Basics only for beginners ZERNIKE MICROSCOPE. PHASE-CONTRAST MICROSCOPE FLUORESCENT MICROSCOPE When a substance absorbs light, the electrons present at the outermost orbit absorbs energy and get excited; on the way back to the ground state, it emits a part of the energy absorbed. This phenomenon is termed as fluorescence.Fluorescent microscopeinvolves staining of specimens with special fluorescent dyes (fluorescein, acridine orange, etc). When a fluorescent dye is applied to a substance, it absorbs a wavelength of light (excitation wavelength) and emits light of different wavelength (emission wavelength).

1. MICROSCOPY 1
PHASE-CONTRAST MICROSCOPE
 The first phase contrast microscope was developed by FRITZ ZERNIKE (FREDRICK
ZERNIKE) in 1933, hence also referred as ZERNIKE MICROSCOPE.
 The phase contrast microscope enables to differentiate transparent, unstained, living
(without killing or altering the living component) structures.
 Phase contrast is an illumination technique provides greater degree differentiation
inside the cells by phase contrast optics.
WORKING PRINCIPLE
Light from the illumination (light) source from the base of the Microscope stand is
aimed at annular diaphragm stop. The annular-diaphragm-stop allows only the hollow
cone of light rays to pass through sub-stage condenser lens. The sub-stage condenser
lens focuses light through slit in the stage onto the sample. The projected light from the
sample is collected by special set of objective lens with phase plate and phase rings
which are placed in the back/rare focal plane of the objective. The direct rays (unaltered
amplitude and phase, but retarded by ¼ wavelength) from transparent sample converge
on the phase ring within the objective and produce phase shift. The most diffracted rays
(altered rays due to difference in density) pass through phase plate by missing phase ring.
The convergence of diffracted and direct rays on the image plane results in image.
ADVANTAGES
 Specimens which have a refractive index similar to their surroundings can be invisible
in Brightfield, but are well defined in Phase Contrast.
 Phase Contrast is normally used to examine unstained biological specimens.
 Living microorganisms and their minute details such as Cilia, flagella can be observed.

2. MICROSCOPY 2
FLUORESCENT MICROSCOPE
When a substance absorbs light, the electrons present at the outermost orbit absorbs
energy and get excited; on the way back to the ground state, it emits a part of the energy
absorbed. This phenomenon is termed as fluorescence.Fluorescent microscopeinvolves
staining of specimens with special fluorescent dyes (fluorescein, acridine orange, etc).
When a fluorescent dye is applied to a substance, it absorbs a wavelength of light
(excitation wavelength) and emits light of different wavelength (emission wavelength).
WORKING PRINCIPLE
Illumination (light) is provided by a bright mercury vapor lamp (very expensive +
harmful), produces light range of 200-400nm and generates considerable amount of heat.
The heat filter absorbs heat, allows UV rays and visible rays by blocking infrared rays.
The exciter filter ensures high energy - short wavelength - monochromatic light towards
dichroic mirror. Dichroic mirror (beam splitter) eliminates visible light and reflects
excited UV light to the dark-field condenser, which provides high contrast for
fluorescence and also deflects majority of UV light.The excitation light is focused on to
the fluorochrome specimen. The fluorescent labeled specimen absorbs light and emits
excitatory light along with florescent light, which reaches objectivelens .As per the
capacity of objective lens, the specimen would be magnified and are directed towards
barrier filter. The additional barrier filter permits only the fluorescent wavelength and
rejects excitation light. The fluorescent light passing through ocular lens creates the
magnified image, which can also be detected by detector to give a photographic image.
APPLICATIONS
 Imaging the genetic material (DNA & RNA) and other structural components.
 Monitoring the environment for microbial contamination.
 Certain micro-organism can be detected and identified only by this microscopy.