Skip to main content

Other Types of Microscopy

Electron Microscopy

Image Source: sv.rkriz.net 
                                        In electron microscope, the source of illumination is electron beam. The construction and principles of electron microscope are easily related to those of light microscope. The range of wavelength of visible light used in light microscope is 4000Å-7000Å, while with an electron microscope employing 60-80KV electron, the wavelength is only 0.05Å. 
In the instrument, the electron gun generates electron beam. These electrons are concentrated by other components of electron gun producing a fast moving narrow beam of electron. Electrons are focused by electromagnetic lenses. Electromagnetic lens consists of wire encased in soft iron casing. When electric current is passed through the coil, it generates an electromagnetic field through which electrons are focused. 
There are three types of electromagnetic lenses. The one is placed between the source of illumination and the specimen. This focuses the beam of electron on specimen functions in a similar manner as that of light microscope. The other two lenses are on the opposite side of specimen which magnify the image in similar fashion as objective and ocular in light microscope. 


TEM (Transmission Electron Microscopy)
Image Source: www.hk-phy.org

The electron source is commonly a tungsten filament of 30-150KV potential. The electron beam passes through a centre of ring like magnetic condenser and becomes converged on the specimen. After being transmitted through the specimen, the magnetic objective focuses the electron into a first image of the object which is enlarged (2000 times). The magnetic projector lens then magnifies a portion of the first image producing magnification up to 240000 or more. 
The final enlarged image can be reviewed by striking a fluorescent screen which makes it visible. The image can also be thrown upon a photographic plate for permanent record. Portions of the photographs may be enlarged four to six times giving the picture in the range of two million times as large as the object. 
Molecules in the microscope interferes with the movement of electrons. To prevent this, interior of the microscope is kept in the state of high vacuum, around 10-4 to 10-6 mm Hg. It is also necessary to have specimen ultra thin. The electron beams has very poor penetrating power, therefore, only small objects or very thin sections of the specimen can be examined. 

(SEM) Scanning Electron Microscopy 

In SEM, electrons are not transmitted through the very thin specimen from below but impinge on its surface from above. The specimen may be opaque and of any manageable thickness and size. if the specimen is an electron conductor, it needs only to be held on an appropriate support. If it is non-conductor, it is allowed to dry but if most, freeze dried in liquid nitrogen is necessary. the specimen is then coated with metal vapour in vacuum. 
The Electrons originate at high energy (20,000 V) from a hot tungsten or lanthanum hexoboride cathode "gun". These electrons are sharply focused, adjusted and narrowed by an arrangement of magnetic fields. Instead of forming a broad inverted cone of rays, in SEM a needle sharp probe is made. This probe acts only as an exciter of image forming secondary electrons emerging from the surface of the specimen. The Probe scans the specimen like that on a blank TV Screen. The probe can impinge on depth and heights with equal speed and accuracy giving great depth of field and producing images with three dimensions. Images are elicited from wherever the probe strikes the metal coated areas of the specimen. Magnification is the ratio of final image to the diameter of area scanned. 
Labels:

Comments

Post a Comment

Popular posts from this blog

Statistics in Flow Cytometry Data and "MFI" values

                                 The speed of the flow cytometry offers wide range of data points and data plots. Due to its sensitivity and versatility, it has been used extensively. Flow cytometer can detect up to 1000s of cells per second.. But, it should be noted that the flow cytometry also involves the statistics, its significance, calculating the fluorescence intensity..                Statistics in flow cytometry involves, total number of data points acquired, percentage population, most importantly mean and median fluorescence intensity, and others. Most of us get confused about the term "MFI", whether it means mean fluorescence intensity or median fluorescence intensity.. Truth is, both.. It means it can be described as either mean or median fluorescence intensity. Some researchers use Mean for MFI and some use Median for MFI values.  (Keeping in mind that, consideration of statistics in flow cyomtery also depends on the type of Application you are interest
Post a Comment
....CONTINUE READING

Protocol: Chromosomal DNA Isolation from Bacteria

1.       Spin down 50-100 ml well-grown bacteria, 3600 rpm,15min. 2.     Resuspend bacteria with 20 ml Buffer S, immediately add 100 µl ProteinaseK (10 mg/ml). Vortex to make sure no chunks. 3.       Add 2 ml of 20% SDS, mix gently by inverting. 4.       Incubate the mixture at 65 o C for 1 hr with inverting every 15 min. 5.     Add 10 ml of phenol and 10 ml of chloroform, mix thoroughly by inverting for 5 min, spin at 3600 rpm for 20 min. 6.       Transfer supernatant to a new tube, add 0.6 volume of isopropanol. Mix gently by inverting. You will see cotton-like genomic DNA. 7.       Hook out the cotton-like DNA to a 1.5 ml tube; wash with cold 70% ethanol. 8.      Dry DNA at RT, dissolve DNA in 500 µl H 2 O (50 o C or 4 o C overnight). 9.      Add 5 µl DNase-free RNase A (20 mg/ml stock) to the DNA. Incubate at 37 o C for 30 min. 10.   Add 500 µl phenol, mix, spin at 3600 rpm for 20 min. Repeat phenol extraction once if necessary. 11.   Tr
Post a Comment
....CONTINUE READING

Gene Cloning Technique

Gene Cloning Technique in Molecular Biology Field Involves the following steps: 1.a) Isolation of Genetic Material and Gene Sequence of Interest.  First, cells are lysed using detergent or lysozyme enzymes which disrupts the plasma membrane and release the genetic material along with the macro molecules such proteins and RNA molecules. Cell contents are then treated with protease to disrupt the proteins and RNase to destroy the RNA. Cell debris are then pelleted using centrifuge and supernatant containing DNA is transferred to a fresh and clean tube. A proper amount of Ethanol is added to this supernatant and precipitated using centrifuge. Supernatant is discarded and the pellet which has DNA is suspended using a proper suitable buffer.  Primers are designed for the specific gene sequence of interest which will be used for the cloning procedure.  Gene sequence is then amplified using PCR (Polymerase Chain Reaction) which will yield in many copies of the gene sequence.
Post a Comment
....CONTINUE READING