B I O 1 5 0 C e l l a n d M o l e c u l a r B i o l o gy Le c t u r e

Techniques in Cell and Molecular Biology

Dr. Jay T. Dalet

Studying Cell Structures

- Cells vary in size, form, and function
- Animal cell is minute measuring 10-20 micrometers in diameter
- 1838 T. Schwann and 1839 M. Schleiden established the cell doctrine
o postulated that animal and plant tissue/organisms are aggregates of cells
- Objective: to understand the structures, to elucidate
- To overcome basic hindrances, they used stains

Use of variety of stains

- To elucidate minute structures, supply sufficient contrast to make these features visible
- They used techniques in specimen preparation
- Cell components drawn in a logarithmic scale
o The range of objects that and be readily resolved in the LM and EM
o Units of length used: micrometer (10-8) and nanometer (10-9) and Angstrom (10-10)
- Important requirement: light
o Without it we cannot see anything
o All colors are transmission of light
o Relative to the properties of the object under observation
Light is a Radiation
- Fundamental limitation of LM/ limit of resolution: light cannot probe details smaller than its own wavelength
o Dictated by the wavelength of visible radiation 0.4 micrometers – 0.7 micrometers (ROYGBIV)
- The wave nature of light affects it so that it does not follow the idealized straight ray path predicted by geometric optics
o Light rays interfere with one another and in this interference, they demonstrate optical diffraction
- Optical diffraction
o When light bends around a barrier and spreads out in multiple directions, become spread out
o At high magnification, when they pass an object, they appear as a set of parallel lines, pag spot, concentric rings
o Imagine two points, the closer the distance, the two points may appear as one depending on the limit of resolution
- Rsin(theta)
o The numerical aperture value increases with increasing magnification

Different Types of Optics (Light Microscopy)

depends on the specimen to be viewed
 Naturally pigmented specimen Spirogyra structures may appear more striking and impressive using Dark Field and DIC,
 Other structures visible during mitosis, pigmented organelles, best viewed under Bright Field, colors are true to nature
 In the case of stained tissue, best choice is Bright Field
 Phase contrast does not contribute additional information, halos can be observed around the specimen of spore forming bacteria advantage
best view of structures that are invisible in Bright Field optics
o Chromosomes
o Bacillus
 Dark field optics mas visible structures na mas malaki but minute features cannot be delineated like spores and cell walls
o Brightly illuminated against a dark background
o Mas kita outlines
 Differential interference optics increase the depth of focus on overlapping structures
o BF Amoeba very flat

31 August 2018
Observing Fluorescence in FM
- In order for an observer to obtain a complete view of fl image I a fl microscope, may 3 basic requirements
o Exciter Filter
o Sample Specimen
o Barrier Filter
- Basic Principle: EF selects appropriate photon of light > excites outermost electron in the probe > SS absorbs and transmits fl > BF captures
emitted appropriate photons of light
- Emission: signal transmitted (fluorescence), perceivable
- Fluorescent dye used must fall within 400-700nm range depending on preference or specification of the MF
- Usually the dye used is commercially available, procurable via supplier
o Specific probes that will target specific molecules in the specimen

- Fluorescent Microscope
o Tungsten halogen lamp or mercury lamp > LED (less heat is given off)
 Light towards specimen
 Passes through filter
 Filter (2 to 3 to 4)
 Ex., UV light to excite, use a UV filter
 o Can double as a compound microscope
o Filter Set: 2 barrier filters
 1st BF: only B light can pass through
 FITC – fluorescein? Isothiocyanate?
 Beam-splitting Mirror
 Reflects light below 510 nm, transmits above 510
 2nd BF: specific green emission between 520-560 nm
 Fluorescein –
o Objective lenses are of high numerical aperture, under a given magnification, brightness is directly proportional to the 4 th power
of the numerical aperture
- How to analyze fm
o Broken lines – emission maximum
o Solid lines – absorption maximum
o Ex. Absorption and emission spectra of CF488A Abs, Alexa Fluor 488, Flurescein on Goat anti mouse IgG

o
- TRITC Tetramethyl rodamine isothiocyanate
o Nucleus is B, stained by DAPI – not targeted by probe only by stain – diamno 2-phenyl indo -used as a counterstain
 Targets DNA
o Acti-stain – targets actin filaments

Fluoresence in situ hybdization

- Before hybridization, probe is labelled with a fl dye, fluorophore
- Conjugated with protein, antibody or nucleic acid = fluorochrome
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