1. Chapter 6 Cellular Measurements in Biomaterials and Tissue Engineering

2. StructureDescription Cytoplasm The inside of the cell not including the organelles. Organelles Membranous sacs within the cytoplasm. Cytoskeleton Structural support made of microtubules, actin and intermediate filaments. Endoplasmic Reticulum (ER) (two types) Site of protein and lipid synthesis and a transport network for molecules. Golgi Apparatus Modifies molecules and packages them into small membrane bound sacs called vesicles. Lysosomes Main point of digestion. Microtubules Made from tubulin and make up centrioles, cilia, cytoskeleton, etc. Mitochondria Site of aerobic respiration and the major energy production center. Nucleus Location of DNA; RNA transcription. Peroxisomes Use oxygen to carry out catabolic reactions. Ribosomes Located on the Endoplasmic Reticulum in the cytoplasm. RNA goes here for translation into proteins. Table 6.1 Typical cell content.

3. 0.1 nm Diameter of hydrogen atom 0.8 nm Amino acid 2 nm Thickness of DNA membrane 4 nm Protein 6 nm Microfilament 7 to 10 nm Cell membranes 17 to 20 nm Ribosome 25 nm Microtube 50 to 70 nm Nuclear pore 100 nm AIDS virus 200 nm Centriole 200 to 500 nm Lysosomes and peroxisomes 1  m Diameter of human nerve cell 2  m Bacteria 3  m Mitochondrion 3 to 10  m Nucleus 9  m Human red blood cell 90  m Amoeba 100  m Human egg Table 6.2 Typical sizes of cellular features.

4. Figure 6.1 (a) Light rays through a lens and the corresponding focal point F. (b) The light rays are bent by the lens and refocus as an image. (a)(b)

5. Figure 6.2 Compound microscope. The compound microscope contains a light source for sample illumination, a field iris to control the light field, a condenser to focus the illuminating light, an objective lens, and an eyepiece.

6. Figure 6.3 Cone Angle 

7. Figure 6.4 In phase contrast microscopy, light from the lower annular ring is imaged on a semitransparent upper annular ring. This microscope uses the small changes in phase though the sample to enhance the contrast of the image.

8. Figure 6.5 The interaction of light waves that meet in phase results in constructive interference. The amplitude of the wave is doubled by this interaction.

9. Figure 6.6 Interaction of light waves that meet one-half wavelength out of phase results in destructive interference. The waves cancel each other when they intersect.

10. Figure 6.7 Example of darkfield illumination. (a) shows the typical brightfield view while (b) shows the darkfield view of the same object. Imagine the object in the darkfield view glowing like the moon at night. (a)(b)

11. Figure 6.8 CCD Array. The CCD array is made up of many light sensing elements called pixels. Each pixel can independently sense light level and provide this information to a computer.

12. Figure 6.9 Comparison of the wide field versus the point scan techniques. (a) widefield collects an entire image while (b) point scan image must be assembled point by point.

13. Figure 6.10 Basic radiation counting system. The power supply has a voltage source and series resistor. Ionization by a radiation particle causes a voltage pulse, which passes through the capacitor and is read by the meter. Freq to volt counter         Plates Particle Ions Voltage supply Meter Pulse counter Resistor + Capacitor

14. Figure 6.11 Confocal laser scanning microscope. The microscope removes out of focus (z-plane) blur by keeping out of focus light from reaching the detector using a pinhole.

15. Two beam excitation Objective Dichroic mirror Focal plane Specimen Detector Figure 6.12 Two photon excitation microscope. This microscope doesn’t require a pinhole but is able to excite single points within the sample by having two photons excite the sample only at the exact location of interest.

16. Figure 6.13 Video enhanced contrast microscope (VECM) system. A light microscope image is recorded by a camera and the image is sent to a recorder and a computer for image processing.

17. Figure 6.14 (a) Original low contrast video image. (b) The threshold is set just under the saturation level. (c) Reducing the background level. (d) Gain is adjusted to amplify dark-low intensity signal.

18. Figure 6.15 (a) An unprocessed photo of cells of the inner epidermis taken through an interference contrast microscope.

19. Figure 6.15 (b) The same image with digital contrast enhancement, the single structures become apparent. The background fault remains.

20. Figure 6.15 (c) Subtraction of the background and resulting with further contrast enhancement.

21. Figure 6.16 Intensified Fluorescence Microscopy system. In this system, the fluorescent signal is received through the microscope by the camera. The video image is typically time stamped and sent to the frame grabber. The frame grabber digitizes the signal and sends it to the computer for image processing. The processed signal is then sent to a monitor for display by the operator and permanently recorded for later playback and analysis.

22. Figure 6.17 A TEM image of skeletal muscle cells.

23. Figure 6.18 A SEM image of stressed liver cells.

24. Figure 6.19 In the micropipet technique, force causes a displacement to determine cell deformation properties.

25. Figure 6.20 Parabolic distribution of velocities and the shear stress it causes on a blood cell.

26. Figure 6.21 Cone and plate system. The cone is immersed in a liquid and then rotated at a constant rate. Since the velocity changes linearly with distance, the shear stress is a function of rotation velocity. shear stress viscosity of the liquid gradient of the velocity between the cone and the plate.

27. Figure 6.22 Time-correlated single photon counting system block diagram. When a light source provides an excitation pulse, some of the light is deflected to the start timer circuit while the rest illuminates the sample. A single fluorescent photon is detected by a photomultiplier tube, which generates a pulse that stops the timer. The time difference is then converted to an amplitude. The various amplitudes are recorded in the multichannel analyzer and a profile of different time intervals versus number of photons in that interval are displayed.

28. Figure 6.23 A sample graph of fluorescence of proteins during FRAP. (a) Before phototbleaching F(-), (b) just after protein photolysed F(+), and (c) long after protein photolysed F( 

29. Figure 6.24 (a) The DNA strand is denatured with heat and put in the same culture as the fluorescent probe. (b) The fluorescent probe binds to the target gene.

30. Figure 6.25 Human chromosomes probed and counterstained to produce a FISH image. The lighter area within each chromosome show the fluorescent probe attached to the target gene. ( From Detecting Nucleic Acid Hybridization. [Online] www.probes.com/handbook/sections.0805.html www.probes.com/handbook/sections.0805.html