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CELL BIOLOGY TECHNIQUES Visualize cells - Microscopy Organelles – Fractionation of subcellular components Culturing cells.

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Presentation on theme: "CELL BIOLOGY TECHNIQUES Visualize cells - Microscopy Organelles – Fractionation of subcellular components Culturing cells."— Presentation transcript:

1 CELL BIOLOGY TECHNIQUES Visualize cells - Microscopy Organelles – Fractionation of subcellular components Culturing cells

2 Light Microscopy

3 Resolution of 0.2µm Magnification – objective and projection lens Resolution – D = 0.61λ/N sin α Resolution is improved by using shorter wavelengths or increasing either N or α.

4 BRIGHT FIELD PATH MICROSCOPY

5 Visualize unstained living cells Phase Contrast microscopy – Thin layers of cells but not thick tissues Differential Interference contrast – Suited for extremely small details and thick objects – Thin optical section through the object

6

7 Microscopy of Live cells

8 Fluorescence Microscopy Major Function: Localization of specific cellular molecules – example proteins Major Advantages: –Sensitivity:glow against dark background –Specificity: immunofluorescence –Cells may be fixed or living Fluorescent dyes or proteins (Flurochromes) –flurochromes may be indirectly or directly associated with the cellular molecule –Multiple flurochromes may be used simultaneously

9 Absorb light at one wavelength and emit light at a specific and longer wavelength

10 HYDRA EXPRESSING GFP Fluorescent protein in live cells

11 FIX EMBED SECTION STAIN

12 Immunofluorescence Microscopy and Specific Proteins Fluorescently tagged primary anti body Fluorescently tagged secondary antibody Fluorescently labelled antibody to tagged proteins such as myc or FLAG

13 RAT INTESTINAL CELL WALL – GLUT 2

14 CONFOCAL AND DECONVOLUTION MICROSCOPY This overcomes the limitations of Fluorescence microscopy – Blurrred images – Thick specimens

15 REMOVES OUT OF FOCUS IMAGES

16 EXAMPLE OF IMAGE RECONSTRUCTED AFTER DECONVOLUTION MICROSCOPY

17 ELECTRON MICROSCOPY

18 Transmission EM – theoretically nm; practically 0.1 nm –1 nm (2000x better than LM) – High – velocity electron beam passes through the sample – nm thick sections – 2-D sectional image – surface details are revelaed – Subcellular organelles Scanning EM – Resolution about 10 nm – Secondary electrons released from the metal coated unsectioned specimen – 3-D surface image

19 GOLD PARTICLES COATED WITH PROTEIN A ARE USED TO DETECT ANTIBODY BOUND TO PROTEIN

20 TEM IMAGE

21 CRYOELECTRON MICROSCOPY HYDRATED, UNFIXED AND UNSTAINED SAMPLES SAMPLES ARE OBSERVED IN ITS NATIVE HYDRATED STATE METHOD - AN AQUEOUS SUSPENSION OF SAMPLE IS APLLIED ON A GRID AND HELP B Y A SPECIAL MOUNT 5 nm RESOLUTION

22 SURFACE DETAILS BY METAL SHADOWING

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24 SEM OF EPITHELIUM LINING THE INTESTINAL LUIMEN

25 PURIFICATION OF CELL ORGANELLES CELL DISRUPTION SEPARATION OF DIFFERENT ORGANELLES USING CENTRIFUGATION PREPARATION OF PURIFIED ORGANELLES USING SPECIFIC ANTIBODIES

26 BREAKING OPEN PLASMA MEMBRANES IN CELLS CELLS ARE SUSPENDED IN ISOTONIC SUCROSE SONICATION HOMOGENIZATION CELLS IN HYPOTONIC SOLUTION – RUPTURE OF CELL MEMBRANES

27 SEPERATING ORGANELLES DIFFERENTIAL CENTRIFUGATION DENSITY GRADIENT CENTRIFUGATION

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30 ANTIBODIES ARE USED TO MAKE HIGHLY PURIFIED ORGANELLES

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32 CELL SORTER – FLOW CYTOMETRY

33 CELL CULTURE REQUIREMENTS SOLID MEDIA – Specially coated plastic dishes or flasks (CAMs) – Agar as the medium GROWTH MEDIA Rich in nutrients- amino acids, vitamins, salts fatty acids, glucose, serum provides the different growth factors,

34 TYPES OF CULTURED CELLS PRIMARY CELL CULTURES – DIFFERENTIATE IN CELL CULTURE CELL STRAIN – ALSO HAVE A FINITE LIFE SPAN (FROM A PRIMARY CULTURE) CELL LINE - INDEFINITE LIFE SPAN

35 PRIMARY CULTURES

36 STAGES IN CELL CULTURE

37 DIFFERNTIATION OF A CELL LINE – C2C12 IN CULTURE

38 HOMEWORK-1 CHAPTER 9 – REVIEW CONCEPTS QUESTIONS -2,5,7,9 – ANALYZE THE DATA DUE NEXT WEEK IN THE WORKSHOPS


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