DIFFERENT TYPES OF MICROSCOPY

BY JAN ROBIN D. NARVAEZ

TYPES OF MICROSCOPY
Fluorescence microscopy

mage from Microscopyu.com

TYPES OF MICROSCOPY
Confocal microscopy

TYPES OF MICROSCOPY
Multiphoton microscopy

Image from Olympus

Microscopy Resource
Center Website

FLUORESCENCE MICROSCOPY
It was devised in the 20th century
August Khler, Karl Reichert, Heinrich

Lehmann

Images from
Wikipedia

FLUORESCENCE MICROSCOPY
Fluorescence is the main component

It is the light produced by a substance

when it is stimulated by another light

FLUORESCENCE MICROSCOPY
How it works:
Cellular components are colorless and

cannot be distinguished under a

microscope
Fluorescence microscopy stains

components with dyes

UV is used to locate certain components

of a cell

FLUORESCENCE MICROSCOPY
How it works:
Image produced is based on the second

light source

Image from Science

Education Resource
Center @ Carleton College

FLUORESCENCE MICROSCOPY
Principles of fluorescence microscopy:
Energy is absorbed by the atom,

which becomes excited

The electron jumps to a higher
energy level
The electron drops back to the
ground state, emitting a photon

Image from the official

website of the Nobel Prize

FLUORESCENCE MICROSCOPY
Fluorochromes- fluorescent stains

Arcidine orange- used in detection of

malaria
Auramine- used in detection of

tuberculosis
Fluorescent antibody (FA) used in

detection of specific antibody

FLUORESCENCE MICROSCOPY
Auramine-rhodamine process
Uses a yellow fluorescent dye to visualize

Mycobacterium tuberculosis

FLUORESCENCE MICROSCOPY
Auramine-rhodamine step-by-step

process:
1. Reagents needed for staining
Auramine-phenol solution
Acid-alcohol for decolorization
Potassium permanganate or acridine

orange for counterstaining

FLUORESCENCE MICROSCOPY
Auramine-rhodamine step-by-step

process continued
2. Examine under the fluorescent

microscope using a low power objective

(100-150x)
Bacilli will appear as slender bright yellow

fluorescent rods

FLUORESCENCE MICROSCOPY
Auramine-rhodamine step-by-step

process continued
3. Patient tests positive for tuberculosis

with the minimum of four AFB in the

entire smear
While negative when there is less than
four bacilli

FLUORESCENCE MICROSCOPY
Advantages:
Has the ability to isolate individual

proteins
Different molecules can be stained with
different colors
Disadvantages:
Not being able to examine specimens in

their live or natural self

CONFOCAL MICROSCOPY
Pioneered by Marvin Minsky in 1955
Minsky wanted to work on living

brain tissues

Image from Wikipedia

CONFOCAL MICROSCOPY
How it works:

Uses optical imaging technique to

increase optical resolution of a micrograph

Uses spatial filtering techniques to

eliminate
out-of-focus light in specimens

CONFOCAL MICROSCOPY
How it works:
A pinhole is place in front of

the illumination source to allow

transmission only through a small area
Light source of very high intensity like

laser light is used

Light reflects off a dichoric mirror,

which directs it to scanning mirrors

CONFOCAL MICROSCOPY
Optical principle

Image from
Creative
Commons,
Rice University

CONFOCAL MICROSCOPY
Can process 3-D images

Image from
Olympusfluoview.com

CONFOCAL MICROSCOPY
Used as a noninvasive imaging technique

that has proved useful for in vivo

real-time cytomorphological analysis of
analysis of bascal cell carcinoma cells
infiltrating the epidermis
Uses a laser source
Water immersion objective lens was used

and attached to the skin

Confocal imaging criteria for BCC was
used

CONFOCAL MICROSCOPY
Advantages:

Illuminates entire view of field uniformly

Does require to process specimen (i.e.,

with freezing, staining)

3-D imaging gives a better view

and resolution of the specimen

CONFOCAL MICROSCOPY
Disadvantages:

Harmful nature of high-intensity

irradiation to living cells and tissues

High cost of purchasing and operating

MULTIPHOTON MICROSCOPY
Pioneered by Winfried Denk
Combined the idea of two-photon

absorption with the use of a laser scanner

Image from Max Planck

Institute Website

MULTIPHOTON MICROSCOPY
How it works:
Multiple absorption from multiple photons

induce a molecular excitation of a

magnitude equivalent to the sum of the
absorbed photon energies
At very high photon densities, it becomes
for two or more photons to be
simultaneously absorbed
Each multiply absorption induces a
molecular excitation of a magnitude
equivalent to
the sum of the absorbed photon energies

MULTIPHOTON MICROSCOPY
Jablonski Energy Diagram

Image from Cornell

University Website

MULTIPHOTON MICROSCOPY
Skin cancer diagnosis and monitoring

Identification of abnormally located

melanocytes
3-D imaging technique is used
Fluorescence was also included in the

technique

MULTIPHOTON MICROSCOPY
Advantages:

Provides 3-D images

Photodamage or photobleaching is greatly

reduced due to decreased excitation volume

Excitations photon energy is inversely

proportional to wavelength
Longer wavelenth light may be used

MULTIPHOTON MICROSCOPY
Photopleaching rates in multiply stained

specimens