Cell imaging techniques detailed presentation

Cell imaging techniques detailed presentation

1. Cell Imaging techniques

2. 1) In vitro and In vivo cell tracking techniques In vivo cell tracking offers insights into underlying biological processes of new cell based therapies, with the aim to depict cell function, migration, homing and engraftment at organ, tissue, cellular and molecular levels Includes fluorescence, reflectance imaging, fluorescence molecular tomography and bioluminescence imaging.

3. Bioluminescence Bioluminescent imaging is based on luc gene and luciferase- mediated oxidation of luciferin intravenous administration of luciferin generates light emission. Photon generation takes place exclusively at the site of luciferase expression

4. Fluorescent dyes- They have proven effective for in-vitro cell labelling and in-vivo cell tracking with Optical imaging. It has been demonstrated that when lipophilic dyes are added to the cell suspensions, they bind to the cell membrane (phospholipid bilayer). E.g. Cyanine dye. Fluorescent proteins (FP) such as green fluorescent protein (GFP), and over 30 different GFP – like proteins monomeric red, orange and yellow Fps have been cloned. GFP have been used for cell tracking with Optical imaging in small animal models, for example, with metastasizing GFP- expressing human adenoid cystic carcinoma cells in vivo

5. Colloidal quantum dots (QD): These inorganic fluorophores are comprised of colloidal semiconductor cores (Au, Cd, S, Se, Te , Zn) surrounded by a chemical coating that defines their surface chemistry Quantum dots have a narrow emission and continuous broad absorption spectrum (i.e., broad excitation spectrum) which allows fluorescent excitation by any wavelength below the emission maximum

6. 2) Immuno-electron microscopy Electron microscopy is an indispensable tool to investigate the intricate structure of the cell and organelles, and also to study the cellular biological processes Immuno-electron microscopy is one of the best methods of detecting and localizing proteins in cells and tissues successful application of immune electron microscopy depends on the preservation of the protein antigenicity

7. an adequate handling of the biological samples is required, which involves fixation, an appropriate selection of an embedding resin and the ready availability of the specific antibodies for the molecules whose ultrastructural location needs to be determined Immunoelectron microscopy technique uses antibodies, or molecules that interact with antibodies in conjugation with electron microscopy to localize ultra-structurally antigens and antibodies in cells and tissues

8. 51 ELECTRON MICROSCOPE Co-invented by Max knoll and Ernst Ruska in 1931. Electron Microscopes uses a beam of highly energetic electrons to examine objects on a very fine scale. Magnification can be upto 2million times while best light microscope can magnify up to 2000 times.

9. TRANSMISSION ELECTRON MICROSCOPE (TEM) Stream of electrons is formed. Accelerated using a positive electrical potential. Focused by metallic aperture and Electro magnets. Interactions occur inside the irradiated sample which are detected and transformed into an image .

10. WORKING OF TEM Electrons possess a wave like character. Electrons emitted into vacuum from a heated filament with increased accelerating potential will have small wavelength. Such higher-energy electrons can penetrate distances of several microns into a solid. If these transmitted electrons could be focused - images with much better resolution. Focusing relies on the fact that, electrons also behave as negatively charged particles and are therefore deflected by electric or magnetic fields.

11. 1: Electron cannon. P A R TS OF TEM focus the electron beam inside the column . . 3: Vacuum pumps system 4: Opening to insert a g with samples into the h rid i g h - vacuum chamber for observation. . 5: Operation panels 6: Screen for menu and image display 7: Water supply to cool the instrument 2. Electro-magnetic lenses to direct an electron

12. Advantages TEMs offer very powerful magnification and resolution. TEMs have a wide-range of applications and can be utilized in a variety of different scientific, educational and industrial fields TEMs provide information on element and compound structure . Images are high-quality and detailed. Disadvantages TEMs are large and very expensive. Laborious sample preparation. Operation and analysis requires special training. Samples are limited to those that are electron transparent. TEMs require special housing and maintenance. Images are black and white . AD V AN T AGES & DISAD V AN T AGES OF TEM

13. 1.Electron cannon. 2. Electro-magnetic lenses to focus the electron beam . 4.Opening to insert the object into the high- . 5. Operation panel with focus, alignment and magnification tools and a joystick for positioning of the sample. vacuum observation chamber. 6. Screen for menu and image display 3. Vacuum pumps system 7.Cryo-unit to prepare frozen material before insertion in the observation chamber in Cryo-SEM mode PARTS OF SEM

14. SEM SAMPLE PREPARATION A spider coated in gold 13mm radius aluminium stubs Sample coated with a thin layer of conductive material. Done using a device called a " sputter coater.” Sample placed in a small chamber that is at a vacuum . Gold foil is placed in the instrument. Argon gas and an electric field cause an electron to be removed from the argon, making the atoms positively charged. The argon ions then become attracted to a negatively charged gold foil. The argon ions knock gold atoms from the surface of the gold foil. These gold atoms fall and settle onto the surface of the sample producing a thin gold coating . Sputter coater

15. SEM WORKING The electron gun produces an electron beam when tungsten wire is heated by current. This beam is accelerated by the anode. The beam travels through electromagnetic fields and lenses, which focus the beam down toward the sample. A mechanism of deflection coils enables to guide the beam so that it scans the surface of the sample in a rectangular frame. When the beam touches the surface of the sample, it produces: Secondary electrons (SE) Back scattered electrons (BSE) X - Rays... The emitted SE is collected by SED and convert it into signal that is sent to a screen which produces final image. Additional detectors collect these X-rays, BSE and produce corresponding images.

16. Advantages It gives detailed 3D and topographical imaging and the versatile information ga thered from different detectors. This instrument works very fast. Modern SEMs allow for the generation of data in digital form. Most SEM samples require minimal preparation actions. Disadvantages SEMs are expensive and large. Special training is required to operate an SEM. The preparation of samples can result in artifacts. SEMs are limited to solid samples. SEMs carry a small risk of radiation exposure associated with the electrons that scatter from beneath the sample surface. ADVANTAGES & DISADVANTAGES OF SEM

17. BIOLOGICA L APPLICATIONS OF SEM Virology - for investigations of virus structure Cryo-electron microscopy – Images can be made of the surface of frozen materials. 3D tissue imaging - Helps to know how cells are organized in a 3D network Their organization determines how cells can interact. Forensics - SEM reveals the presence of materials on evidences that is otherwise undetectable SEM renders detailed 3-D images extremely small microorganisms anatomical pictures of insect, worm, spore, or other organic structures

18. Differences between SEM and TEM TEM SEM Electron beam passes through thin sample. Electron beam scans over surface of sample. Spe c i a l l y prep a red t h in sa m p l es are supported on TEM grids. Sample can be any thickness and is mounted on an aluminum stub. Specimen stage halfway down column. Specimen stage in the chamber at the bottom of the column. Image shown on fluorescent screen. Image shown on TV monitor. Image is a two dimensional projection of the sample. Image is of the surface of the sample

19. SEM IMAGES Vibrio cholerae with polar flagella Treponema pallidum

20. IN VIVO CELL TRACKING TECHNIQUES MICROARRAYS

21. The la r g e -scale gen o m e se q uencing e f f o rt and t h e ability to immobilize thousands of DNA fragments on coated glass slide or membrane, have led to the development of microarray technology. A m icroa r ray is a p a ttern o f ssDNA p r o b es which are immobilized on a surface called a chip or a slide. Microarrays use hybridization to detect a specific DNA or RNA in a sample. DNA microarray uses a million different probes, fixed on a solid surface.

22. An array is an orderly arrangement of samples where matching of known and unknown DNA samples is done based on base pairing rules. An array experiment makes use of common assay systems such as microplates or standard blotting membranes. Fig-01 Robotic arm with spotting slides

23. Microarray technology evolved from Southern blotting . The concept of microarrays was first proposed in the late 1980s by Augenlicht and his colleagues. They spotted 4000 cDNA sequences on nitrocellulose membrane and used radioactive labeling to analyze differences in gene expression patterns among different types of colon tumors in various stages of malignancy.

24. The core principle behind microarrays is hybridization between two DNA strands. Fluorescent labeled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases. Fi g - 02 Ar r ay h y bridizati o n

25. The principle of DNA microarray technology is based on the fact that complementary sequences of DNA can be used to hybridise, immobilised DNA molecules. There are four major steps in performing a typical microarray experiment. Sample preparation and labeling Hybridisation W ashing Image acquisition and Data analysis

26. Isolate a total RNA containing mRNA that ideally represents a quantitative copy of genes expressed at the time of sample collection. Preparation of cDNA from mRNA using a reverse- transcriptase enzyme. Short primer is required to initiate cDNA synthesis. Each cDNA (Sample and Control) i s l a b e l l ed with f l uoresc e nt cyanine dyes (i.e. Cy3 and Cy5). Fig-03 Sample labeling

27. Here, the l a b e lled cDNA (Sa m ple a n d Contr o l) a r e mixed together. Purification 🞂 A f t e r pur i fi c a t i on, t h e m ix e d l a b e lled cDNA is competitively hybridised against denatured PCR product or cDNA molecules spotted on a glass slide. Fi g - 04 Array H y brid i sat ion

28. Slide is dried and scanned to determine how much labelled cDNA (probe) is bound to each target spot. Hybridized target produces emissions. Microarray software often uses green spots on the microarray to represent upregulated genes. Red to represent those genes d o wnre g ulated present in and eq u al that are y ellow to ab u n d ance Fig-05 Gene chip showing different type of color spots

29. Glass cDNA microarrays which involves the micro spotting of pre-fabricated cDNA fragments on a glass slide. High-density oligonucleotide microarrays often referred to as a "chip" which involves in situ oligonucleotide synthesis.

30. Glass cDNA microarrays was the first type of DNA microarray technology developed. It was pioneered by Patrick Brown and his colleagues at Stanford University. Produced by using a robotic device which deposits (spots) a nanoliter of DNA onto a coated microscopic glass slide (50-150 µm in diameter) . Fig-06 Contact printer with robotic pins

31. FIG-07 Spotting of slides Selection of the material to spot onto the microscope glass surface. Preparation and purification of DNA sequences representing the gene of interest. Spotting DNA solution onto chemically modified glass slides via a contact printing or inkjet printing.

32. Advantages of Glass cDNA microarrays include their relative affordability with a lower cost. Its accessibility requiring no specific equipment for use such that hybridisation does not need specialised equipment. Data capture can be carried out using equipment that is very often already available in the laboratory.

33. Glass cDNA microarray have a few disadvantages such as intensive labour requirement for synthesizing, purifying, and storing DNA solutions before microarray fabrication. They may hybridise to spots designed to detect transcript from a different gene.

34. MICR O ARRA Y AS A GE N E EXPRESSION PROFILING TOOL MICROARRA Y AS A CO M P AR A TIV E GENOMICS TOOL DISEASE DIAGNOSIS DRUG DISCOVERY T OXICOLOG I C AL RES E ARCH