BRIGHT FIELD MICROSCOPES

REPORTER:CASIDO, NICASIO JR. S.SCHEDULE: MWF 3:00pm-4:00pm

Importance of the Microscope• Important for hematology, microbiology,

TB, and malaria testing• Compound microscope used in

bacteriology, biology, and medicine to examine minute objects such as bacteria, other unicellular organisms, and plant and animal cells and tissue

• Advances in fluorochrome stains and monoclonal antibody techniques caused growth in use of fluorescence microscopy in both biomedical analysis and cell biology

ADVANTAGES

Brightfield microscopy is very simple to use with fewer adjustments needed to be made to view specimens.

Some specimens can be viewed without staining and the optics used in the bright-field technique don’t alter the color of the specimen.

It is adaptable with new technology and optional pieces of equipment can be implemented with bright-field illumination to give versatility in the tasks it can perform.

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DISADVANTAGES

Certain disadvantages are inherent in any optical imaging technique.

By using an aperture diaphragm for contrast, past a certain point, greater contrast adds distortion. However, employing an iris diaphragm will help compensate for this problem.

Bright-field microscopy can’t be used to observe living specimens of bacteria, although when using fixed specimens, bacteria have an optimum viewing magnification of 1000x.

DISADVANTAGES

Bright-field microscopy has very low contrast and most cells absolutely have to be stained to be seen; staining may introduce extraneous details into the specimen that should not be present.

Also, the user will need to be knowledgeable in proper staining techniques.

Lastly, this method requires a strong light source for high magnification applications and intense lighting can produce heat that will damage specimens or kill living microorganisms.

TERMINOLOGIES

Resolution Ability to distinguish (resolve) two close-

together points as separate. Contrast Differences in intensity between two

objects, or between an object and background

Important in determining resolution Staining increases contrast

LIGHT MICROSCOPY

A. Bright-field microscopes

B. Dark-field microscopes C. Phase microscopes D. Fluorescent microscopes

WHAT ARE BRIGHT-FIELD

MICROSCOPES ??

DESCRIPTION

Brightfield microscopy is the most elementary form of microscope illumination techniques and is generally used with compound microscopes.

The name "brightfield" is derived from the fact that the specimen is dark and contrasted by the surrounding bright viewing field. Simple light microscopes are sometimes referred to as brightfield microscopes. 

WHEN TO USE BRIGHT FIELD MICROSCOPY

Bright field microscopy is best suited to viewing stained or naturally pigmented specimens such as

stained prepared slides of tissue sections or living photosynthetic organisms.

It is useless for living specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections.

OBSERVED USING BRIGHT-FIELD MICROSCOPY, AND APPROPRIATE MAGNIFICATIONS

Prepared slides, stained - bacteria (1000x), thick tissue sections (100x, 400x), thin sections with condensed chromosomes or specially stained organelles (1000x), large protists or metazoans (100x).

Smears, stained - blood (400x, 1000x), negative stained bacteria (400x, 1000x).

Living preparations (wet mounts, unstained) - pond water (40x, 100x, 400x), living protists or metazoans (40x, 100x, 400x occasionally), algae and other microscopic plant material (40x, 100x, 400x). Smaller specimens will be difficult to observe without distortion, especially if they have no pigmentation

Using a bright field microscope

STEPS

Mount the specimen on the stage Optimize the lighting Adjust the condenser Think about what you are looking for Focus, locate, and center the specimen Adjust eyepiece separation, focus Select an objective lens for viewing Adjust illumination for the selected

objective lens

HOW DOES IT WORKS???

In bright-field microscopy a specimen is placed on the stage of the microscope and incandescent light from the microscope’s light source is aimed at a lens beneath the specimen. This lens is called a condenser.

The condenser usually contains an aperture diaphragm to control and focus light on the specimen; light passes through the specimen and then is collected by an objective lens situated in a turret above the stage.

The objective magnifies the light and transmits it to an oracular lens or eyepiece and into the user’s eyes. Some of the light is absorbed by stains, pigmentation, or dense areas of the sample and this contrast allows you to see the specimen.

For good results with this microscopic technique, the microscope should have a light source that can provide intense illumination necessary at high magnifications and lower light levels for lower magnifications

BASIC COMPONENTS OF THE MICROSCOPE

AND THEIR FUNCTIONS

Power switch

Light intensity control

Condenser

Stage

Objectives

Eyepiece lens

Stage motion control knobs

Course and fine adjustments knobs

Field diaphragm ring

Aperture diaphragm

Care and Use of the Bright Field Microscope

Malaria parasite

Mycobacterium tuberculosis

CARE AND MAINTENANCE OF THE MICROSCOPE

Good preventive maintenance and care includes: Regular cleaning of oculars and objectives Avoid damaging oculars and other optics with eye

make-up or other debris Careful handling to avoid abrupt motions Protect from direct sunlight, high temperature,

humidity, dust and vibration Use appropriate materials to clean the lenses Cover when not in use with vinyl or plastic dust cover

CLEANING THE MICROSCOPE

Routine Cleaning Supplies: Commercial lens tissue for optics Caution: Do not use paper

towels or other rough paper products

Cotton swabs with wooden shaft (optics)

70% isopropyl alcohol Dilute methanol is satisfactory Mild detergent and soft cloth for

stage and base of microscope

CLEANING OCULARS AND OBJECTIVES Unplug the microscope Wash hands Remove dust from optical glass

surfaces Carefully remove eyepieces,

objectives, condenser, and filters–one at a time

Excessive rubbing can cause damage to iridescent coating on lens

Clean and replace as completed Do Not take eyepiece or objectives

apart

Unplug microscope and allow bulb to cool

Carefully place microscope on its side

Open bulb house; use tissue to remove bulb

Use tissue (to avoid fingerprints) to pick up new bulb

Insert new bulb and close bulb house

Replacing Microscope Bulb

Plug in microscope and turn on illuminator. Rotate nosepiece to lock 10X objective in place

Place smear on stage and center it under the 10X objective

Open the field diaphragm all the way and close condenser diaphragm all the way

Move up (rack up) stage to its highest position

Adjust the oculars for interpupillary distance so that only one circle of light is seen

Rack up condenser as high as possible

Setting the Koehler Illumination

• Close field diaphragm half way and focus smear at 10X

• Close field diaphragm until diameter of illuminated image is smaller than the field of view

• Lower condenser with positioning knob until you have a sharp, focused image of the edges of the field diaphragm

• Adjust condenser using centering screws so that the circle of light is centered in field

• Open field diaphragm until illuminated image is just larger than the field of view. If more light is needed, use the transformer.

• Koehler illumination is now set. It is important not to move the condenser up or down or change the field diaphragm.

Setting the Koehler Illumination (continued)

OPERATION OF THE MICROSCOPE – EXAMINING SMEARS

Put smear on stage and center it under the 10X objective

Adjust intensity of the light to a comfortable level with the transformer

Open condenser diaphragm about 70% to achieve a good balance of resolution and contrast

Adjust oculars for interpupillary distance so that when looking with both eyes only one circle of light is seen

Examining Smears (continued)• Adjust sharpness of image by moving

adjustment ring on adjustable ocular• Once 10X focus is achieved, rotate

nosepiece so that the 40X objective is in place

• Readjust the intensity of light to a comfortable level using the transformer

• Use the fine adjustment knob to focus up and down through the different planes of the field

Microscope Problems – Troubleshooting 1 Problem: Black Field

Possible Causes:• Microscope not plugged in• Power not available at

outlet• Illuminator not turned on• Bulb burned out• Objective not clicked into

place• Condenser too low with

diaphragms closed

Microscope Problems – Troubleshooting 2

Problem: Field only partially illuminated

Possible Causes:• Objective not clicked into

position• Condenser not centered

correctly• Condenser too low• Field diaphragms closed too

much

Problem: Difficulty focusing with 10X

objective

Possible Causes:• Wrong objective in place• Objective not screwed into

place• Not in correct plane of focus

Microscope Problems – Troubleshooting 3

Microscope Problems – Troubleshooting 4

Problem: Difficulty focusing with 40X

objective

Possible Causes:• Not in correct plane of focus • Not initially focused at 10X

Microscope Problems – Troubleshooting 5

Problem: Blurry image at 10X or 40X

Possible Causes:• Dirty objective• Dirty slide• Dirty coverslip

Problem: Ground glass appearance

Possible Causes: • Condenser too high

REFERENCES:

http://www.microscopemaster.com/brightfield-microscopy.html

Advanced Light Microscopy vol. 1 Principles and Basic Properties by Maksymilian Pluta, Elsevier (1988)

Advanced Light Microscopy vol. 2 Specialised Methods by Maksymilian Pluta, Elsevier (1989)

Introduction to Light Microscopy by S. Bradbury, B. Bracegirdle, BIOS Scientific Publishers (1998)

Microbiology: Principles and Explorations by Jacquelyn G. Black, John Wiley & Sons, Inc. (2005)

Microscopy and Imaging Literature http://www.ruf.rice.edu/~bioslabs/methods/microscopy/

microscopy.html