chapter 6 cellular measurements in biomaterials and tissue engineering

Chapter 6

Cellular Measurements in Cellular Measurements in Biomaterials and Tissue EngineeringBiomaterials and Tissue Engineering

StructureStructure DescriptionDescription

Cytoplasm Cytoplasm The inside of the cell not including the organelles. The inside of the cell not including the organelles.

Organelles Organelles Membranous sacs within the cytoplasm.Membranous sacs within the cytoplasm.

Cytoskeleton Cytoskeleton Structural support made of microtubules, actin and Structural support made of microtubules, actin and intermediate filaments. intermediate filaments.

Endoplasmic Reticulum Endoplasmic Reticulum (ER) (two types)(ER) (two types)

Site ofSite of protein and lipid synthesis and a transport network for protein and lipid synthesis and a transport network for molecules. molecules.

Golgi ApparatusGolgi Apparatus Modifies molecules and packages them into small membrane Modifies molecules and packages them into small membrane bound sacs called vesicles.bound sacs called vesicles.

LysosomesLysosomes Main point of digestion.Main point of digestion.

MicrotubulesMicrotubules Made from tubulin and make up centrioles, cilia, Made from tubulin and make up centrioles, cilia, cytoskeleton, etc.cytoskeleton, etc.

MitochondriaMitochondria Site of aerobic respiration and the major energy production Site of aerobic respiration and the major energy production center.center.

NucleusNucleus Location of DNA; RNA transcription.Location of DNA; RNA transcription.

PeroxisomesPeroxisomes Use oxygen to carry out catabolic reactions.Use oxygen to carry out catabolic reactions.

RibosomesRibosomes Located on the Endoplasmic Reticulum in the cytoplasm. Located on the Endoplasmic Reticulum in the cytoplasm. RNA goes here for translation into proteins.RNA goes here for translation into proteins.

Table 6.1 Typical cell content.

0.1 nm0.1 nm Diameter of hydrogen atomDiameter of hydrogen atom

0.8 nm0.8 nm Amino acidAmino acid

2 nm2 nm Thickness of DNA membraneThickness of DNA membrane

4 nm4 nm ProteinProtein

6 nm6 nm MicrofilamentMicrofilament

7 to 10 nm7 to 10 nm Cell membranesCell membranes

17 to 20 nm17 to 20 nm RibosomeRibosome

25 nm25 nm MicrotubeMicrotube

50 to 70 nm50 to 70 nm Nuclear poreNuclear pore

100 nm100 nm AIDS virusAIDS virus

200 nm200 nm CentrioleCentriole

200 to 500 nm200 to 500 nm Lysosomes and peroxisomesLysosomes and peroxisomes

1 1 mm Diameter of human nerve cellDiameter of human nerve cell

2 2 mm BacteriaBacteria

3 3 mm MitochondrionMitochondrion

3 to 10 3 to 10 mm NucleusNucleus

9 9 mm Human red blood cellHuman red blood cell

90 90 mm AmoebaAmoeba

100 100 mm Human eggHuman egg

Table 6.2 Typical sizes of cellular features.

O bject

Im age

F

L F

LO

L I

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)

Eyepiece

Objective

Eye

SampleSlide

Condenser

Field iris

Light source

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.

Objective

Specimenr

Figure 6.3 Cone Angle

Slide

Condenser

Sample

Objective

Lower annular ring

Upper annular phase changing ring

Deflected light

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.

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.

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.

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)

S ing le p ixe l

C C D array

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.

(a) W ide fie ld (b) Point scan

O bjective

Sam ple

O bjective

Sam ple

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.

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

Voltagesupply

Meter

Pulsecounter

Resistor

+

Capacitor

Pinhole

illumination aperture

or laser

Pinhole

detector aperture

Objective

Dichroic

mirror

Focal plane Specimen

Out of focus

In focus

Detector

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.

Two beam excitation

Objective

Dichroicmirror

Focal plane

Specimen

DetectorFigure 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.

Light

microscope

Video camera

Monitor and

computer

Frame grabber

Video

recorder

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.

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.

Intensity Intensity

D istance DistanceA B

Intensity Intensity

D istance DistanceC D

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

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

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

Microscope CameraTime codegenerator

Framegrabber

Imageprocessingcomputer

Recordingdevice

Monitor

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.

Figure 6.17 A TEM image of skeletal muscle cells.

Figure 6.18 A SEM image of stressed liver cells.

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

F

C ell M icropipet

D isplacem ent

F

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

Vessel wall

B lood

B lood C ells

V 1

V 2

R ange ofveloc ities

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.

C oneLiqu id

P late (m icroscopic s lide) vm axv = 0

dv/dx

Lightsource

Beamsplitter

Microscope Photomultipliertube

Sample

Stop timercircuit

Start timercircuit

TimerTime-to-

amplitudeconverter

Multichannelanalyzer

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.

B

F(¥)

Leve l o ffluorescence

tPhotobleach ing

F( )

F (+)

A

B

C

A

¥

FF

FFRm

:proteins mobile of Percentage

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(¥

C

C hrom osom e target

F luorescent probeA

B

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.

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