Brightfield microscope Darkfield microscope Phasecontrast microscope Fluorescence microscopes
������ �� • ���������� – – Bright-field microscope Dark-field microscope Phase-contrast microscope Fluorescence microscopes
Eyepiece Revolving nosepiece Objective Stage กลองจลทรร ศน Binocular tube Arm Mechanical stage Fine adjustment Course adjustment Condenser Base Illuminator
Working distance Low power High power
Working distance Oil Immersion
• ����������� Property Objective lens Scanning Low power High power Oil Immersion magnification 4 x 10 x 40 -45 x 90 -100 x Numerical aperture 0. 1 0. 25 0. 55 -0. 65 1. 25 -1. 4 40 mm 16 mm 4 mm 1. 8 -2. 0 mm Working distance 17 -20 mm 4 -8 mm 0. 5 -0. 7 mm 0. 1 mm Resolution power 2. 3 m 0. 9 m 0. 35 m 0. 18 m Focal length (WL= 450 nm; blue light) • working distance - ���������������� ����
Mechanical Lengths
Objective Specifications
The 3 Classes of Objectives Chromatic and Mono-Chromatic Corrections
Plan Objectives
Dark field microscope
Phase contrast microscope
Figure 2. 10
Fluorescence Microscope
��������� Fluorescence Microscope
Transmission Electron Microscope • electrons scatter when they pass through thin sections of a specimen • transmitted electrons (those that do not scatter) are used to produce image • denser regions in specimen, scatter more electrons and appear darker
EM
Ebola
The Scanning Electron Microscope • uses electrons reflected from the surface of a specimen to create image • produces a 3 -dimensional image of specimen’s surface features
Fly head
Confocal microscopy • have extremely high resolution • can be used to observe individual atoms
Confocal Microscopy • confocal scanning laser microscope • laser beam used to illuminate spots on specimen • computer compiles images created from each point to generate a 3 dimensional image
Figure 2. 30
Homogenizer http: //www. freewebs. com/ltaing/chpt 7. 3 Cellfractionation. gif
Ultrasonic vibration
Deep freezer
Gel Filtration http: //fig. cox. miami. edu/~cmallery/150/proteinsb. htm
. 2��������� )Separation of the homogenate(. 1������� (Centrifugation( - Gravity (g( -Relative centrifugal force (RFC( -Rate of sedimentation of components ) size, shape, density of particle and solvent, speed) - Differential centrifugation -Rate-zonal or density-gradient centrifugation
Differential Centrifigation http: //www. freewebs. com/ltaing/chpt 7. 3 Cellfractionation. gif
Differential Centrifigation http: //www. freewebs. com/ltaing/chpt 7. 3 Cellfractionation. gif
. 3���������� (Separation of biomolecules(. 1��������� (Solubility(. 2���� (size( -Centrifugation -Dialysis -Ultrafiltration -Gel filtration -SDS -PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis(
Ultrafiltration
. 3����� (Charge( Ion exchange chromatography Isoelectric focusing Electrophoresis. 4 Biological properties Affinity of chromatography Immunoelectrophoresis Blotting - Southern blot (DNA( - Northern blot (RNA( - Western blot (Protein( Chromatography ����� (paper. Gas , Thinlayer(
Ion Exchange http: //fig. cox. miami. edu/~cmallery/150/protein/ion_exchange.
Immunoelectrophoresis http: //www. haps. nsw. gov. au/edrsrch/edimages/myeloma 2. jpg
Southern blot ��������� DNA ��������� agarose gel electrophoresis
Northern blot ��������� RNA
Western blot
Buffer Optimalization • • • 10 m. M Tris HCl, p. H 8. 3 50 m. M KCl 1. 5 - 2. 5 m. M Mg. Cl 2 50 - 200 m. M d. NTP DNA template concentration Optional compounds
Primer Considerations • • • Primer lengths GC contents Melting temperature – Tm = 2(T+A)+4(G+C) Tm difference 3’ and 5’ end considerations
PCR Principle
- Slides: 60