ntroductionMorphological observations have driven the course of biology ever since the first microscope was built in the late sixteenth century. Molecular imaging is a rapidly emerging biomedical research discipline that extends such observations in living subjects to a more meaningful dimension. It may be defined as the visual representation, characterization, and quantification of biological processes at the cellular and subcellular levels within intact living organisms. It is a novel multidisciplinary field, in which the images produced reflect cellular and molecular pathways and in vivo mechanisms of disease present within the context of physiologically authentic environments. The term “molecular imaging” implies the convergence of multiple image-capture techniques, basic cell/molecular biology, chemistry, medicine, pharmacology, medical physics, biomathematics, and bioinformatics into a new imaging paradigm.GoalsThis now creates the possibility of achieving several important goals in biomedical research, namely,? To develop noninvasive in vivo imaging methods that reflect specific cellular and molecular processes, for example, gene expression, or more complex molecular interactions such as protein–protein interactions? To monitor multiple molecular events near-simultaneously? To follow trafficking and targeting of cells? To optimize drug and gene therapy? To image drug effect at a molecular and cellular level; (6) to assess disease progression at a molecular pathological level.TYPES OF TECHNIQUES AND MICROSCOPY USED FOR LIVE-CELL IMAGINGWhen choosing an optical microscopy system for live-cell imaging, the following three variables should be considered:? Sensitivity of the detector (signal-to-noise)? Specimen viability? The speed required for image acquisition considered Light microscopy ? The light microscope remains a basic tool of cell biologists, with technical improvements allowing the visualization of ever-increasing details of cell structure. Contemporary light microscopes are able to magnify objects up to about a thousand times. ? Since most cells are between 1 and 100 ?m in diameter, they can be observed by light microscopy, as can some of the larger subcellular organelles, such as nuclei, chloroplasts, and mitochondria. One of the primary and favorite techniques used in all forms of optical microscopy for the past three centuries, brightfield illumination relies upon changes in light absorption, refractive index, or color for generating contrast.Principle? As light passes through the specimen, regions that alter the direction, speed, and/or spectrum of the wavefronts generate optical disparities (contrast) when the rays are gathered and focused by the objective.? Resolution in a brightfield system depends on both the objective and condenser numerical apertures, and an immersion medium is often required on both sides of the specimen (for numerical aperture combinations exceeding a value of 1.0).? It Provides a limited degree of information about the cell outline, nuclear position, and the location of larger vesicles. However, the general lack of contrast in brightfield mode renders this technique relatively useless for serious investigations of cell structure and function.ExampleFigure : analysis of stained cells Four images are shown of the same fibroblast cell in culture. All four types of images can be obtained with most modern microscopes by interchanging optical components. (A) Bright-field microscopy. (B) Phase-contrast microscopy. (C) Nomarski differential-interference-contrast microscopy. (D) Dark-field microscopy.Figure : showing analysis of fibroblast cellsDifferential interference contrast? Differential interference contrast is an important technique for imaging thick plant and animal tissues because, in addition to the increased contrast, DIC exhibits decreased depth of focus at wide apertures, creating a thin optical section of the thick specimen. This effect is also advantageous for imaging adherent cells to minimize blur arising from floating debris in the culture medium.Principle: It requires plane-polarized light and additional light-shearing prisms to exaggerate minute differences in specimen thickness gradients and refractive index.? Lipid bilayers, for example, produce excellent contrast in DIC because of the difference in refractive index between aqueous and lipid phases of the cell.? In addition, cell boundaries in relatively flat adherent mammalian and plant cells, including the plasma membrane, nucleus, vacuoles, mitochondria, and stress fibers, which usually generate significant gradients, are readily imaged with DIC.Example: Squash preparation of stained onion root tip cells showing various stages of mitosis. Differential interference contrast optics on a Leica microscope,taken with the X40 objective lens.Figure : showing analysis of onion root cellsFigure: working of differential contrast microscopeFluorescent microscopy? It is a widely used and very sensitive method for studying the intracellular distribution of molecules.PrincipleA fluorescent dye is used to label the molecule of interest within either fixed or living cells. The fluorescent dye is a molecule that absorbs light at one wavelength and emits light at a second wavelength.? This fluorescence is detected by illuminating the specimen with a wavelength of light that excites the fluorescent dye and then using appropriate filters to detect the specific wavelength of light that the dye emits.? Fluorescence microscopy can be used to study a variety of molecules within cells. One frequent application is to label antibodies directed against a specific protein with fluorescent dyes, so that the intracellular distribution of the protein can be determined. Proteins in living cells can be visualized by using the green fluorescent protein (GFP) of jellyfish as a fluorescent label.? GFP can be fused to a wide range of proteins using standard methods of recombinant DNA, and the GFP-tagged protein can then be introduced into cells and detected by fluorescence microscopy.Table showing fluorescent proteins and their advantagesFluorescent proteinsAdvantages and uses EGFP Fluorescent proteins are useful for live-cell imaging applications because of their unique chemical structure; for example, GFP family proteins are compact, relatively small, and chemically inert mKO2Fluorescent proteins are minimally disruptive to most proteins when attached to the N or C terminus mOrange2 Fluorescent proteins can mature quickly and remain fluorescent in many subcellular compartmentstd-Tomatofold well within biological temperature ranges hcRed -as genetically encoded fluorescence markersmPlumtrack cells in tissuemonitor protein–protein interactions Dronpa, PS-CFP2, PA-mCherry, and PA-GFP as biological sensors to monitor biological events and signalsExample: Observing Mitosis with Fluorescence Microscopy ? Living epithelial kidney cells, derived from the rat kangaroo (Potorous tridactylus) and grown in culture, are often used to visualize mitosis in the microscope because they contain only a few large chromosomes and the cells remain relatively flat throughout all of the division stages. ? Termed PtK2, the marsupial kidney cells afford clear visualization of the chromosomes, mitotic spindle, nucleoli, and other components during mitosis. ? At 37 degrees Celsius, PtK2 cells undergo mitosis in approximately 2 to 3.5 hours and a typical healthy, growing culture can easily contain cells at every stage. Figure 1 presents images from five of the mitosis stages, as well as the interphase period. ? The cellular DNA was stained with DAPI (blue), while the mitochondria were stained with MitoTracker Red CMXRos (red) and the microtubules stained green with Alexa Fluor 488 attached to secondary antibodies.Figure: showing mitosis in Rat kangrooConfocal microscopy Principle ? It combines fluorescence microscopy with electronic image analysis to obtain three-dimensional images. A small point of light, usually supplied by a laser, is focused on the specimen at a particular depth. ? The emitted fluorescent light is then collected using a detector, such as a video camera. Before the emitted light reaches the detector, however, it must pass through a pinhole aperture (called a confocal aperture) placed at precisely the point where light emitted from the chosen depth of the specimen comes to a focus. Consequently, only light emitted from the plane of focus is able to reach the detector. Scanning across the specimen generates a two-dimensional image of the plane of focus, a much sharper image than that obtained with standard fluorescence microscopy Moreover, a series of images obtained at different depths can be used to reconstruct a three-dimensional image of the sample.Figure : showing working of confocal microscopyTwo-photon excitation microscopy ? It is an alternative to confocal microscopy that can be applied to living cells. The specimen is illuminated with a wavelength of light such that excitation of the fluorescent dye requires the simultaneous absorption of two photons. Principle ? The probability of two photons simultaneously exciting the fluorescent dye is only significant at the point in the specimen upon which the input laser beam is focused, so fluorescence is only emitted from the plane of focus of the input light. ? This highly localized excitation automatically provides three-dimensional resolution, without the need for passing the emitted light through a pinhole aperture, as in confocal microscopy. Moreover, the localization of excitation minimizes damage to the specimen, allowing three-dimensional imaging of living cells.Figure : working of two contrast phase microscopyRadionuclide ImagingRadionuclide imaging:”Radionuclide Imaging is a test which produces the scans of the internal body parts by using the small amount of a radioactive material.”Introduction:The test is used for providing the images of the organs and different areas of body which cannot be examined well with the standard X rays. Different abnormal tissues growth such as the tumors are specifically visible by using the nuclear imaging.In the analytical imaging, two types of modalities may be differentiated.1. Anatomical imaging: This provides a very accurate and clear visualization of internal structures of human body.2. Functional imaging: That is aimed for the quantification of the physiological processes that take place inside human body without any influence or side effect.Principle:”The non-invasive resolving of the physiological phenomena. A radio-pharmaceutical is Radioactive-Drug that is used for the Diagnosis or for Therapy in minute quantities by no pharmacological impact. This tracer is scattered, absorbed, and evacuated in accordance to their chemical structure. Their biological functions may be displayed in the form of images, time-activity curves or numerical data.”i. Radio-labelled tracers are administered.ii. ? rays are emitted by radioisotopes and are detected external to the body on a gamma cameraiii. lead collimators that are used to absorb the scattered ? rays.iv. ? rays intrude on the sodium iodide crystals and converted them into the light that is detected by the photo-multipliers.Types of Radionuclide Imaging:a) PETb) SPECTPosition Emission Tomography:Positron emission tomography also called as “PET” imaging or “PET scan”. It is a kind of the Radio-nuclide imaging.Positron emission tomography uses the small amounts of the radioactive materials that are called as Radiotracers, special camera and a computer that helps to evaluate organs and tissues functions. By analyzing body changes at cellular level PET can detect early start of the disease before the evidence by other imaging tests. ? PET scan measures the important functions of body like oxygen use, blood flow and glucose metabolism to help the doctors to evaluate the functioning of organs and tissues. ? Now almost all PET scans are completed on those instruments that contain combination of PET and CT-scanners. These combined PET-CT scans give images that may pinpoint anatomic location of the abnormal metabolic functioning within body. These combined scans provide more precise and accurate diagnostic results.Example of PET Scan:Brain PET Scan:? Brain positron emission tomography scan is a scan test which allows the doctors to examine how the brain is working.? Scan captures the images of activity of brain after the radioactive „tracers? have absorbed in the blood-stream. These tracers are then attached to the compounds like sugar. Glucose is the main fuel of brain.? Different active areas of brain will utilize the glucose at higher rate than the inactive areas. When they are highlighted under PET scanner, it will allow doctors to examine how brain is functioning and helps them to detect abnormalities if any.How a Brain PET Scan Is Performed:1. Patient is brought into procedure room and then seated in a chair.2. Technician would insert an intravenous catheter into patient?s arm. A specific dye with the radio-active tracers will then be injected into patient?s veins by this IV. Human body needs sometime to absorb these tracers as the blood flows through brain. It typically requires about an hour.3. Further scan will be undergone. It involves lying on narrow table that is attached to PET machine, that looks like giant paper roll. This table glides gradually and smoothly in the machine so that the scan can be completed.4. Patient must lie still during this scan. This scan records brain activity as such. This activity may be recorded as a video or in the form of still images. The tracers are in more amount in the areas of improved blood flow.5. When the chosen images are stored in computer, patient will exit the machine. Test is completed.Benefits of PET SCAN: ? Provides details on both the functions and anatomic structures of body. ? For several disease PET scans yield the useful information regarding a diagnosis or for determining an appropriate treatment. ? PET Scan is less expensive and can give more accurate information than examining surgery.Risks: ? Radiation exposure is very low to patient for examination so the radiation risk is low as compared with the potential benefits. ? There are no long-term side effects of these low exposure radiations. ? Rare allergic reactions may occur. ? Injection of radiotracer can cause slight pain and the redness that must be rapidly resolved.Single photon emission computed tomography:SPECT scan involves a gamma camera that rotates around patient to produce images of many angles to form a tomographic image by computer.Principle:SPECT scanning provides a new 3-D view with improved scratch detectability resulting by the elimination of the over-lying and under-lying source activity.Example:Brain perfusion SPECT scanning is a “functional” radionuclide imaging technique that is performed to evaluate the regional cerebral perfusion.Because CBF is closely related to the neuronal activity in different areas of brain.A lipophilic PH neutral radiopharmaceutical 99m Tc HMPAO and 99m Tc ethylene cysteine diethyl ester is injected in the patient that crosses the blood brain barrier and then continues to emit gamma rays. It can be detected using gamma detectors.Benefits of SPECT:More often point out the site of abnormalityIf there are some problems due to the location changes in body like it is difficult to find the abnormality in feet, fusing a SPECT with the CT can detect abnormality.Risks:? Small dose of radiation is injected so there are no risks.Optical imaging: Optical imaging is a process for the non-invasive looking in the body by using visible light and other properties of photons to get detailed images of the organs and tissues and many other small structures like molecules and cells.Bioluminescence Imaging:”Bioluminescence imaging is used mostly for the preclinical molecular and cellular imaging of small animals. It refers to the light produced by enzymatic reaction of luciferase enzyme with substrate.”luciferase is the most commonly used luciferase for the molecular imaging. It oxidizes its substrate that is luciferin that emits light with an emission spectrum and peak at 560 nm.Example:Firefly luciferase needs D luciferin to be injected in the body prior to the imaging.Its emission peak wavelength is almost 560 nm. Due to the weakening of blue green light in the tissues, the red shift of this emanation makes the detection of the firefly luciferase more sensitive in vivo.Benefits:? Helps in the substrate biosynthesis mostly by bacterial substrate.? Develops bioluminescent pathogens in prokaryotes that helps in the genetical engineering of it into mammalian expression system.Fluorescence Imaging:”Fluorescence imaging involves the external light of an accurate wavelength to excite a targeted molecule (Fluorescent protein, endogenous molecules or other optical contrast agents) that gives the release of longer wavelength for imaging (low-energy light).”Example:Epi fluorescent scanning involves three components in a dividing cancer cell of human.DNA is marked blue -a protein called “INCENP” is green and the micro-tubules are red. Every fluorophore is scanned separately by using a different amalgamation of excitation and the emission filters. The images are then captured serially using a digital CCD camera then over-laid to form a complete image.Benefits of Optical Imaging:? Reduces exposure to harmful radiations by the usage of non-ionizing radiations.? Visualizes soft tissues.MAGNETIC RESONANCE IMAGING? An MRI and CT scanner are similar in a way that it provides cross-sectional images of the body. X-rays are used for CT scan instead of MRI. Rather, a strong magnetic field and radio waves are used in MRI for producing very clear and detailed high-tech images of the inner side of the body.? An abnormality detected on a CT scan can be further evaluated by MRI.? Spine MRI is commonly used for a herniated disk or narrowing of the spinal canal in people with arm, neck, back or leg pain.? Bone and joint MRI can also be used to check the bones, joints, and soft tissues. It can be used to detect injured tendons, ligaments, cartilage, muscles and bones. Moreover, it can also be used to detect infections and masses.? For women, Pelvic MRI in women provides a comprehensive look at the ovaries and uterus and is frequently used to follow up any abnormality found in ultrasound. For men, pelvic MRI is to check for prostate cancer. Pelvic MRI is also used to check at the bones and muscles of the pelvis.PrincipleMRI makes use of the magnetic properties of certain atomic nuclei. Hydrogen nucleus or single proton present in the water molecules, and consequently in all body tissues. The hydrogen nuclei are then partially aligned by the strong magnetic field in the scanner. Radio waves are used to rotate the nuclei, and they later oscillate in the magnetic field while returning to equilibrium. Simultaneously, a radio signal is emitted. This is detected using antennas or coils.What an MRI Scan can diagnose?• Most of the ailments of brain• Sport injuries• Musculoskeletal problems• Most of the spinal injuries• Vascular abnormalities• Pelvic problems in females• Prostate problems in males• Gastrointestinal tract conditions• ENT conditions• Soft tissue and bone pathologyWho can’t have an MRI Scan?• A cardiac pacemaker• Certain clips in the head due to brain operations• A cochlear implant• A metallic foreign body in your eye• Had surgery in the last 8 weeksMRI Preparation? Depending on which part of the body is being imaged, it is necessary to wear a hospital gown. Clothes with metal snaps should be replaced with a gown.? No such preparation is needed. The only exception is the study of bile ducts, called an Magnetic Resonance Cholangiopancreatography, in which eating or drinking is not allowed for 2 to 3 hours before the test.? A contrast or dye may be needed to inject into a vein through an IV. This helps the doctor to see the inner side of the body.When are MRI scans used?Neurosurgeons uses MRI not only in explaining brain anatomy but also evaluates the integrity of the spinal cord after trauma. It is also associated with the vertebrae or intervertebral discs of the spine. It can evaluate the structure of the heart and aorta, where it can detect aneurysms or tears. MRI scans are not the first line of imaging test for these issues or in cases of trauma. It provides valuable information on glands and organs within the abdomen, and accurate information about the structure of the joints, soft tissues, and bones of the body.COMPUTED TOMOGRAPHY? Computer tomography also known as computed axial tomography and body section rentenography.? It was designed by Godfrey N. Hounsfield? Plain radiography includes X-rays which pass through the patient, and then create an image directly on a photographic film. Image is mainly a shadow. An incomplete picture of an object’s shape is obtained.? Thus, a three-dimensional structure is represented on a two-dimensional plane, that gives rise to disturbing superimposition.? CT eradicates the superimposition of structure images outside the area of interest. The “tomography” word is basically derived from the Greek word tomos meaning „slice? and graphein that means „to write?.? CT is generally more widely available and is cheaper.PrincipleX-rays are attenuated, on their way through tissues because of absorption of the energy. Different tissues give different degrees of X-ray attenuation. The X-ray tube emits a thin collimated fan-shaped X-rays beams that are weakened when they pass through the tissues. They are generally detected by a series of special detectors. X-ray photons create electrical signals, which are then converted into the images. High density areas appear as white whereas low density areas appear as black.CT technology generations• First generation: These CT scanners used a pencil-thin radiation beam directed at one or two detectors. The images were attained by a “translate-rotate” technique in which the x-ray source and detector in a static relative position passes across the patient followed by a rotation of the x-ray source/detector combination (gantry) by one degree.• Second generation:This design amplified the number of detectors and altered the shape of the radiation beam. The x-ray source changed to a fan shaped beam. Rotation was amplified from one degree to thirty degrees.• Third generation:In the third generation, a fan shaped beam of x-rays is directed to an array of detectors that are fixed in position comparative to the x-ray source.• Fourth generation:The 4th generation scanners used a stationary 360-degree ring of detectors. The fan shaped x-ray beam moves around the patient focused at detectors in a non-fixed relationship.What are CT scans used for?? CT scans are generally more detailed than regular X-rays.? The information obtained from the two-dimensional images of computer can be recreated to build three-dimensional images by modern CT scanners.? take pictures of the brain and virtually any part of the body.? finest and fastest tools for examining the chest, abdomen and pelvis.? It detects different types of cancer such as in the lung, bowel, liver or kidney, examining patients with certain injuries and provides the cause for sudden rapid onset symptoms.? CT is used for the detection, analysis and treatment of vascular disease, that ultimately lead to a stroke, kidney failure or blood clots in the lungs.? CT diagnoses many spinal problems & injuries that may occur to the hands, feet and other skeletal structures.? CT is good for providing detailed images of even very small bones.