Imaging Retreat - UMASS 2012 Customized real-time confocal and 2-photon imaging Mike Sanderson Department of Microbiology and Physiological Systems University of Massachusetts Medical School Thanks for the invite Allan and Silvia: i organizing ii committee
Seeing is believing Intercellular Ca 2+ waves Image Stack High Speed recordings of cilia 3D Image reconstruction Time-lapse of cell motility Ca 2+ oscillations in lung slices Trans-illumination of airways Ca 2+ sparks in Atrial cells
Talk Outline Theme: Less focus on what we do (biology) - more focus on how we do it (technology) Our is a non-commercial, build your own approach more flexibility, less expensive. Image capture and Storage High Speed and wide field Imaging Principles and need for confocal microscopy Our scanning approach speed v image size Photodetectors and pin holes - spectral scanning Principles of 2-photon microscopy Implementation of 2-photon microscopy laser power dye excitation photodetector position
Image Capture, Storage and Access/Analysis Easy to acquire many hundreds if not thousands of images. Smallest image usually = 512 x 512 x 8 bit = ¼ Mbyte ( 4 images per Mbyte) But single color image has RGB plane (3 times the size) More often: Imaging experiment record RGB channels for different fluorophores and a normal light image (4 images simultaneously = 1 Mbyte) With present day computers (64 bit) Problem has greatly eased; several Gbytes of memory now available. I gbyte = 4000 images but at high speed of 200 fps = 20 secs of data Important: Capture to memory, Save to disk between samples. Access file format - proprietary or generic ( commercial instruments use own software). Play back of data slow, fast, backward forward etc
Our solution; Recording software VIDEO SAVANT (digital VCR). Direct recording to striped hard drive at 120 Mbytes/sec limited only by size of hard disc (Terabyte). Image Data Stream Striped Disk Array 120 Mb s -1 Compatible with most frame grabbers and cameras
High-speed imaging: becomes easy Digital Frame Grabber 200 FPS CCD Progressive scan camera Striped Disk Array 120 Mb s -1 Microscope: Differential interference optics Camera sees the image Cilia recorded at 200 fps; replay at 30 fps as mpeg mpeg movie created by VS.
Other Current Solutions: MicroManager μmanager is a software package for control of automated microscopes. Together with the image processing application ImageJ, μmanager provides a comprehensive, freely available, imaging solution. www.micro-manager.org/ http://rsbweb.nih.gov/ij/ We use VS as acquisition and some analysis with ImageJ for faster analysis
Wide-field Florescence Imaging Use Same approach: 30 fps with low-light sensitive camera Important: Thin cell preparation What about thicker tissues?
Wide-field image formation Excitation Light Light compression Thick specimen objective Image plane camera Image contrast is low low resolution
Basic idea of confocal image formation Only excite a single plane or spot lens Image plane Dichroic Mirror Em Filter cube Ex specimen Laser Light Problem: Excitation is still not limited to the optical plane
Basic idea of confocal image formation Scan the specimen achieved by 2 mirrors lens No 2D Image formed Image plane Dichroic Mirror Em PMT Filter cube Ex specimen Laser Light
PMT Filters Dichroic i Laser Mirrors (Scan and descan) Scanning Design Determines the imaging speed mirror inertia Influences exposure time (dwell time) Influences Light sensitivity Zoom/magnification Microscope Line Scanners using a slit, faster but some loss of resolution Practical llayout Acoustic Optical Device - AOD varies the refractive index to alter the light pathway Spinning Disc Microscope
Our approach for Scanning Use a resonance scanner for fast horizontal direction low inertial oscillates like a tuning fork (8kHz) Each line occurs in 125 usec video rate Use conventional galvanometer scanner for slow vertical direction Disadvantage: Image distortion (horizontal plane only) Bi-directional scan speeds up scanning a (1200 x 200) 1 0.5 0-0.5 05-1 b c Forward mirror Scan Image center = End of scan 0 Rastor line 600 Reverse mirror scan 0 45 90 135 180 225 270 315 360 d e 0 0 600
Image correction Cosine function of mirror velocity any size image h (420 x 400) corrected (600 x 400) reversed, interlaced 1.6 1.2 0.8 Radian 0.4 De gree s 0-90 -60-30 -0.4 0 30 60 90-0.8-1.2-1.6 Image formation PMT only gives intensity i Use the scanning mirrors to determine - pixel, line position Fast A/D on a frame grabber build up the picture provide sync signals point by point, line by line, transfer to computer repeat the process (30 fps)
Imaging speed vs Image Size with any objective Line scan remains the same = 480 corrected pixels = 62.5 usec: dwell time ~ 87 nsec Dwell time = time to excite and detect photons 480 480 480 480 800 400 60 fps 200 120 fps 100 30 fps 15 fps Increased frame rate by reducing vertical line number Centered around center line
Automatic Zoom control with scanning mirrors with any objective (x40 x100) 100 um 50 um Reduce angle of scan 100 um (for both x and y mirror) 50 um
Multi-wavelength Imaging : differences in dye excitation and emission spectra 2. Multiple emissions Align of image is exact as all 3 PMTs are 3 PMTs synchronized by the scanning mirrors Adjustable Confocal Iris Top Adjustable Mirror Inverted Microscope Dichroic Mirrors Barrier Filter Objective/Specimen Solid State Laser (635 nm) (592 nm) (488 nm) Dichroic Mirror Side Port Solid State Laser Shutter (S) Solid State Laser Scanner V Bracket Scanner h Eye Piece Internal Microscope Mirror 1. Laser Wavelength Excitation Align incoming lasers to same excitation spot ( or via optic fiber)
Spectral Scanning Emission From microscope Diffraction grating Confocal Aperture Movable slit PMT Multiple PMTS
Transmitted Image very useful for seeing where you are and for non-fluorescence areas Not confocal more conventional PMT Scattered, absorbed Objective Emitted fluorescence Excitation laser light Alignment exact as synced by the mirrors
400nm Principles of Two-Photon Microscopy Excitation of fluorophore (Jablonski diagrams) One Photon Excitation Two-photon Excitation 10-11 10-15 10-9 Absorb 1 photon 550 nm Emit one photon Relaxation Intermediate state 800 nm 10-18 Absorb 2 photons 550 nm 10-17 Emit one photon 2 photons Simultaneously absorbed Approximately twice the wavelength required but not a rule
2 Photon 1 photon Excitation cone of light confocal Large zone >1um Thin plane No aperture e needed Fluorescence Depth controlled by an Aperture Density of photons quadratic function of distance from plane of focus Emission proportional to square of excitation intensity Two-photon requires both spatial and temporal compression of photons
Two-Photon Requires: A high photon density Both photons must arrive within 10-18 secs Requires a pulsed laser: 532 nm Green Pump Laser (5-18 W) Ti:Sapphire 100 femto-second pulses Mode-locked 12.5 ns (80 Mhz) Multiple microscope elements 100 fs pulse Group Velocity Dispersion (GVD) Reduces power and penetration
Advantages of two-photon Penetration depth increased important for thick tissues Red light (longer wavelength) less scattered than blue (shorter wavelength) light Thin optical slice Requires no aperture Image formation simplified All photons useful if can be collected Good for caged compounds Excitation Beam 780 1040 nm Fluorescence only occurs at focal plane Bleaching and photo-toxicity toxicity decreased narrow plane only Microscope construction easier no need to de-scan Disadvantages Cost - expensive laser Little light obtained slower 2P Fixed focus depth
2-photon microscope layout Inverted Microscope Optio onal Beam Splitt ter 2nd System Neutral Den nsity Objective/Specimen Top adjustable Mirror Top Adjustable Mirror Dichroic Mirrors Shutter (S) Side Port Two- Photon Laser M3H/M3S Bracket Eye Piece Internal Microscope Mirror 3 PMTs CRS PMT Detectors near the objective
Dye Excitation Spectra Tunable range power varies depend on Pump power Mikhail Drobizhev, Nikolay S Makarov, Shane E Tillo, Thomas E Hughes & Aleksander Rebane Nature Methods 8, 393 399 (2011)doi:10.1038/nmeth.1596
Transmitted image and second harmonic Imaging Transmitted Fluorescence or 2nd Harmonic pmt Transmitted light image pmt DM Specimen secondary objective Added images Primary objective DM pmt Single Image 2-photon laser
Our resources Scanning Confocal 3 lasers 2/3 channels Transmitted channel Air table/ 37 C incubator Scanning Confocal 2 lasers 2 channels Air table Scanning microscope Scanning microscope 37 C incubator 37 C incubator Shared Ti:sapphire Laser (5W) Big Air table