Microscopes: Types, Features, and Cell Observation

Microscopes Overview

• Microscopes magnify small objects or fine details of larger objects that would be invisible to the naked eye.
• Compound, hand lens, transmission electron, and scanning electron microscopes are different types.
• A compound microscope uses ocular (eyepiece) and objective lenses.

Cell Observation

• Microscopes needed because cells are too small to see with the naked eye.
• The light microscope is common in laboratories and can reveal the cytoplasm and nucleus of a cell.
• Jansen invented inverted the compound light microscope in 1590.
• The more detailed electron microscope was developed in the 1950s.
• Electron microscopes use electron beams to flood specimens.

Key Features

• Magnification and resolving power are the main features.
• Magnification refers to a microscope's ability to enlarge a specimen.
• Light microscopes can magnify cell structures about 1,000 times their normal size.
• Electron microscopes can magnify images 250,000 times or more

Laboratory Types

• Resolving power allows microscopes to reveal fine specimen details and separate closely positioned points
• Transmission and dissecting microscopes are two types used in laboratories.

Microscope Operation

• Light waves go through the the specimen in transmission microscopes
• Electrons are transmitted through specimens in electron microscopes.
• Light reflects off the specimen in dissecting microscopes to show surface features.
• SEM, a dissecting microscope variant, shows surface features.
• TEM, a transmission microscope variant, shows internal structures.

Compound Microscope Structure

• The diagrams labels the eye piece, draw tube, body tube, coarse and fine adjustment, nose piece, objective, stage, slide, clip, diaphragm and filter, condenser, light reflector and foot.

Compound Microscope Components

• The structural parts are the head, base, and arm.
• The head, or body tube, contains the optical parts.
• The base provides support and houses the illuminator.
• The arm connects the base to the head and enables carrying.

Optical Elements

• The optical systems consist of eyepiece and objective lenses.
• Eyepiece, or ocular, lenses are looked through at the top of the microscope.
• Eyepiece lenses typically magnify at 10x, with others ranging from 5x to 30x.

Eyepiece Functions

• The eyepiece tube secures the eyepiece lenses.
• The eyepiece normally contains a 10X or 15X power lens is the lens the viewer looks through to see the specimen
• Binocular microscope heads often have a diopter adjustment ring to account for vision inconsistencies.
• Diopter adjustment changes focus on one eyepiece to correct vision differences between eyes.

Body and Arm

• The body tube joins the eyepiece to the objective lenses.
• The arm connects the body tube to the microscope base.
• Focusing the specimen is done with the coarse adjustment.
• Objective lenses are primary optical lenses on a microscope and range from 4x to 100x.
• Standard objectives include 4x, 10x, 40x, and 100x.

Nosepiece Purpose

• The nose piece holds and rotates the objective lenses.
• Fine adjustment increases specimen detail with fine focus
• Fine and coarse adjusting knobs are coaxial, built on the same axis with the fine adjustment knob on the outside.

Stage and Light

• The stage is the platform for specimen placement.
• Stage clips secure the slide with manual movement when there is no mechanical stage.
• The aperture is the hole in the stage for light from the base to reach the stage.
• An illuminator is a light source, commonly in the microscope base.
• Adequate light ensures proper focusing of the magnified object.

Iris Role

• Located above the condenser and below the stage, it manages light levels reaching the specimen.
• Iris diaphragm controls both focus and light quantity.
• Condenser focus knob adjusts the condenser to control lighting focus on the specimen.

How Images Form

• Light from the illuminator passes through the aperture, slide, and objective lens.
• The objective lens magnifies the specimen image.
• The magnified image is further magnified in the eyepiece.

Prep steps

• Turn on the illuminator gradually increase the light intensity.
• Center and secure the specimen on the stage over the aperture.
• Fully open the iris diaphragm to allow maximum light.
• Position the lowest power objective lens over the specimen to find the area of iterest.

Alignment steps

• To the viewers comfort view the lenses to ensure the light is on and clear.
• Use the course adjustment knot for focusing of lenses and slides, be aware that the two parts do not collide.
• Use the finer adjustment to properly view the specimen.

Magnification Details

• Magnification is the apparent enlargement of an object via an optical instrument.
• It represents the ratio of an images dimensions to the objects actual dimensions.
• Calculate total magnification by multiplying ocular lens power by objective lens power.
• A 10x eyepiece with a 40x objective results in 400x magnification.

Drawing Magnification

• First view and measure the object.
• Calibrate the microscope by viewing a known size and compare to its actual size on the slide.
• Magnification of the illustration calculates as the illustration size divided by the speciman size:

Additional Factors

• Round off the final magnification result to the nearest number
• Estimated magnification includes a third factor: the investigators perceived magnification on a drawing.
• Total magnification writes as x10x10x4, with x10 eyepiece, x10 objective lens, and four times in image magnification.

Image Resolution

• Resolution is the ability to differentiate close objects.
• It denotes the shortest resolvable distance between two points as distinguished by observer or system.
• Aperture, illumination and speciman properties determine resolution.

Immersion lenses

• Some monocular and all binocular compound microscopes use 100x oil immersion lenses, identified by a red or white band.
• Above 500x, light refracts too much in air, reducing resolution.
• Higher magnifications use mineral oil to better transmit light to resolve the image at a 400x centre.

Oil Applying Procedure

• Raise objective lens to its limit and move the lens halfway of of it's position.
• Apply Immersion oil directly to the middle of lens to enable focusing of the oil drop.
• Rotate the oil and view to see the focus.
• Clean the lens and save the slide if neccessary.

Mounting Methods

• The speciman has to be thin enough to allow light through it.
• During examination water protects the protoplasm.
• For a wet mount place and cover in a clean grease free micro slide.
• Use a lughol or iodine solution.

Mounting Position

• Hold the grease free micro slip to one side.
• Ensure the micro cover holds an angle between 45 and 60 degress to eliminate air bubbles.
• Lower the micro slip gently as support with a needle, ensure you do not cause air bubbles.
• Ensure the lenses can focus at the course and fine settings, producing the clearest shot.

Parfocal Image Usage

• This parfocal setup produced a clear shot, the lower the power the clearer
• Use a x10 objective at a 2mm diameter field of view.
• Calculate cells by dividing their number from their length.
• 0.25mm is determined mm divided by 10000 um.
• Lens size depends on the objective used.

Transmission Details

• Transmission electron microscopes visualize samples with a beam of electrons, creating high-magnification black and white images.
• They can magnify objects by up to 2 million times.
• Air needs removal creating a space in a vacuum chamber, where beams of electrons produce magnified images.
• View thin specimens for a produced image.

TEM Drawbacks

• Electron and electromagnetic fields must be in a protected area.
• Operation requires upkeep of maintaining voltage, currents and cooling water.
• TEM SEM differ as SEM focuses more to a point as opposed to a Static beam found in TEM, that is more suited to linear scanning