Bioimaging 2 Notes PDF

Module structure  Fundamentals of light  Propagation of light in waveguides  Fundamentals of matter  Light interaction with matter  Laser, LED and photodetector basics  Photobiology basics  Biophotonics applications  Bioimaging  Optical Biosensors  Flow cyt...

Module structure  Fundamentals of light  Propagation of light in waveguides  Fundamentals of matter  Light interaction with matter  Laser, LED and photodetector basics  Photobiology basics  Biophotonics applications  Bioimaging  Optical Biosensors  Flow cytometry  Light activated therapy  Tissue engineering  Topics to be covered  Introduction  Optical and Non-Optical  Overview of methods  Transmission, reflection, fluorescence  Imaging techniques  Phase contrast microscopy  Dark-field microscopy  Fluorescence microscopy  Confocal microscopy  Two-photon fluorescence light microscopy  Optical coherence tomography  Fluorescence resonance energy transfer  Introduction  Bioimaging is a major branch of Biophotonics  Where as X-ray, computerised (axial) tomography (CT) scans, ultrasound and magnetic resonance imaging (MRI) are geared towards structural images at an organ level, diagnosis and treatment of diseases often requires imaging at a cellular and sub-cellular level  Optical techniques allow the study of a wide range of specimens in vivo, ex vivo and in vitro  Different techniques cover a broad range of sample types and sizes  Bioimaging relies on an optical contrast in light transmission, reflection or fluorescence between the area of interest and the background  Various techniques have been developed over the years to improve this contrast, thus improving the image quality  Non-Optical vs. Optical imaging Non-Optical methods Optical methods  X-ray and CT scans cause  Not harmful (above UV) ionisation - can be harmful  Imaging of objects as small  X-rays unsuitable for very as 100nm (using near-field) young patients  Multidimensional imaging  X-rays unable to distinguish possible between benign and  Imaging of in vivo, in vitro, malignant tumours specimens possible  MRI cannot provide real time  Fluorescence imaging can changes at a cellular level monitor spectra, quantum  Ultrasound resolution is efficiency, lifetime and poor. Unable to distinguish polarisation between benign and  Optical imaging can be malignant tumours combined with other techniques, e.g. ultrasound  Optical methods of imaging Optical Imaging Transmission Reflection Fluorescence (Transillumination) (Back Scattering) P.N. Prasad, Introduction to Biophotonics, Wiley-Interscience, 2003  Transmission P.N. Prasad, Introduction  A tissue is a highly to Biophotonics, Wiley- Interscience, 2003 scattering media  When a sample is illuminated by a pulse of light, the photons that emerge at the end can be categorized into three types  Coherently scattered photons (ballistic photons) slightly longer to arrive and are called snake do not scatter and thus take photons the shortest route to the  Most photons undergo severe scattering and detector. A small pulse after so traverse longer paths before arriving at the a very short time lapse detector. These are called diffuse photons indicate their arrival  Ballistic photons carry the most information  about the medium, followed by snake photons Photons that are scattered slightly, but mainly in the  Diffuse photons carry very little information and usually have to be removed from the forward direction take measurement Why?  Techniques for transmission microscopy  Spatial filtering – a confocal aperture (pinhole) on the central axis rejects a substantial amount of off-axis photons. Since diffused photons are more likely to be spread out off-axis, spatial filtering with a pinhole collects mainly ballistic photons and some snake photons. This is the most simple form of filtering and is used in reflection and fluorescence imaging  Polarisation gating – the sample is illuminated with linearly polarised light. Ballistic photons will maintain the same polarisation when they exit the sample. Snake photons will be partially depolarised. Diffuse photons will be highly depolarised. If light is collected through a linearly polarised window that has the same polarisation as the original light source, then diffuse light will be filtered out  Time gating – the sample is illuminated by a short pulse of light. An optical gate at the receiver end opens to allow ballistic and/or snake photons through and shuts immediately after. A number of established techniques are available for synchronising the gate to the ballistic photons  Optical methods of imaging Optical Imaging Transmission Reflection Fluorescence (Transillumination) (Back Scattering) P.N. Prasad, Introduction to Biophotonics, Wiley-Interscience, 2003  Reflection  Reflection imaging collects back-scattered light from the sample  As with transmission imaging, coherently back- scattered light needs to be discriminated against multiply back-scattered light  Two techniques used are confocal and interferometric  Confocal – spatial filtering is carried out using a confocal aperture (pinhole) on the central axis  Interferometric – a technique called optical coherence tomography (OCT) uses the interference between a reference signal and back-scattered light to image a surface  Optical methods of imaging Optical Imaging Transmission Reflection Fluorescence (Transillumination) (Back Scattering) P.N. Prasad, Introduction to Biophotonics, Wiley-Interscience, 2003  Fluorescence  Widely used optical bioimaging technique  Allows detailed probing of structure and dynamics, both in vitro and in vivo, for widely varying tissue dimensions  REMEMBER energy of excitation is greater than the energy of emission  Has very high signal-to-noise ratio so even small samples can be used  A number of fluorophores (molecule responsible for fluorescence) have been developed which can be added to biological samples  These fluorophores bind with specific molecules in the sample (tagging) so that specific organelles can be observed under fluorescence microscopy  Optical methods of imaging Optical Imaging Transmission (Transillumination) Spatial Filtering Polarisation Time Confocal Gating Gating Microscopy Reflection (Back Scattering) Spatial Filtering Interferometric Confocal Optical Coherence Microscopy Tomography Fluorescence Spatial Filtering Spatially Polarisation Fluorescence Confocal Resolved Resolved Resonance Microscopy Localised Energy Transfer Spectroscopy (FRET) P.N. Prasad, Introduction to Biophotonics, Wiley-Interscience, 2003  Effects of refractive index variations  Many biological samples such as single cells introduce very little amplitude change in transmitted light making it difficult to observe under traditional bright field illumination  However, light passing through the sample can under go diffraction, refraction and phase changes because of minute refractive index changes between regions  The human eye is not suitable for detecting phase changes and so a number of techniques have been developed to make use of the information in the received signal’s phase, thus improving the image contrast http:// www.microscopyu.com/ Standard image (Bright field) Using Phase Information articles/phasecontrast/ phasemicroscopy.html  Phase contrast microscopy  Light passing through the condenser annulus forms a hollow light cone which is focused on the sample  The objective lens collects all the light (which includes diffracted light as well as direct light) and focuses it onto a phase plate, Diffracted light lags by 90°  The phase plate changes the amplitude & phase of only the direct light by 90°, bringing the direct and diffracted light into phase (or completely out of phase)  At the image plane, the interference between the direct and refracted light has a bigger impact on the light intensity http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html because the they are both in phase (or completely out of phase)  Modern contrast microscopes use  Therefore, the phase difference has been electronic adjustments and signal translated into a amplitude difference processing to further improve the image  Dark-Field microscopy  In transparent samples, direct transmitted light drowns out the diffracted/refracted light  Here, the objective lens is designed such that only the diffracted/refracted light is transmitted through to the eyepiece  The hollow cone of light which is focused on the From Wikipedia sample does not enter the objective lens  Only light  This technique allows images to be diffracted/refracted from formed without staining the sample the cone by the sample will  May require very strong illumination enter the objective lens since received light intensity will be very low – photo-oxidation side effect  Epi-fluorescence microscopy  The sample is excited with short wavelength light (e.g. UV) through the same objective lens that collects the fluoresced light (e.g. green) for imaging  The beam splitter, which is a dichromatic mirror, only reflects light with a short wavelength. Longer wavelength light passes P.N. Prasad, Introduction to Biophotonics, Wiley-Interscience, 2003 straight through  Since fluoresced light always has a longer wavelength, only the fluoresced image will be seen at the eyepiece  Different fluorescent dies that attach to specific molecules can be added to the sample so that the image forms in different colours From Wikipedia  Scanning optical microscopy  In fluorescence imaging, the out-of-focus regions of the sample appear as “flare” which reduces the signal-to-noise ratio  High intensity excitation on the whole sample may induce photo- oxidation of the fluorochrome  In scanning optical microscopy, the sample is illuminated point- by-point by a fast scanning laser in a X-Y raster pattern  The laser beam direction is controlled by movable mirrors  Intensity information from each point is recorded on a computer, which forms a complete image with all the data points  The resolution of the image is limited by the laser spot size  With the low intensities used in this technique, flare and photo- oxidation are reduced  Confocal microscopy  A thick sample in a wide-field microscope will have a finite section that falls within the objective lens’s depth of field  This section will appear as a sharp image  However, sections of the sample that fall outside the depth of field will appear blurred  The observed image will be a combination of both focused and blurred components  High magnification requires a high numerical aperture which results in a limited depth of field  Therefore, at high magnifications (> x10), the blurring becomes an issue  The blurred background diminishes the contrast of the in- focus image  Confocal microscopy - 2  Confocal microscopy uses a pinhole in the path of the image forming beam to reject all the out of focus light  Light from the focal plane on the sample focuses exactly onto the hole in confocal aperture  This technique is combined with scanning optical microscopy to form a complete image from P.N. Prasad, Introduction to Biophotonics, Wiley-Interscience, 2003 individual data points  By moving the sample  When layers are combined together in a closer or further away from computer, a 3D image of the sample can be the objective lens, the laser produced scanning beam can image  Since very little light reaches the eyepiece, thin layers of the whole high optical intensities are required, which sample may photo-oxidise any fluorochromes  Confocal microscopy - 3 Widefield Confocal http://www.olympusfluoview.com/theory/confocalintro.html  Note the sharp focus and 3D nature of the formed image in confocal microscopy  Two-photon fluorescence light microscopy  Uses simultaneous excitation of a fluorophore by two low- energy photons (typically in IR spectrum)  Lens focuses light to a single point in the tissue to produce very high intensity light in a very small region  Fluorescence is limited to this very small region (), which allows 3D imaging and removes need for a pinhole  irradiance needed  Typically femto or picosecond laser pulses used to minimise thermal damage  Suitable for thick materials as less tissue absorption and scattering in IR spectrum Peter TC Sc, Two-photon Fluorescence Light Microscopy, Secondary article, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA  Optical coherence tomography (OCT)  OCT uses light (in the IR or near-IR range) to form reflection images in a similar way to sound in ultrasound imaging  OCT forms an image by measuring the “echo time-delay and intensity of back-scattered or back-reflected light”1 from the sample under investigation  Where as ultrasound requires contact, OCT can be carried out at a distance  OCT offers considerably higher resolution than ultrasound but the light cannot penetrate as far as into the sample  A typical resolution of 100μm for ultrasound requires a time resolution of approximately 100ns. This can be easily detected by modern electronic circuits and components  The speed of light in water is ~200,000 larger than that of sound so the time resolution required to detect a light “echo” in a 10μm sample is in the 30fs range  This is outside the range of modern electronics and so a different detection method is used based on the Michelson Interferometer 1 B.E. Bouma and G.J. Tearney, Handbook of optical coherence tomography, Marcel Dekker Inc, 2002  Optical coherence tomography (OCT) - 2 P.N. Prasad, Introduction to Biophotonics, Wiley-  Light from the source is Interscience, 2003 split two ways equally by the beamsplitter  The reference beam heads towards a movable reference mirror  The sample beam heads towards the sample under observation  Following reflection (and backscatter), both beams recombine back at the beamsplitter and  The interference caused by the combining is collected by the beams is dependent on the difference in detector distances travelled by both paths  Optical coherence tomography (OCT) - 3  Interference fringes will be seen at the detector when the reference mirror is moved up or down (i.e. changing the reference beam path length)  If a highly coherent source (e.g. laser) is used, the same interference fringes will be observed for a wide range of path length differences. There will be no noticeable change in intensity at the detector with increasing reference mirror displacement  If the source has low coherence (e.g. LED), constructive interference will be detected but only when the path length difference is within the coherence length of the light source  The coherence length is the distance over which the light wave has temporal coherence – shorter the length the better the resolution.  The beam splitter (e.g. half-silvered mirror) will only split the beam equally at a specific wavelength, so a large portion of the LED band will not be used  A superluminescent light source is needed  Optical coherence tomography (OCT) - 4  For a fixed sample position, the reference mirror is scanned up and down whilst the received optical intensity is recorded at the detector  For any given location of the reference mirror, only light from a certain depth range within the sample (unique to that particular reference mirror location) will constructively interfere with the reference beam  Therefore the magnitude of the backscattered sample beam and the echo time can be measured by scanning (moving up and down) the reference mirror and demodulating the interference signal  The depth resolution into the sample is governed by the coherence length. The less coherent the source is, the better the resolution  The transverse resolution across the sample is governed by the size of the optical beam spot falling on the sample  If the sample is mounted on a XYZ stage, a 3D image can be formed using computer controlled scanning and image processing  Depth resolution can be further improved by using a confocal aperture (pinhole)  Optical coherence tomography (OCT) - 5  Advantages include  High resolution – OCT can achieve 4-10μm compared with 110 μm for ultrasound  Real-time imaging  Catheter/Endoscopes – fibre optic based designs can be easily integrated with catheters and endoscopes  OCT of retina From https://epsomeyecare.co.nz/optical-coherence-tomography/  Spectral and time-resolved imaging  Microscopy methods described so far have been able to provide spatial resolutions fine enough to image sub-cellular structures  Spatial imaging by itself is insufficient to study biological functions – to observe process in real-time  This requires additional information from spectral and time-resolved imaging  Spectral and time-resolved imaging is primarily used for detecting fluorescence  Therefore, these techniques can be used in conjunction with epi-fluorescence and confocal microscopy  Spectral imaging  In fluorescence-based imaging techniques discussed earlier, the spatial imaging provides structural information about cells and tissues  Spectral imaging provides additional information by examining the frequency content of the fluoresced light  This allows the use of more than one fluorescent marker and the spatial tracking of different markers within the sample (different markers will fluoresce at different frequencies)  Shifts in the emission profiles also provide information on the biological processes taking place (used widely in drug-organelle interaction studies)  Spectral imaging techniques can involve:  Bandpass filtering – if the fluorescence spectra of the different markers are widely spaced, simple bandpass filters can be used to isolate each emission band  Excitation wavelength – if the excitation spectra are widely spaced, the illumination wavelength can be tuned for each marker in turn  Fluorescence resonance energy transfer (FRET) imaging  This spectral imaging technique makes use of two different fluorescent markers (fluorophores) to study molecular reactions (such as protein-protein interactions and calcium metabolism)  To study protein-protein interactions, one protein is tagged with a fluorophore that needs short wavelength energy to fluoresce (donor)  The other protein is tagged with a fluorophore that needs longer wavelength to fluoresce (acceptor) www.nature.com  The donor and acceptor fluorophores are  If the acceptor fluorophore is within carefully selected so that the emission 10nm of the donor, a non- band of the donor overlaps the radiative energy transfer occurs absorption band of the acceptor from the donor to the acceptor  The sample is illuminated with a narrow  This causes the decrease in donor bandwidth of light corresponding to the fluorescence and increase in donor absorption band acceptor fluorescence Fluorescence resonance energy transfer (FRET) imaging -2  Imaging is completed by measuring the intensities of donor emission (ID) and acceptor emission (IA) at all XY locations across the sample and calculating the IA/ID ratio at each location  The dipole-dipole interaction between fluorophores causing non-radiative energy transfer occurs only at very close proximities  The distance dependence for the process falls at a rate of R-6 where R is the separation distance  This allows very fine resolution imaging (