Towards 3-Dimensional and time sequenced (4D) live cell imaging
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1 Towards 3-Dimensional and time sequenced (4D) live cell imaging Colloquium presented by Heather Campbell Supervisor: Alan Greenaway (Waves and Fields Group) In collaboration with the HW Nano-Optics Group; particularly Richard Warburton and Paul Dalgarno. Undergraduate students: Aurelie Putoud, Robert Lambert, Killian Weiss, Sebastian Kirsch, Scott Aitken, Carola Diez and Alan Baird. Additional Collaborators: Dave and Cathy Towers (UoLeeds), David Stephens (Bristol), Illan Davis (UoE, Wellcome Trust).
2 Introduction Outline Current State-of-the-Art, concentrating on: Conventional and Fluorescence microscopy Confocal and Multi-Photon microscopy Our method Multi-layer imaging by use of a DOE Application to bio-imaging Experimental Details Imaging at the nano-scale and practical particle tracking Experimental data using nano-holes and nano-spheres The Future Extension to 3D Phase Contrast Imaging Inclusion of Wavefront Sensing Towards 4D Live Cell Imaging 2
3 Goals in Biomedical Imaging Microscopy Dynamic processes of proteins & molecules Imaging of DNA (small scale) Virus tracking 4D problem (3D + time) Nanometer accuracy required Listeria invading cells Towards 4D Live Cell Imaging 3
4 Important Properties of the Microscope Resolution Successful Imaging Resolution The least separation between two separate points at which they are distinguished as separate. Magnification Contrast This should be chosen to capture the finest specimen detail Towards 4D Live Cell Imaging 4
5 Important Properties of the Microscope Resolution Successful Imaging Magnification Enlarging the image sufficiently for it to be accurately sampled by the eye/camera. Magnification Contrast Intensity M Towards 4D Live Cell Imaging 5
6 Important Properties of the Microscope Resolution Successful Imaging Contrast The ability to distinguish the imaged object from other objects and the background. Infinite resolution without contrast is useless. Magnification Contrast Idea taken from SUPA Biophotonics lecture by John Girkin Towards 4D Live Cell Imaging 6
7 Requirements for Live-Cell Imaging Primary Considerations: Specimen viability (and consideration of the specimen s natural environment) Signal to Noise (SNR) Required Speed/Time Window (to image dynamic processes) Ideally we want highly sensitive imaging, fast capture rates, high resolution and a wide dynamic range Towards 4D Live Cell Imaging 7
8 Fluorescence A large number of bioimaging applications use auto or induced fluorescence. GFP and its colour-shifted genetic derivatives allows multicolour imaging. Issues: Photobleaching, Fluorophore saturation, ph. Aequorea Victoria the bioluminescent jellyfish that gave us GFP Human Lung Tissue Towards 4D Live Cell Imaging 8
9 Imaging in 3D Achieving Depth Resolution To build 3D images we need: To be able to distinguish the signal coming from our plane of interest (focal plane) and the background. To image planes at various depths within the sample (called optical sectioning). Confocal and Multi-Photon Microscopy both achieve this and are widely used as tools for live-cell imaging Towards 4D Live Cell Imaging 9
10 Confocal Microscopy Spatial filtering eliminates/reduces secondary fluorescence. LSCM Scan the sample or beam to build a 2D slice. Collect serial z images to build the 3D image. From Booth et al SPIE Vol.5894 p26-34 (2005) Towards 4D Live Cell Imaging 10
11 Confocal Widefield Comparison of a widefield and confocal image of the same neonatal rat neuron Towards 4D Live Cell Imaging 11
12 Multiple Beam Confocal Microscopy Spinning Disc systems: Numerous apertures illuminate hundreds of spots simultaneously. Faster scan rates (video rate) Higher transmission Larger pinholes/slits = thicker optical sections Rapid live-cell imaging at reasonable cost. Image from Towards 4D Live Cell Imaging 12
13 Confocal Microscopy Pro s and Con s Advantages: The ability to produce thin optical sections. Increased SNR (reduction of background fluorescence). Zoom factor adjust spatial resolution by altering the scanning laser sampling period. Non-invasive, allows examination of live and fixed specimens. Disadvantages: Limited number of excitation wavelengths (laser lines) available. Scanning speed limits data acquisition rates. Photodamage risks with high powered lasers. Time delay in obtaining 3D images Towards 4D Live Cell Imaging 13
14 Multi-Photon Microscopy Two photons arrive simultaneously and combine their energies to excite the fluorophor. NIR excitation wavelengths mean: Higher depth penetration Less scattering Reduced photo-damage Towards 4D Live Cell Imaging 14
15 One photon Comparison of one-photon and two-photon imaging Two photon Towards 4D Live Cell Imaging 15
16 Multi-Photon Microscopy Key Property: Excitation confined almost exclusively to the focal plane (no pinholes required, increased efficiency, greatly reduced photo-damage). Lasers High peak power (ultrashort pulses), low average power. Single photon excitation and heating effects are reduced Towards 4D Live Cell Imaging 16
17 Multi-Photon Microscopy This image demonstrates that one-photon excitation creates fluorescence throughout the focal illumination cone. Two-photon on the other hand only excites fluorophors at the exact focus. One Photon Two Photon Towards 4D Live Cell Imaging 17
18 Advantages: Multi-photon Microscopy Pro s and Con s Uses longer friendlier wavelengths. Deeper penetration, less scattering. No pinholes, greater efficiency. Photobleaching and photo-damage minimised, and localised to the focal region. Dramatically increased SNR. Disadvantages: Significant work still required to profile common flurophors and develop new ones for multi-photon work. Time delay in obtaining 3D images. Complexity and cost Towards 4D Live Cell Imaging 18
19 Proposing a new method. We have used a specially designed DOE to: Provide snapshot imaging of multiple object planes simultaneously onto a single image plane. Applications: M 2 measurements, wavefront sensing, particle tracking, and now bioimaging So how does it work? Towards 4D Live Cell Imaging 19
20 D.O.E s The Basics In its simplest form a diffraction grating is a grid of multiple slits. Amplitude grating ~ formed by alternate opaque/transparent regions Phase grating ~ variation in optical thickness provides a phase delay Transmitted beams interfere to produce the familiar alternating pattern of maxima and minima Towards 4D Live Cell Imaging 20
21 Imaging with a straight grating Straight ruled grating Image Plane Object Plane f A The lens equation: A u A A = + f u v v Towards 4D Live Cell Imaging 21
22 Detour Phase By bending the straight rulings the grating we can change its imaging properties. Detour phase is the phase shift added to each diffraction order by the distortion. Distorting the diffraction grating to produce a quadratic (defocus) detour phase results in an important property: The focal length in each diffraction order is different Towards 4D Live Cell Imaging 22
23 Properties of the QD grating The focal length in each diffraction order is determined by the amount of detour phase added. The grating is used as a tool to artificially propagate the plane which is in focus in the 0 th order by ± f G in the ±1 orders, ± 2f G in the ±1 orders.. Can be set up to capture images about the focal plane of the lens or about a pupil plane. Dynamic range largely determined by the distance between the imaged planes (i.e. f G ) Towards 4D Live Cell Imaging 23
24 Multi-Plane Imaging with a QD grating Multiple Object Planes A B C f QD grating Image Plane A B f G = R 2 2mW 20 f c = L f f L G f + f s G v C Towards 4D Live Cell Imaging 24
25 Imaging at the nano-scale Moving from imaging on the macroscopic scale to the nano-scale is not a trivial problem! The system must now incorporate: high magnification high-precision positioning control vibration isolation make every photon count! Samples: Nano-holes and Nano-spheres (210nm). Simulate fluorescently tagged bio-particles. Chosen for their size and point source behaviour Towards 4D Live Cell Imaging 25
26 Image of one nanohole f c = 215mm The Microscope f=400mm CCD f=200mm iris corrected Objective 100x NA 1.3 Magnified Image Fibre illumination (633nm) Piezo Positioners (x,y,z) Nanoholes Towards 4D Live Cell Imaging 26
27 Through focal (z) series Towards 4D Live Cell Imaging 27
28 Measuring Performance Measure the FWHM and compare to the diffraction limit: Single image cross section In focus image Intensity (counts) FWHM = 233 nm Sparrow s Limit = 244nm Distance (µm) Towards 4D Live Cell Imaging 28
29 Adding the grating QD DOE f c = 215mm CCD 215mm f=200mm iris Imaging on 3 object planes 100x NA 1.3 Fibre illumination (633nm) Piezo Positioners (x,y,z) Towards 4D Live Cell Imaging 29
30 Imaging on 3 planes -1 order in focus 0th order in focus +1 order in focus Measured Resolution: Without grating = 233nm With grating = 226nm and 231nm (for 0 th and ±1orders respectively) Towards 4D Live Cell Imaging 30
31 Practical Particle Tracking Aim: to track particles in real-time in 3 dimensions (4D). A Centre Of Mass calculation on each intensity image provides the x,y co-ordinate. We have developed a method of determining the position in z using the concept of Image Sharpness. For another application of this system to Particle Imaging Velocimetry see: Towers et al Opt.Lett. 31(9) p (2006) Towards 4D Live Cell Imaging 31
32 Image Sharpness Images from Optical Transfer Function Image Sharpness is given by integrating the normalised MTF plot Towards 4D Live Cell Imaging 32
33 Calculating Image Sharpness Image Fourier Take the Modulus Transform OTF MTF Normalise Image Sharpness Integrate In 2D Normalised MTF Towards 4D Live Cell Imaging 33
34 Image Sharpness A Single Focal Series Normalised Sharpness Distance (µm) Ambiguity in z when using only a measure of the Image Sharpness. Our Solution? Compare the Sharpness curves for two (or more) different orders Towards 4D Live Cell Imaging 34
35 Image Sharpness Plots (±1 orders) 1 Normalised Sharpness Distance (µm) Towards 4D Live Cell Imaging 35
36 Modelling the Sharpness Simulated data is created to match the experimental data as closely as possible using: The defocus added by the grating. The additional defocus due to the movement of the source. Linear relationship between the Spherical Aberration (SA) in the system and the defocus from the source movement. N.B. Simulated data is noiseless and contains less aberrations. The Image Sharpness is then calculated in exactly the same way, but this time using the simulated data Towards 4D Live Cell Imaging 36
37 Modelling the Sharpness = Experiment = Model Towards 4D Live Cell Imaging 37
38 Accuracy in z ~ Measuring the Ratio linear monotonic Towards 4D Live Cell Imaging 38
39 Accuracy Initial analysis to asses the accuracy of the position measurement: Accuracy in x,y (COM) 50 snapshots taken at each position. Standard deviation gives repeatability error of better than 40nm. Accuracy in z (Sharpness measurement) Sub-nanometer over the central linear region. Region of maximum accuracy could be increased by decreasing the depth of focus Towards 4D Live Cell Imaging 39
40 The Quest for Brownian Motion Aim: To study single particles moving under Brownian motion. Fluorescent nanospheres used to simulate moving bio-particles. Seisenberger et al Science 294 p1929 (2001) Challenge: To maximise use of the available light (without bleaching) and with fast exposure times. Fluorescing Nanospheres Towards 4D Live Cell Imaging 40
41 Tracking a moving particle Towards 4D Live Cell Imaging 41
42 Transmitted Light Contrast Imaging Transmitted light techniques are often used to study cell shape, position and motility (i.e. during mitosis). Darkfield Intensity contrast is achieved in unstained specimens by manipulating/removing the un-diffracted portion of the transmitted light. Phase Contrast Diatom (unicellular algae) Male Mosquito Images from Towards 4D Live Cell Imaging 42
43 Achieving Contrast Manipulating the Fourier Plane Red = light scattered by sample Black = un-diffracted beam Image Plane Phase Object Schlieren Knife edge Dark Field Occulting Spot Fourier Plane Phase Contrast λ/4 Phase Spot Towards 4D Live Cell Imaging 43
44 3D Phase Contrast Imaging QD DOE f c = 215mm CCD Phase Spot 215mm f=200mm iris 100x NA 1.3 Magnified Image Piezo Positioners (x,y,z) Towards 4D Live Cell Imaging 44
45 Initial Results - Schlieren Imaging Results taken by Carola Diez Contrast improves as the knife edge is stepped further into the focal spot Towards 4D Live Cell Imaging 45
46 Extension to Wavefront Sensing The shape and phase of the input wavefront can be calculated using the same intensity images we use to perform the particle tracking. The difference of the ±1 order images is an approx. of the axial intensity gradient. Options for phase retrieval: Iterative solution (Gerchberg-Saxton, Simulated Annealing etc) Greens function (reported accuracy λ/1000) Analytically using the SAE (still in development) Towards 4D Live Cell Imaging 46
47 Wavefront Sensing Specimen-induced aberrations reduce signal levels and cause image degradation. Wavefront Sensing would allow: Characterisation of system aberrations. Characterisation of specimen-induced aberrations. Possibility of aberration correction (AO) or precompensation (in the grating). For more information about the problems caused by specimen-induced aberrations see: Booth et al SPIE Vol.5894 p26-34 (2005) Towards 4D Live Cell Imaging 47
48 References Live-Cell Imaging: Seisenberger, G., et al., "Real-Time Single Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus". Science. Vol. 294, p (2001) Stephens, D.J. and V.J. Allan, "Light Microscopy Techniques for Live Cell Imaging". Science. Vol. 300, p (2003) [N.B. this was a special issue containing several interesting papers on bio-imaging] Booth, M.J., M. Schwertner, and T. Wilson, "Specimen-induced aberrations and adaptive optics for microscopy" in Advanced Wavefront Control: Methods, Devices, and Applications III(Proc.SPIE, 2005). Vol p Microscopy Resources: Bradbury, S. and B. Bracegirdle, eds. Introduction to Light Microscopy. Microscopy Handbook Series. Vol , Bios Scientific Publishers. Bradbury, S. and P.J. Evennett, eds. Contrast Techniques in Light Microscopy. Microscopy Handbook Series. Vol , Bios Scientific Publishers. Eisenstein, M., "Something to See". Nature. Vol. 443, p (2006) Davidson, M.W. and M. Abramowitz, "Optical Microscopy", Available for free download from: Websites: [this is a very extensive source of information on all aspects of microscopy!] Towards 4D Live Cell Imaging 48
49 References (cont ) The Quadratically Distorted (QD) grating multi-plane imaging method: Blanchard, P.M. and A.H. Greenaway, "Simultaneous multiplane imaging with a distorted diffraction grating". Applied Optics, 38, p (1999) Blanchard, P.M. and A.H. Greenaway, "Broadband simultaneous multiplane imaging". Optics Communications, 183, p (2000). Wavefront sensing with the QD grating: Blanchard, P.M., et al., "Phase-diversity wave-front sensing with a distorted diffraction grating". Applied Optics, 39, p (2000). Campbell, H.I., et al., "Generalised Phase Diversity for Wavefront Sensing". Optics Letters, 29, p (2004). Djidel, S. and A.H. Greenaway, "Nanometric wavefront sensing" in 3rd International Workshop on Adaptive Optics in Industry and Medicine(Starline Printing Inc., Woods, S.C. and A.H. Greenaway, "Wave-front sensing by use of a Green's function solution to the intensity transport equation". JOSA A, 20, p (2003). Particle tracking with the QD grating: Towers, C.E., et al., "Three-dimensional particle imaging by wavefront sensing". Optics Letters, 31, p (2006) Towards 4D Live Cell Imaging 49
50 Please visit our website: This presentation is available to download from the Waves and Fields group website: Towards 4D Live Cell Imaging 50
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