Imaging Techniques in Pharmacology MAIN.pptx

Pharmacolgoy skills

1. Imaging Techniques in Pharmacological Research Mr. Payaam Vohra M.S.(Pharma) Pharmacology and Toxicology NIPER MOHALI

2. HIGH THROUOPUT SCREENING

3. TYPES OF IMAGING TECHNIQUES

4. WHAT CAN BE OBSERVED IN IMAGING TOOLS

5. Digital DIAGNSOIS: RADIOMICS AI-powered applications provide personalized therapeutic interventions and diagnosis for various medical conditions, offering virtual support and guidance.

6. Routine pinhole collimator fitted with custom-made plastic adaptor. Using dorsal skin grasp and immobilization against adaptor makes high-quality imaging possible without need for sedation and with standardization of distance and orientation toward pinhole aperture.

7. SHARED DECISION MAKING IN EARLY DIAGNOSIS OF DISEASES

8. https://youtu.be/iiRlVM_M7i0

9. COMPANIES UTILIZING AI POWERED IMAGING IBM Watson Health: Description: IBM Watson Health offers AI-powered solutions for medical image analysis and interpretation. Applications: Oncology imaging, neuroimaging, and general medical imaging. Siemens Healthineers : Description: Siemens Healthineers integrates AI into its imaging and diagnostics solutions to improve efficiency and accuracy. Applications: AI-powered tools for CT, MRI, X-ray, and ultrasound . Description: Canon Medical Systems utilizes AI for image reconstruction, analysis, and diagnostic support. Applications: AI-powered solutions for CT, MRI, and ultrasound . Nuance Communications : Description: Nuance provides AI-driven solutions for speech recognition, medical transcription, and radiology reporting. Applications: AI-powered radiology reporting and workflow optimization .

10. Chest X-ray and CT Scan Analysis : Application: Detection of Pulmonary Abnormalities AI Functionality: Algorithms can analyze chest X-rays or CT scans to identify patterns indicative of conditions like pneumonia, lung nodules, or other pulmonary diseases. MRI Brain Image Analysis: Application: Neurological Disorders Diagnosis AI Functionality: AI algorithms can assist in the analysis of brain MRI images to detect abnormalities such as tumors , aneurysms, or signs of neurodegenerative diseases. Mammography and Breast Ultrasound : Application: Breast Cancer Screening AI Functionality: early detection of breast cancer by analyzing mammograms and breast ultrasound images, identifying potential lesions Dermatology Imaging: Application: Skin Cancer Detection AI Functionality: Photographs or dermoscopy images, can be analyzed by AI algorithms to detect features associated with skin cancer. Ophthalmic Imaging (Retinal Scans): Application: Diabetic Retinopathy Detection AI Functionality: AI-powered tools can analyze retinal scans to identify signs of diabetic retinopathy, macular degeneration, or other eye conditions, supporting early intervention.

11. Application: Organ Abnormalities and Tumor Detection AI Functionality: AI algorithms can analyze abdominal imaging to detect abnormalities in organs such as the liver, kidneys, or pancreas, as well as identify tumors or cysts. Cardiac Imaging (Echocardiography, CT, MRI): Application: Cardiovascular Disease Diagnosis AI Functionality: AI assists in analyzing cardiac imaging data to assess heart function, detect structural abnormalities, and identify signs of cardiovascular diseases. Musculoskeletal Imaging (MRI, X-ray): Application: Bone and Joint Disorders AI Functionality: AI algorithms can analyze musculoskeletal imaging to identify fractures, arthritis, or other bone and joint disorders, assisting in diagnosis and treatment planning. Computed Tomography Angiography (CTA): Application: Vascular Abnormalities AI Functionality: AI can analyze CTA scans to detect vascular abnormalities, such as aneurysms, stenosis, or other conditions affecting blood vessels. Prostate Imaging (MRI, Ultrasound): Application: Prostate Cancer Diagnosis AI Functionality: AI-powered imaging analysis can assist in the detection and characterization of prostate cancer using MRI or ultrasound data.

12. Histology Histos : Tissue Logia to study or learn Histology, branch of biology concerned with the composition and structure of tissues in relation to their specialized functions Histological Studies examine quantities of tissue that have been removed from the living body; these tissues are cut into very thin, almost transparent slices using a special cutting instrument known as a microtome. Bears a close relationship to several other biological sciences like anatomy , physiology, biochemistry Bears a close relationship to severalother biological and medical science suchas anatomy, biochemistry, physiology • Basic medical science                                                                                                                                                                                                          

13. There are multiple steps involved in Tissue preparation followed by sectioning and staining. After staining we counter stain the sections and observe under microscope

14. Tissue Preparation

15. Mouse Brain with Barium sulfate

16. Fluorescent Microscopy Fluorescence emission (this light is non-coherent and is emitted over a spherical volume surrounding the fluorophore) is captured by the objective and directed back through the dichromatic mirror, which in turn reflects most of the contaminating excitation light back toward the light source. Emission wavelengths passing through the dichromatic mirror are further purified by another filter of defined bandpass , the  emission  filter, before traveling to the eyepieces or the camera image plane.

17. fluorochrome  refers to a molecule that exhibits fluorescence, whereas  fluorophore  is used to identify a fluorochrome that is attached to a binding partner that enables it to target a specific biological entity.  The key to fluorescence microscopy is the use of appropriate filters to segregate the intense excitation light from the much weaker secondary emission generated by fluorophores. Fluorescent filters are used

18. A basic principle in fluorescence microscopy is the highly specific visualization of cellular components with the help of a fluorescent agent. This can be a  fluorescent protein  – for example  GFP  – genetically linked to the protein of interest. If cloning is impossible – for instance in histologic samples – techniques such as  immunofluorescence staining  are used to visualize the protein of interest. For this purpose, antibodies are utilized, which are linked to distinct fluorescent dyes and bind to the adequate target structure either directly or indirectly. With the help of fluorescent dyes, fluorescence microscopy is not only restricted to proteins but can also be used to detect nucleic acids, glycans and other structures. You can even use an application-specific variety of  live cell dyes  available that allow for organelle visualization with organelle-selective stains (e.g. ER, mitochondria, Golgi) or function assays like, e.g. live cell tracking, labeling, cell proliferation, or live dead assays, where fluorescence is the way of read-out. Even non-biological substances like Calcium ions can be detected. This article provides an introduction to the commonly used fluorescent agents.

19. FITC Fluorescein isothiocyanate (FITC) is an organic fluorescent dye and probably one of the most commonly used in immunofluorescence and flow cytometry. It has an excitation/emission peak at 495/517 nm and can be coupled to distinct antibodies with the help of its reactive isothiocyanate group, which is binding to amino, sulfhydryl, imidazoyl , tyrosyl or carbonyl groups on proteins. 

20. A dye very often used in combination with FITC is TRITC (Tetramethylrhodamine-5-(and 6)- isothiocyanate ). In contrast to FITC, TRITC is not a fluorescein but a derivate of the Rhodamine family. Rhodamines also have a large conjugated aromatic electron system, what leads to their fluorescent behavior. TRITC is excited with light in the green spectrum with a maximum at 550 nm. Its emission maximum is lying at 573 nm. The bond to proteins (e.g. antibodies) is also based on a reactive isothiocyanate group.

21. Cyanines Alexa Fluor® dyes are a big group of negatively charged and hydrophilic fluorescent dyes, frequently used in fluorescence microscopy. All the Alexa Fluor® dyes are sulfonated forms of different basic fluorescent substances like fluorescein, coumarin , cyanine or rhodamine (e.g. Alexa Fluor®546, Alexa Fluor®633). The respective laser excitation wavelength is mentioned in their labeling. For example, Alexa Fluor®488, one of the most commonly used dyes, has an excitation maximum at 493 nm, which allows excitation with a standard 488 nm laser, and an emission maximum at 519 nm Alexa Fluor®488 is a fluorescein derivate and has similar properties than FITC. However, it shows better stability, brightness and lower pH sensitivity.

22. DNA staining DAPI (4',6-diamidino-2-phenylindole) which binds to A-T rich regions of the DNA double helix. DAPI fluorescence intensity increases if attached to DNA compared to its unbound state. It is excited by UV-light with a maximum at 358 nm. Emission spectrum is broad and peaks at 461 nm. A weak fluorescence can also be detected for RNA binding. In this case, emission shifts to 500 nm. Interestingly, DAPI is able to permeate an intact plasma membrane which makes it useful for fixed and living cells.

23. A membrane-impermeable DNA stain is  Propidium-Iodide  which is often used to differentiate between living and dead cells in a cell culture because it cannot enter an intact cell. Propidium -Iodide is also an intercalating agent but with no binding preference for distinct bases. In the nucleic acid bound state, its excitation maximum is at 538 nm. Highest emission is at 617 nm.

24. A dye which is capable to make a difference between DNA and RNA without previous manipulation is  Acridine Orange . Its excitation/emission maximum pair is 502 nm/525 nm in the DNA bound version and turns to 460 nm/650 nm in the RNA bound state. Furthermore, it can enter acidic compartments like lysosomes where the cationic dye is protonated. In this acidic surrounding Acridine Orange is excited by light in the blue spectrum, whereas emission is strongest in the orange region. It is often used to identify apoptotic cells, as they have a lot of engulfed acidic compartments

25. Compartment and organelle specific dyes To observe mitochondria is the utilization of  MitoTracker ® . This is a cell permeable dye with a mildly thiol-reactive chloromethyl moiety used to bind to matrix proteins covalently by reacting with free thiol groups of cysteine residues. LysoTracker  is a group of dyes available in different colors used to stain acidic compartments such as lysosomes. These are membrane permeable weak bases linked to a fluorophore. The Endoplasmic Reticulum (ER) is usually stained when studying protein secretion. One classical dye to stain this compartment is  DiOC6(3)  which has a preference for the ER but still binds to other membranes like those of mitochondria. Another way to specifically stain the ER is to use  ER-Trackers  like ER-Tracker Green and Red. LysoTracker ® Blue DND-22 is a blue fluorescent dye that stains acidic compartments in live cells

26. Ion Imaging I n the case of neuronal studies, gene activity or cellular movement it is of interest to study the ion concentration of the cell. Sodium, calcium, chloride or magnesium ions have a deep impact on many different cellular events. Typically, ions can be trapped with the help of fluorescently labeled chelators that change their spectral properties when bound to the appropriate ions. One example of labelled chelators are the calcium indicators  fura-2, indo-1, fluo-3, fluo-4  and  Calcium-Green . For sodium detection,  SBFI  (sodium-binding benzofurzanisophthalate ) or  Sodium Green  are commonly used.  PBFI  (potassium-binding benzofurzanisophthalate ) detects potassium ions.

27. Confocal microscopy is widely used for fluorescence imaging in the life sciences. In last few years we have seen advances in illumination sources, detectors, fluorescent probes, optics, and sample preparation techniques, which provide improvements in different combinations of speed, depth, and resolution.

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