NRSG 205 Lecture 3 - Microscopy and Staining

1 cm = ____ m

10ˆ-3 m

1 μm = ____ m

10ˆ-6 m

1 nm = ____ m

10ˆ-9 m

1 pm = ____ m

10ˆ-12 m

2 qualities of older microscopes

1. Simple

2. 1 lens

2 qualities of modern microscopes

1. Compound

2. Multiple lenses

How to find magnification of a light microscope

objective lens * ocular lens

Ocular lens

lens by the eye that is typically 10X

Objective lens

lens by the specimen that can be 1X, 10X, 40X, 100X

Resolution

ability to distinguish 2 points (clarity)

Refraction

bending of light

What determines refraction?

The medium which the light passes through

Function of refraction

creates contrast because of the bending of light in different directions

8 compound light microscopes

1. Brightfield microscopes

2. Darkfield microscopes

3. Phase contrast

4. Differential Interference Contrast (DIC)

5. Fluorescent light microscope

6. Confocal microscope

7. Two photon microscope

8. Super resolution

Brightfield microscope view

specimen appears against a white background

Magnification of brightfield microscope

Magnification can reach up to 1000X-2000X which can require the use of oil immersion

Function of oil immersion

prevents light from escaping

What 2 processes must occur to a specimen before it is viewed with a brightfield microscope?

1. Fixation

2. Staining

What type of light does the brightfield microscope use (and what wavelength)?

0.2 μm

Darkfield microscope view

bright specimen appears against a dark background

Structure of darkfield microscope

Opaque disk placed over light source

Function of darkfield microscope

Can look at unstained specimens and live specimens

Phase contrast microscope

uses 2 sets of light that are brought together

2 light sources of phase contrast microscopes

1. Light source

2. Light refracted from structures in specimen

Function of phase contrast microscope

Used to look at sharp images of internal structures of a specimen

DIC microscope

2 beams of light create a 3D image

Function of DIC microscope

Creates an almost 3D image of the specimen that can be in color

DIC microscope benefit over phase contrast microscope

Higher resolution than phase contrast microscope

Fluorescent light microscope

Specimen absorbs short wavelengths of light, and emit at a longer wavelength at a certain wavelength

Fluorescent light microscope light

fluorescent light is used instead of white light

Function of fluorescent light microscope

Used for fluorescent antibodies (diagnostic tool, visualization of structures)

Confocal microscope

type of fluorescent microscope that uses short wavelengths of light, and a pinhole aperture reduces and eliminates blurring of light

Benefit of confocal microscope over fluorescent light microscope

Increased resolution compared to fluorescent microscope

Function of confocal microscope

3D image creation - looks at the images in slices and looks at it in all 3 planes

Two photon microscope

uses longer wavelengths (red)

Function of two photon microscope

Creates a deeper image for thicker specimens (up to 1 mm deep)

Super resolution microscope

creates resolution on a nanometer scale

Function of super resolution microscope (practical application)

Looks at small specimen and viruses – high resolution

What is one example of a sound microscope?

Scanning acoustic microscope

Scanning acoustic microscope

uses sound waves to create an image

Scanning acoustic microscope function

Can study living specimens that are attached to a biofilm

Downside of using scanning acoustic microscope

Poor resolution

2 types of electron microscopes

1. Transmission electron microscope (TEM)

2. Scanning electron microscope (SEM)

Transmission electron microscope (TEM)

uses electrons to create an image

Magnification of a transmission electron microscope

Magnification can reach up to 10,000-10,000,000X and works on the picometer scale

Function of transmission electron microscope

Focuses on internal structures of cells

Requirement of a specimen being viewed by a transmission electron microscope

Specimen must be extremely thin (<100 nm thick)

Scanning electron microscope (SEM)

uses electrons to create an image

Magnification of a scanning electron microscope (SEM)

Magnification reaches 1000X-500,000X and works on the nanometer scale

Function of scanning electron microscope (SEM)

Visualizes surfaces structures (on cells) and creates close up 3D images

Function of scanning probe microscopes

Use a probe to examine the surface of specimens using an electric current

2 types of scanning probe microscopes

1. Scanning tunneling microscope

2. Atomic force microscope

Scanning tunneling microscope

uses a tungsten probe that reveals bumps and depressions of atoms on the specimen

Scanning tunneling microscope resolution

1/100 size of an atom

Function of scanning tunneling microscope

Provides a detailed view of molecules like DNA

Atomic force microscope

uses a metal and diamond probe to scan the surface of the specimen

Atomic force microscope view

Creates a 3D image

Atomic force microscope function

Used to view molecular processes and biological substances

Staining function

used to create contrast between specimen and background

Which occurs first: fixing or staining?

Fixing

Fixing funcftion

uses heat or alcohol to kill and attach microorganism to slide

Positive stain

bacteria are negatively charged, so basic (positively charged) dyes can stain them

Negative stain

acidic (negatively charged) dyes can be repelled by the bacteria, so it can stain the background

4 examples of basic dyes

1. Crystal violet

2. Methylene blue

3. Malachite green

4. Safranin

3 examples of acidic dyes

1. Eosin

2. Fuchsin

3. Nigrosin

3 main types of stains

1. Simple stain

2. Differential stain

3. Negative stain

Simple stain function

used to simple visualize the specimen

2 substances used in a simple stain

1. Single basic dyes

2. Mordant

Mordant

Improves bond between specimen and stain

What mordant is used in the gram stain?

Iodine

Differential stain function

differentiates between different types of organisms

2 types of differential stains

1. Gram stain

2. Acidic-fast stain

Gram stain

distinguishes between different types of bacteria's cell walls (gram positive or gram negative)

Stain and counterstain in gram stain

• Stain: crystal violet (+)

• Counterstain: safranin (-)

What color does the specimen turn in a gram positive result?

Purple

What color does the specimen turn in a gram negative result?

Red/pink

Acidic-fast stain

acid-fast bacteria are stained

What 2 conditions does mycobacterium cause?

1. Tuberculosis

2. Leprosy

Cell wall of mycobacterium

have a mycolic acid in their cell wall that makes it waxy

Stain and counterstain in gram stain

• Stain: carbofuschin (red)

• Counterstain: methylene blue (blue)

What color does the specimen turn if it is an acid-fast bacterium

Red

What color does the specimen turn if it is a non acid-fast bacterium?

Blue

Negative stain function

used to look at structures that cannot handle the harsh staining process

What specific structure does a negative stain stain?

Capsules

Capsule

layer of sugar outside of the cell wall that makes substances more virulent

Function of negative stain (practical application)

Can look for endospores and flagella