Optical Path of Confocal Microscope Requirements: 1) Laser path has same conjugate planes as intermediate, detector, eyepieces 2) Laser Scans undeviated around pivot point: Stays on optical axis 3) Back aperture of objective is always filled For highest resolution Consequences: 1) Pupil transfer lens 50-100 mm fl to fill lens 2) Max scan angle ~7 degrees while still filling lens 3) Position of pupil lens is critical for parfocality with Kohler illumination Brightfield and epi-fluorescence Scanning Galvanometers Much faster than stage scanning (1000x) Point Scanning Mirrors on magnets x y Laser out To Microscope Laser in Olympus Fluoview Scan Time Issues Typical scan rate 1s /scan 512X 512 Faster is not stable with galvos, but can reduce #pixels t=0 X = 512 X = 128 Y = 128 Y = 512 t=0 t = 0.25 sec t = 1 sec Scan Time Issues Two scan types: 1. 2. 1) Bidirectional: Resonant galvos, Very fast Require post imaging Processing, cannot change Speed for zoom 2)Unidirectional:flyback Normal for galvo scanners: Have hysteresis, settling time: 30% duty cycle Digital Zoom: Reducing scan angle, higher pixel density per area Not equivalent to changing objective lens magnification 1x 1024 points 2x 1024 points 4x 1024 points Note that we have reduced the field of view of the sample linearly Note: There will only be a single zoom value where optimal resolution can be collected : Nyquist Criterion zoom 2-3 Confocal Parameters and Intensity, Resolution Point Scan Detection: Photomultiplier (PMT) Photocathode creates Secondary electrons -1000V Usually 12-13 dynodes in practice Gain ~105-7 detected at anode PMT APD Both can work under Single-photon Counting mode Spectral Response and QE of PMTs 1,2,3: Alkali Photocathodes 4: GaAS Photocathode PMTs best in UV Alkali lousy in visible 10-15% QE: probably optimistic weakest link on Confocal Scope Silicon Response for (Avalanche) Photodiodes Avalanche Photodiodes: used sometimes in imaging 1) 75% efficiency at 700-800 nm 2) Better than PMTs in visible, near IR 3) Very small areas ~200 microns: difficult to align in confocal 4) Low max count rates (small dynamic range) Efficiency & Signal/Noise? • • • • • Collection efficiency of microscopy: ~25% Detector quantum yield: ~70-90% Thermal noise Shot noise (quantum noise): Read noise (A/D conversion) Typical Dark Counts CCD Temperature Sensitive Area Dark Counts -70 C 10-20 m 0.001 e/sec/pixel APD -20 C 100-500 m 10-100 e/sec/pixel Gain of PMTs with Applied Voltage 2 Modes: 1) Analog Detection 2) Single Photon Counting Analog (<1000 V) : linear regime, integrated current~number photons, higher voltage=bigger current (until dark current takes over) Match gain (voltage) with dynamic range of integration electronics, for each sample For best S/N. used in commercial instruments 2) Counting (>1200V): detector in saturation: Every photon produced same voltage pulse Increased sensitivity, but smaller range Poisson statistics~ 1/n1/2 More complex, more expensive Not used commercially Photodiode PMT: photomultiplier CCD APD: Avalanche Photodiode