Microscopes

An early microscope was made in 1590 inMiddleburg, The Netherlands Three eyeglass makers are variously given credit: Hans Lippershey (who developed an early telescope); Zach arias Jansen, with the help of his father, Hans Janssen. Giovanni Faber coined the name for Galileo Galilee's compound microscope in 1625 (Galileo had called it the "occhiolino" or "little eye".)

The first detailed account of the interior construction of living tissue based on the use of a microscope did not appear until 1644, in Giambattista Odierna's L'ochio della mosca, or The Fly's Eye.

It was not until the 1660s and 1670s that the microscope was used seriously in Italy, Holland and England. Marcelo Malpighi in Italy began the analysis of biological structures beginning with the lungs. Robert Hooke's Micrographia had a huge impact, largely because of its impressive illustrations. The greatest contribution came from Antoni van Leeuwenhoek who discovered red blood cells and spermatozoa. On 9 October 1676, Leeuwenhoek reported the discovery of micro-organisms.

The most common type of microscope---and the first invented---is the optical microscope. This is an optical instrument containing one or more lenses producing an enlarged image of an object placed in the focal plane of the lenses

piece, objective, mechanical stage, condenser, coarse and fine focusing

knobs, and light source.

Historians credit the invention of the compound microscope to the Dutch spectacle maker, Zacharias Janssen, around the year 1590. The compound microscope uses lenses and light to enlarge the image and is also called an optical or light microscope (vs./ an electron microscope). The simplest optical microscope is the magnifying glass and is good to about ten times (10X) magnification. The compound microscope has two systems of lenses for greater magnification, 1) the ocular, or eyepiece lens that one looks into and 2) the objective lens, or the lens closest to the object. Before purchasing or using a microscope, it is important to know the functions of each part.

Eyepiece Lens: the lens at the top that you look through. They are usually 10X or 15X power.

Tube: Connects the eyepiece to the objective lenses

Arm: Supports the tube and connects it to the base

Base: The bottom of the microscope, used for support

Illuminator: A steady light source (110 volts) used in place of a mirror. If your microscope has a mirror, it is used to reflect light from an external light source up through the bottom of the stage.

Stage: The flat platform where you place your slides. Stage clips hold the slides in place. If your microscope has a mechanical stage, you will be able to move the slide around by turning two knobs. One moves it left and right, the other moves it up and down.

Revolving Nosepiece or Turret: This is the part that holds two or more objective lenses and can be rotated to easily change power.

Objective Lenses: Usually you will find 3 or 4 objective lenses on a microscope. They almost always consist of 4X, 10X, 40X and 100X powers. When coupled with a 10X (most common) eyepiece lens, we get total magnifications of 40X (4X times 10X), 100X , 400X and 1000X. To have good resolution at 1000X, you will need a relatively sophisticated microscope with an Abbe condenser. The shortest lens is the lowest power, the longest one is the lens with the greatest power. Lenses are color coded and if built to DIN standards are interchangeable between microscopes. The high power objective lenses are retractable (i.e. 40XR). This means that if they hit a slide, the end of the lens will push in (spring loaded) thereby protecting the lens and the slide. All quality microscopes have achromatic, parcentered, parfocal lenses.

Rack Stop: This is an adjustment that determines how close the objective lens can get to the slide. It is set at the factory and keeps students from cranking the high power objective lens down into the slide and breaking things. You would only need to adjust this if you were using very thin slides and you weren't able to focus on the specimen at high power. (Tip: If you are using thin slides and can't focus, rather than adjust the rack stop, place a clear glass slide under the original slide to raise it a bit higher)

Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen. Condenser lenses are most useful at the highest powers (400X and above). Microscopes with in stage condenser lenses render a sharper image than those with no lens (at 400X). If your microscope has a maximum power of 400X, you will get the maximum benefit by using a condenser lenses rated at 0.65 NA or greater. 0.65 NA condenser lenses may be mounted in the stage and work quite well. A big advantage to a stage mounted lens is that there is one less focusing item to deal with. If you go to 1000X then you should have a focusable condenser lens with an N.A. of 1.25 or greater. Most 1000X microscopes use 1.25 Abbe condenser lens systems. The Abbe condenser lens can be moved up and down. It is set very close to the slide at 1000X and moved further away at the lower powers.

Diaphragm or Iris: Many microscopes have a rotating disk under the stage. This diaphragm has different sized holes and is used to vary the intensity and size of the cone of light that is projected upward into the slide. There is no set rule regarding which setting to use for a particular power. Rather, the setting is a function of the transparency of the specimen, the degree of contrast you desire and the particular objective lens in use.

How to Focus Your Microscope: The proper way to focus a microscope is to start with the lowest power objective lens first and while looking from the side, crank the lens down as close to the specimen as possible without touching it. Now, look through the eyepiece lens and focus upward only until the image is sharp. If you can't get it in focus, repeat the process again. Once the image is sharp with the low power lens, you should be able to simply click in the next power lens and do minor adjustments with the focus knob. If your microscope has a fine focus adjustment, turning it a bit should be all that's necessary. Continue with subsequent objective lenses and fine focus each time.

The one-celled organism

amoeba proteus

A single-celled bacteria

of the type: E. coli

A human red blood cell

A plant cell from the leaf

of a poplar tree

The cell is one of the most basic units of life. There are millions of different types of cells. There are cells that are organisms onto themselves, such as microscopic amoeba and bacteria cells. And there are cells that only function when part of a larger organism, such as the cells that make up your body. The cell is the smallest unit of life in our bodies. In the body, there are brain cells, skin cells, liver cells, stomach cells, and the list goes on. All of these cells have unique functions and features. And all have some recognizable similarities. All cells have a 'skin', called the plasma membrane, protecting it from the outside environment. The cell membrane regulates the movement of water, nutrients and wastes into and out of the cell. Inside of the cell membrane are the working parts of the cell. At the center of the cell is the cell nucleus. The cell nucleus contains the cell's DNA, the genetic code that coordinates protein synthesis. In addition to the nucleus, there are many organelles inside of the cell - small structures that help carry out the day-to-day operations of the cell. One important cellular organelle is the ribosome. Ribosomes participate in protein synthesis. The transcription phase of protein synthesis takes places in the cell nucleus. After this step is complete, the mRNA leaves the nucleus and travels to the cell's ribosomes, where translation occurs. Another important cellular organelle is the mitochondrion. Mitochondria (many mitochondrion) are often referred to as the power plants of the cell because many of the reactions that produce energy take place in mitochondria. Also important in the life of a cell are the lysosomes. Lysosomes are organelles that contain enzymes that aid in the digestion of nutrient molecules and other materials. Below is a labelled diagram of a cell to help you identify some of these structures.

There are many different types of cells. One major difference in cells occurs between plant cells and animal cells. While both plant and animal cells contain the structures discussed above, plant cells have some additional specialized structures. Many animals have skeletons to give their body structure and support. Plants do not have a skeleton for support and yet plants don't just flop over in a big spongy mess. This is because of a unique cellular structure called the cell wall. The cell wall is a rigid structure outside of the cell membrane composed mainly of the polysaccharide cellulose. As pictured at left, the cell wall gives the plant cell a defined shape which helps support individual parts of plants. In addition to the cell wall, plant cells contain an organelle called the chloroplast. The chloroplast allow plants to harvest energy from sunlight. Specialized pigments in the chloroplast (including the common green pigment chlorophyll) absorb sunlight and use this energy to complete the chemical reaction:

6 CO2 + 6 H2O + energy (from sunlight) C6H12O6 + 6 O2

In this way, plant cells manufacture glucose and other carbohydrates that they can store for later use.

Organisms contain many different types of cells that perform many different functions. In the next lesson, we will examine how individual cells come together to form larger structures in the human body.

Animal cells are typical of the eukaryotic cell, enclosed by a plasma membrane and containing a membrane-bound nucleus and organelles. Unlike the eukaryotic cells of plants and fungi, animal cells do not have a cell wall. This feature was lost in the distant past by the single-celled organisms that gave rise to the kingdom Animalia. Most cells, both animal and plant, range in size between 1 and 100 micrometers and are thus visible only with the aid of a microscope.

The lack of a rigid cell wall allowed animals to develop a greater diversity of cell types, tissues, and organs. Specialized cells that formed nerves and muscles-tissues impossible for plants to evolve-gave these organisms mobility. The ability to move about by the use of specialized muscle tissues is a hallmark of the animal world, though a few animals, primarily sponges, do not possess differentiated tissues. Notably, protozoans locomote, but it is only via nonmuscular means, in effect, using cilia, flagella, and pseudopodia.

The animal kingdom is unique among eukaryotic organisms because most animal tissues are bound together in an extracellular matrix by a triple helix of protein known as collagen. Plant and fungal cells are bound together in tissues or aggregations by other molecules, such as pectin. The fact that no other organisms utilize collagen in this manner is one of the indications that all animals arose from a common unicellular ancestor. Bones, shells, spicules, and other hardened structures are formed when the collagen-containing extracellular matrix between animal cells becomes calcified.

Animals are a large and incredibly diverse group of organisms. Making up about three-quarters of the species on Earth, they run the gamut from corals and jellyfish to ants, whales, elephants, and, of course, humans. Being mobile has given animals, which are capable of sensing and responding to their environment, the flexibility to adopt many different modes of feeding, defense, and reproduction. Unlike plants, however, animals are unable to manufacture their own food, and therefore, are always directly or indirectly dependent on plant life.

Most animal cells are diploid, meaning that their chromosomes exist in homologous pairs. Different chromosomal ploidies are also, however, known to occasionally occur. The proliferation of animal cells occurs in a variety of ways. In instances of sexual reproduction, the cellular process of meiosis is first necessary so that haploid daughter cells, or gametes, can be produced. Two haploid cells then fuse to form a diploid zygote, which develops into a new organism as its cells divide and multiply.

The earliest fossil evidence of animals dates from the Vendian Period (650 to 544 million years ago), with coelenterate-type creatures that left traces of their soft bodies in shallow-water sediments. The first mass extinction ended that period, but during the Cambrian Period which followed, an explosion of new forms began the evolutionary radiation that produced most of the major groups, or phyla, known today. Vertebrates (animals with backbones) are not known to have occurred until the early Ordovician Period (505 to 438 million years ago).

Cells were discovered in 1665 by British scientist Robert Hooke who first observed them in his crude (by today's standards) seventeenth century optical microscope. In fact, Hooke coined the term "cell", in a biological context, when he described the microscopic structure of cork like a tiny, bare room or monk's cell. Illustrated in Figure 2 are a pair of fibroblast deer skin cells that have been labeled with fluorescent probes and photographed in the microscope to reveal their internal structure. The nuclei are stained with a red probe, while the Golgi apparatus and microfilament actin network are stained green and blue, respectively. The microscope has been a fundamental tool in the field of cell biology and is often used to observe living cells in culture. Use the links below to obtain more detailed information about the various components that are found in animal cells.

• Centrioles - Centrioles are self-replicating organelles made up of nine bundles of microtubules and are found only in animal cells. They appear to help in organizing cell division, but aren't essential to the process.
• Cilia and Flagella - For single-celled eukaryotes, cilia and flagella are essential for the locomotion of individual organisms. In multicellular organisms, cilia function to move fluid or materials past an immobile cell as well as moving a cell or group of cells.
• Endoplasmic Reticulum - The endoplasmic reticulum is a network of sacs that manufactures, processes, and transports chemical compounds for use inside and outside of the cell. It is connected to the double-layered nuclear envelope, providing a pipeline between the nucleus and the cytoplasm.
• Endosomes and Endocytosis - Endosomes are membrane-bound vesicles, formed via a complex family of processes collectively known as endocytosis, and found in the cytoplasm of virtually every animal cell. The basic mechanism of endocytosis is the reverse of what occurs during exocytosis or cellular secretion. It involves the invagination (folding inward) of a cell's plasma membrane to surround macromolecules or other matter diffusing through the extracellular fluid.
• Golgi Apparatus - The Golgi apparatus is the distribution and shipping department for the cell's chemical products. It modifies proteins and fats built in the endoplasmic reticulum and prepares them for export to the outside of the cell.
• Intermediate Filaments - Intermediate filaments are a very broad class of fibrous proteins that play an important role as both structural and functional elements of the cytoskeleton. Ranging in size from 8 to 12 nanometers, intermediate filaments function as tension-bearing elements to help maintain cell shape and rigidity.
• Lysosomes - The main function of these microbodies is digestion. Lysosomes break down cellular waste products and debris from outside the cell into simple compounds, which are transferred to the cytoplasm as new cell-building materials.
• Microfilaments - Microfilaments are solid rods made of globular proteins called actin. These filaments are primarily structural in function and are an important component of the cytoskeleton.
• Microtubules - These straight, hollow cylinders are found throughout the cytoplasm of all eukaryotic cells (prokaryotes don't have them) and carry out a variety of functions, ranging from transport to structural support.
• Mitochondria - Mitochondria are oblong shaped organelles that are found in the cytoplasm of every eukaryotic cell. In the animal cell, they are the main power generators, converting oxygen and nutrients into energy.
• Nucleus - The nucleus is a highly specialized organelle that serves as the information processing and administrative center of the cell. This organelle has two major functions: it stores the cell's hereditary material, or DNA, and it coordinates the cell's activities, which include growth, intermediary metabolism, protein synthesis, and reproduction (cell division).
• Peroxisomes - Microbodies are a diverse group of organelles that are found in the cytoplasm, roughly spherical and bound by a single membrane. There are several types of microbodies but peroxisomes are the most common.
• Plasma Membrane - All living cells have a plasma membrane that encloses their contents. In prokaryotes, the membrane is the inner layer of protection surrounded by a rigid cell wall. Eukaryotic animal cells have only the membrane to contain and protect their contents. These membranes also regulate the passage of molecules in and out of the cells.
• Ribosomes - All living cells contain ribosomes, tiny organelles composed of approximately 60 percent RNA and 40 percent protein. In eukaryotes, ribosomes are made of four strands of RNA. In prokaryotes, they consist of three strands of RNA.

In addition the optical and electron microscope, scientists are able to use a number of other techniques to probe the mysteries of the animal cell. Cells can be disassembled by chemical methods and their individual organelles and macromolecules isolated for study. The process of cell fractionation enables the scientist to prepare specific components, the mitochondria for example, in large quantities for investigations of their composition and functions. Using this approach, cell biologists have been able to assign various functions to specific locations within the cell. However, the era of fluorescent proteins has brought microscopy to the forefront of biology by enabling scientists to target living cells with highly localized probes for studies that don't interfere with the delicate balance of life processes.osmosis as a type of diffusion

Pre-lab Reading: Maintaining the steady state of a cell is achieved only through regulated movement of materials through cytoplasm, across organelle membranes, and across the plasma membrane. This regulated movement facilitates communication within the cell and between cytoplasm and the external environment. The cytoplasm and extracellular environment of the cell are aqueous solutions. They are composed of water, which is the solvent, or dissolving agent, and numerous organic and inorganic molecules, which are the solutes, or dissolved substances. Organelle membranes and the plasma membrane are selectively permeable, allowing water to freely pass through but regulating the movement of solutes.

Water and selected solutes move passively though the cell and cell membranes by diffusion, a physical process in which molecules move from an area where they are in high concentration to one where their concentration is lower. The energy driving diffusion comes only from the intrinsic kinetic energy in all atoms and molecules. If nothing hinders the movement, a solute will diffuse until it reaches equilibrium.

Osmosis is a type of diffusion, the diffusion of water through a selectively permeable membrane from a region where it is highly concentrated to a region where its concentration is lower. The difference in concentration of water occurs if there is an unequal distribution of at least on dissolved substance on either side of a membrane and the membrane is impermeable to that substance. For example, if a membrane that is impermeable to sucrose separates a solution of sucrose from distilled water, water will move from the distilled water (where it is in higher concentration) through the membrane into the sucrose solution (where it is in lower concentration). Because water is such an important molecule in living things (making of about 70% of the human body), changes in the amount of water in living tissues can have a severe impact on all aspects of physiology.

Hypertonic, hypotonic, and isotonic are used when referring to two solutions separated by a selectively permeable membrane. The hypertonic solution has a greater concentration of solutes than the solution on the other side of the membrane. It is described as having a greater osmolarity(solute concentration expressed as molarity). The hypotonic solution has a lower concentration of solutes than the solution on the other side of the membrane. When the two solutions are in equilibrium, the solute concentration is equal on both sides of the membrane. The osmolarities are equal and the substances are said to be isotonic. The net flow of water is from the hypotonic to the hypertonic solution. When the solutions are isotonic, there is not net flow of water across the membrane.

Objectives:

1. Explain why diffusion and osmosis are important to cells.

2. List several factors that influence the rate of diffusion.

3. Describe a selectively permeable membrane, and explain its role in osmosis.

4. Define hypotonic, hypertonic, and isotonic in terms of relative concentration of osmotically active substances.

5. Discuss the influence of the cell wall on osmotic behavior in cells.

Sample illumination is achieved in different ways in a light and electron microscope.

In a light microscope, the sample is illuminates with light (photon energy)

In an electron microscope, the sample is illuminated by a beam of electrons.

Devon