Microscopes and Microscopy Concepts

Microscopes

• An instrument that helps view objects too small for the unaided eye

Microscopy Concepts

• Magnification is the ratio of the image size to the object's actual size
• Total magnification is determined by multiplying the magnification of the ocular lens by the magnification of the objective lens
• Contrast enables you to see particular details against a background, and can be improved using stain
• Resolution refers to the ability to distinguish two closely positioned points as distinct entities and it improves with magnification

Microscopic Field

• It is the portion of the specimen visible under a microscope
• It decreases with magnification
• Formula: M1 x DFV1 = M2 x DFV2

Microscopic Field Values

• M1: Magnification of the first objective lens
• DFV1: Diameter of the field of view with the first objective lens
• M2: Magnification of the second objective lens
• DFV2: Diameter of the field of view with the second objective lens

Working Distance

• The space between the objective lens and the specimen
• It decreases with magnification

Simple Microscopes

• Considered the first microscopes
• Use a convex lens and a specimen holder
• Magnify objects between 200 to 300 times

Compound Microscopes

• Use two lenses where the second magnifies the image from the first, and it can be binocular or monocular
• Magnify objects up to 1200 times, but their resolution is low

Types of Microscopes

• Microscopes are categorized based on the interaction with the sample to produce an image

Optical Microscopes

• Use visible or invisible light to create an image

Compound Microscopes

• Magnify the specimen through a series of lenses, including an objective and eyepiece lens
• Light illuminates the specimen from underneath
• Produce a two-dimensional image and magnify up to 2500x
• Have low resolving power

Stereo Microscopes

• Light typically illuminates the specimen from above
• Used with opaque specimens
• Create three-dimensional images, but offer low magnification, ranging from 10X to 50X
• Feature lower resolution compared to compound microscopes

Phase Contrast Microscope

• Displays light objects against a dark background
• Feature low magnifications of up to 100x
• Can view everything in a liquid sample including debris
• Light illuminates the specimen from underneath, but a special condenser blocks this light so that only oblique rays hit the object

Laser Scanning Confocal Microscope

• Uses laser beams emitted from a laser light source that pass through the objective lens to scan a sample
• The fluorescence of the sample is reflected from the focal plane, and then visualize the resulting data with a computer
• High-resolution and high-contrast images are produced

Dark Field Microscope

• Best suited for live, unstained biological samples, like tissue culture smears
• Requires strong illumination, which can potentially damage the sample, to see the sample clearly

Dark Field vs. Phase Contrast Microscope

• Dark Field microscopes are best suited for unstained transparent cells
• Live cells can be observed because phase contrast can be converted into brightness differences

Scanning Helium Microscope (SHeM)

• Uses low-energy (5-100 meV) neutral helium atoms to image a sample's surface without causing damage
• Forms an atomic beam through supersonic expansion
• Use helium atom scattering
• Gas scatters from the surface and is collected into a detector to measure the flux of neutral helium atoms

Neutron Microscope

• Uses neutrons, generated by nuclear fission of lithium-6, and small angle neutron scattering to create images

Digital Microscope

• Uses a computer to collect images in place of ocular lenses or uses both ocular lenses and computer
• Software builds three-dimensional images and reduce blurring

Fluorescence Microscope

• Uses invisible light, such as UV light which, when hits a specimen stained with fluorescent dye, emit visible light, increasing resolution
• Enables the observation of fluorescence images and the observation of very specific areas by using different fluorescent labels

Electron Microscope

• Uses a beam of electrons to create an image, using electromagnetic lenses instead of glass ones
• Features very high resolving power
• Two types: Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM)

Transmission Electron Microscope (TEM)

• Gives the ultrastructure of a cell
• Gives a 2D image
• Has a total magnification of over 20 million X

Scanning Electron Microscope (SEM)

• Gives a topography image for the surface of a cell
• Gives a 3D image
• Has a total magnification of over 20,000X

X-ray Microscope

• Uses a beam of X-rays to create the image
• Offer higher resolution than optical microscopes

Scanning Acoustic Microscope

• Uses sound waves to create images and to view samples internally without staining or damage
• Scans and penetrates specimen in water
• Detects sub-micron features, such as popcorn cracking
• Measures the depth of internal layers

Microscope Handling

• Always carry the microscope with two hands with one hand under the base and the other holding the arm, keeping it upright
• Put the microscope gently on the bench, away from the edges
• Do not drag or shove the microscope across the lab bench
• Switch the microscope on or off using the microscope switch, avoiding the main electrical switch
• Clean the microscope before and after each use using lens paper only

Cleaning Microscope Lenses

• First, wipe all optical parts except the oil immersion lens in this order: eyepiece, objectives, condenser and light source
• Then, clean the oil immersion lens only

Storing the Microscope

• Clean the microscope
• Lower the stage to its lowest position
• Position the scanning objective lens (4X) over the condenser
• Coil the electric wire neatly around the arm
• Return the microscope to the cabinet with the microscopic arm facing out

Parts of a Compound Microscope

• Eyepiece / Ocular
• Body tube or Head
• Arm
• Objective Lenses
• Course Focus Knob
• Fine Focus Knob
• Mechanical Stage Knob
• Base
• Brightness adjustment
• Interpupillary distance
• Diopler adjustment
• Revolving Nosepiece
• Stage Clips
• Condenser Lens
• Stage
• Diaphragm
• Light Bulb / Light Source

Eyepiece (Ocular Lens)

• It is the lens through which the viewer looks to see the specimen
• It typically contains a 10X or 15X power lens

Body Tube (Head)

• Connects the eyepiece to the objective lenses

Arm

• Connects the body tube to the base of the microscope and is used to carry the microscope

Coarse Adjustment

• Brings the specimen into general focus

Fine Adjustment

• Fine-tunes the focus and increases the detail of the specimen

Nosepiece

• Rotating turret that houses the objective lenses
• Viewers spins this to select different objective lenses

Objective Lenses

• The most important part of a compound microscope because they are closest to the specimen
• Standard microscopes feature four, ranging from 4X to 100X in power

Specimen (Slide)

• The object being examined
• Most are mounted on slides, which are flat rectangles of thin glass

Stage

• Flat platform where the slide is placed

Stage Clips

• Metal clips that hold the slide in place

Mechanical Stage Knobs

• Control the movement of the stage left, right, up, or down

Illumination

• The light source for the microscope

Iris Diaphragm

• Adjusts the amount of light reaching the specimen

Condenser

• Gathers and focuses light from the illuminator onto the specimen

Base

• Supports the microscope and houses the illuminator

Objective Lenses Types

• Dry objective lenses are Scanning (4X), 10X, and 40X
• Wet objective lenses are one or two (100X) using oil

Viewing a Specimen

• Instructors should demonstrate how to focus the specimen under the microscope

Specimen Focusing Steps

• Fix the slide to the stage
• Put the light spot at the center of the specimen
• Lower the stage to the lowest position
• Start with the lowest light intensity and the scanning objective lens (4X)
• Move the slide using the adobe knob after seeing the image under a certain lens to ensure you are viewing the specimen
• Don't lower the stage from the starting point of focusing when using oil immersion

Aseptic Technique

• Prevents infections of yourself and classmates
• Prevents outdoor and indoor air pollution
• Prevents culture contamination

Culture Mediums

• Pure media are special nutrients where bacteria can grow, but contain no bacteria themselves
• Cultures are media that do contain bacteria
• Prevents laboratory accidents

Aseptic Conditions

• Ensures microbiological work is organized
• Requires researchers know what they will be doing and prepared by researching the experiment before entering the lab

Post-Lab Procedure

• Turn off the Bunsen burner
• If you worked with a microscope, clean it and return it to the cabinet correctly
• Organize the workplace
• Sanitize the bench top with disinfectant or 75% alcohol
• Wash hands with liquid soap and tap water
• Sterilize with antiseptic before leaving the lab
• Take off the lab coat before going out of the lab

Bacteria Wet Mount Procedure

• Requires a sterile, cleaned microscopic slide, coverslip and distilled water

Slide Cleaning Steps

• Wash the slide with liquid soap and tap water
• Rinse the slide with tap water
• Dip the slide in absolute alcohol for 15 minutes to remove soap residue and sterilize by killing bacteria
• Let the slide dry near a Bunsen burner

Sterile Distilled Water

• Free of microorganisms, chloride ions, suspended particles and salt traces

Testing Water Sample

• Water sample can be tested to determine if it is tap, distilled or deionized water
• Add AgNO3 to the sample; a milky suspension indicates chloride ions (Cl-)

Reagents

• AgNO3 + AgCl + NO3 (white precipitate)

Water Testing Comparison

• Milky suspension and conducts electricity indicates tap water
• Won't conduct electricy indicates, a deionized sample, free of ions - Conducts electricity indicates distilled water and contains ions except chloride

Inoculation Loop Sterilization

• Loops are sterilized in the hottest part of the flame (colorless region)
• The loop must be at an angle when in the flame

Wet Mount Slide Preparation

• Place eight loopfuls of sterile distilled water at the center of a sterile cleaned slide
• Add a loopful of bacteria to the distilled water using a sterile cooled loop
• Mix until you get a milky suspension
• Place the edge of a coverslip on the slide so it touches the edge of the water to slowly lower it
• This prevents trapping air bubbles
• Examine under the microscope using the oil immersion lens (100X)

Conclusions on Microscopy

• Microscopic field is the visible part of the specimen under specific magnification
• The microscopic field decreases with magnification
• Working distance is the distance between the objective lens and the specimen
• The working distance decreases with magnification
• Light intensity must be increased as magnification increases
• Images appear smaller and darker at low magnification compared to high magnification
• Microscope resolving power also increases with magnification
• Bacterial cells are minute and colorless, making them hard to see with the naked eye and under a microscope

Bacterial Motility

• Organisms are categorized by either motile or non-motile responses
• Non-motile bacteria can move in one direction (liquid movement) or vibrate at the same place (Brownian movement)
• Motile bacteria can move in all directions