# The Infinitesimally Small

Viewing tiny objects, like cells, under a microscope is a real game of hide-and-seek with the light.  It follows that the specimen must be carefully prepared, or ‘mounted’ on a slide.  Here we get a little closer to the eukaryotic cell.  The building block of life itself…

# Once There Was A Cell…

Cells emerged on Earth at least 3.5 billion years ago.

The almost improbable beginning of life as we know it – the humble cell…

The “Father of Microbiology” is no doubt Dutch scientist Antonie van Leeuwenhoek (1632-1723) who first teaches himself to make optical lenses, then constructs his own basic optical microscopes.

He explores the microscopic world, reporting his amazing findings to the Royal Society, and draws protozoa, such as Vorticella from rain water, and even bacteria from his own mouth.

When Robert Hooke discovers cells in cork, and living plant tissue, with an early compound microscope, he coins the term cell in his ‘Micrographia or Some Physiological Descriptions of Minute Bodies made by Magnifying Glasses with Observations and Inquiries thereupon’ (1665).

But it isn’t until 1839 that German physiologist Theodor Schwann and botanist Matthias Jakob Schleiden elucidate the principle that plants and animals are made of cells, a common unit of structure and development.

### Cell Theory

Indeed the modern interpretation of cell theory states that:

1. All known living things are made up of one or more cells.
2. All living cells arise from preexisting cells by division.
3. The cell is the fundamental unit of structure and function in all living organisms.
4. The activity of an organism depends on the total activity of independent cells.
5. Energy flow occurs within cells.
6. Cells contain the hereditary information necessary for transmitting information to the next generation of cells, and regulating cell functions: DNA found within the chromosome, and RNA found in the cell nucleus and cytoplasm.
7. All cells are basically the same in chemical composition in organisms of similar species.

# Units of Life

The cell is the smallest unit of life, consisting of cytoplasm enclosed within a membrane, which contains bio-molecules: proteins and nucleic acids (DNA and RNA).

Cytoplasm is about 80% water and usually colourless.  The membrane contain a variety of biological molecules, notably lipids and proteins.  Material is incorporated into the membrane, or deleted from it, by a variety of mechanisms.  And inside the cell, a whole lot more is going on…

Be it prokaryotic or eukaryotic, the cell is seemingly the basic structural, functional, and biological unit common to all known living organisms.  The basic unit of structure and the basic unit of reproduction.

## Wall or No Wall?

Many types of cells also have a cell wall that acts to protect the cell mechanically and chemically from its environment, and is an additional layer of protection to the cell membrane.  Different types of cell have cell walls made up of different materials, like cellulose in plants.

Unlike plant cells and bacteria, however, animal cells have no cell wall to support them structurally.  And this interesting fact is well worth remembering when ‘mounting’ your specimen on microscope slides because of a cellular process called osmosis.

## Cell Division

Cells divide.

Eukaryotic cells undergo two distinct types of division:

• mitosis – a vegetative division, whereby each daughter cell is genetically identical to the parent cell and
• meiosis – a reproductive cell division, whereby the number of chromosomes in the daughter cells is reduced by half to produce haploid gametes.

Prokaryotic cells undergo a vegetative cell division: binary fission, whereby their genetic material is segregated equally into two daughter cells.

## DNA Replication

Regardless of organism, all cell divisions are preceded by DNA replication – the biological process of producing two identical replicas of DNA from one original molecule.

Replication is initiated at particular points in the DNA, known as “origins“.  As DNA synthesis proceeds, the original DNA strands continue to unwind on each side of the bubble, forming a replication fork with two prongs.

Eukaryotic DNA strands have long linear chromosomes and initiate replication at multiple origins within these.  Bacterial DNA has a single origin of replication on the circular chromosome, and their replication creates a “theta structure“.

# Multicellular Organisms

Organisms can be classified as unicellular, i.e. consisting of a single cell.  This includes bacteria.

They can be multicellular, like plants and animals.

While the number of cells in plants and animals varies from species to species, human beings contain over 10 trillion (1013) cells.  In fact, humans are an excellent example of multi-cellular organisms because

#### 100 trillion bacteria!

With dimensions between 1 to 100 micrometres (μm), most eukaryotic cells (plants and animals) are visible only under a microscope.

### Putting Microscopic Sizes into Perspective

1 to 10 μm: bacterium (diameter)

3 to 4 μm : yeast cell

7 μm: human red blood cells

9 μm : thickness of the tape in a 120-minute compact cassette (ancient technology, predates MP3)

10 μm: fog water droplet

170 μm: glass cover slip (thickness)

These are the very basics about the ‘humble’ (or rather ‘mighty’) cell.

The study of very small objects, like cells, with a compound light microscope, requires the object, or specimen under scrutiny, to be carefully prepared, and sometimes stained to increase the contrast between the object and its background.

We now look at mounting specimens on slides.

And why we need to do it that way.

# Preparing Slides for the Microscope

## Dry Mount

This technique is only really suitable for looking at macroscopic objects: small insects, crystals, coin inscriptions, hallmarks…

## Wet Mount

The wet mount technique is used for preparing eukaryotic cells, such as the cells of plants and animals for the microscope.
To view bacteria or prokaryotes, which are even smaller than plant and animal cells, a different preparation of the specimen is involved, called a bacterial smear.  But it won’t be described here.

## Slide Preparation

#### Step 1 – Obtain the specimen to be used

What are you going to mount?  Select the specimen to be used.

Specimens for use in Biology 101 often include: onion skin, Elodea leaves or epithelial cells…

#### Step 2 – Prepare thin sections

Prepare thin sections of plant or vegetable matter using a microtome or a very sharp cutting blade.  Keep your thin sections well hydrated.  You can try different techniques and see what works best, e.g. freezing the specimen, before slicing it.  Most of the time, practice makes perfect.

#### Step 3 – Obtain a clean microscope slide

You can use the standard:

• microscope slide (25.4 x 76.2 mm) (1 – 1.2 mm thick)
• glass cover slip (22 x 22 mm) (0.13 – 0.16 mm thick)

Or you can procure concave depression slides if examining a drop of fluid.

#### Step 4 – Place a drop of liquid at the centre of the slide.

That’s the “wet” part of the wet mount.  The liquid used depends on the type of cell being viewed (see below).  Use tweezers to transfer the thin section into the liquid drop on the slide.

#### Step 5 – Stain the specimen to be used

If the specimen is transparent, a drop of iodine or methylene blue can improve the contrast.

Do not use stain if you’re viewing photosynthetic cells (already green with chlorophyll), or living organisms (because it will kill them).

## Osmotic Pressure

Osmosis is a vital process in biological systems.

Biological membranes are semi-permeable.

Generally, these membranes are:

• permeable to non-polar and hydrophobic molecules like lipids and small molecules: oxygen, carbon dioxide, nitrogen and nitric oxide,
• impermeable to large and polar molecules, such as ions, proteins, and polysaccharides.

Osmosis (osmotic pressure) is an important factor affecting cells.  It is the force that a dissolved substance exerts on that semipermeable membrane, through which it cannot penetrate, when separated by it from pure solvent.

You must bear it in mind when mounting your microscope slides.

For example, if you choose to look at animal cells, a physiological saline (contact lens solution) or distilled water must be used.  If ordinary water is used, the cell will explode due to the osmotic pressure.  Remember, animal cells have no cell wall unlike plant cells.

Osmotic pressure is the basis for filtering (or “reverse osmosis”) – a process commonly used in water purification.  Reverse osmosis desalinates fresh water from ocean salt water.

The osmotic pressure of ocean water is about 27 atm.  The water to be purified is placed in a chamber and put under an amount of pressure greater than the osmotic pressure exerted by the water and the solutes dissolved in it.  Part of the chamber opens to a differentially permeable membrane that lets water molecules through, but not the solute particles.

#### Step 6 – Place a cover slip on

To avoid trapping air bubbles, set one edge of the cover slip on the large microscope slide, and let the rest of the cover slip drop to sandwich the specimen.  As the cover slip drops from one side to the other, air will be pushed out and reduce the number of bubbles.

Now that you’ve got a slide ready for viewing, remember you are playing a game of hide-and-seek with light!

The reason why you need to make very thin sections is to allow as much light as possible to travel through the specimen.  And that’s also why you need to use a glass microscope slide and cover slip.

## Refraction Index Through a Microscope Slide

A compound microscope uses a lens close to the object being viewed to collect light (objective lens) which focuses a real image of the object inside the microscope (image 1).

That image is then magnified by a second lens or group of lenses (eyepiece) that gives the viewer an enlarged inverted virtual image of the object (image 2).

Using a compound objective/eyepiece combination allows for much greater magnification and more advanced illumination setups, such as phase contrast.

In optical microscopy, the refractive index is an important variable in calculating numerical aperture, which is a measure of the light-gathering and resolving power of an objective.

The refractive index (or index of refraction) is a value calculated from the ratio of the speed of light in a vacuum to that in a second medium of greater density.  The refractive index variable is symbolised by the letter n in mathematical equations.

Snell’s Law describes the relationship between the angles of incidence and refraction, when light or any other electromagnetic wave passes through the boundary between two media, like glass or water.

$\frac {\sin(\theta_{1})}{\sin(\theta_{2})} = \frac {v_1}{v_2} = \frac {n_2}{n_1}$

It provides a tool for predicting the amount by how much a light ray bends.

In most cases, the imaging medium for microscopy is air.

$n_{vacuum} = 1.000$

$n_{air} = 1.000$

$n_{water} = 1.333$

$n_{oil} = 1.515$

High-magnification objectives employ oil or a similar liquid between the objective front lens and the specimen to improve the resolution.

The numerical aperture equation is given by:

$NA (numerical aperture) = n \times sin(\theta)$

As the refractive index of a material increases, the greater the extent to which a light beam is deflected (or refracted) upon entering or leaving the material.

The refractive index of a medium is dependent (to some extent) upon the frequency of light passing through, with the highest frequencies having the highest values of n.

In ordinary glass, the refractive index for violet light is about 1% greater than that for red light.

A consequence of this phenomenon is that each wavelength experiences a slightly different degree of refraction when a heterogeneous light beam containing more than one frequency enters or leaves the medium.

This effect is called dispersion.  It is responsible for chromatic aberration in microscope objectives.

Thus, it is advisable to use a suitable imaging medium (water) to reduce this phenomenon.

Cells are often called the “building blocks of life“.  This article may have just whet your curiosity about the invisibly small, and perhaps it will see you off on a personal voyage into microscopy.  Then, it’s a good thing.  The World needs more scientists.  Because there are many more building blocks to life than meets the eye, Horatio…