# Basics of Microscopy

## Magnification

Magnification is the process of enlarging the appearance of an object. We calculate the magnification of an object by indicating the fold change in size. So if something appears to be double the size of the real item, then it is obviously magnified 2X. Because there is a magnification by the eye-piece (ocular lens), as well as the objective lenses, our final magnification of an item is the product of those two lenses.

We can calculate that as:
Magnificationtotal = Magnificationobjective X Magnificationocular

## Field of View (FOV)

In a microscope, we ordinarily observe things within a circular space (or field) as defined by the lenses. We refer to this observable area as the field of view (FOV). Understanding the size of the FOV is important because actual sizes of object can be calculated using the Magnification of the lenses. What are the effects of magnification on FOV?

1) Lowest Magnification

2) Low Magnification

3) High Magnification

4) Highest Magnification

In image 1, we can see a model of DNA on a table with a water bottle and a large area of the room. Image 2 displays less of the room in the background but the DNA model is larger in appearance because the magnification is greater. In image 3, we no longer see evidence of a door and the DNA model is much larger than before. In image 4, we no longer see the table the model and water bottle rest upon. While the last image is largest, we see less of the surrounding objects. We have higher magnification at the cost of field of view. FOV is inversely related to the magnification level.

## Depth of field

We notice that when we observe 3 overlapping threads of different color under a microscope, we can focus on one thread at a time. Similarly, when we zoom in a great deal on the DNA model above, we notice that the print on the water bottle is not sharp.

Highest Magnification with shallow depth of field. Notice how the label on the water bottle is blurry while the lettering on the DNA model is sharp.

We know that the water bottle is behind the DNA molecule. Under the microscope, the threads of differing color are also stacked on top of each other. We recognize that they are in different planes because they are three dimensional. Each thread has depth and do not occupy the same exact space. If we focus on the print of the water bottle on the image above, we would no longer see the lettering on the DNA molecule sharply. We refer to this concept as Depth of Field (DOF). Under the microscope, at a low magnification, we can make out fewer finer details. However, most items appear on the same plane in this case and or comparably sharp. But as we increase the magnification and see finer details, the distances between the various planes in view become more apparent. We can see a similar phenomenon at low magnification of the DNA model. At the low magnification, we may not be able to read the print on the water bottle, but the bottle and DNA molecule are of a similar distance from our view that the small difference in apparent depth is not as noticeable. We can still draw on other visual cues to know that the bottle is behind the model, but the sharpness of both items are equivalent.

## Real biological examples

We can see the concepts of FOV and DOF in the following pictures.

In an even more extreme close-up (higher magnification), we would have difficulty focusing on both the eyes and beak since there is depth and distance between those features.

## How do we use microscopes

In our lab, we look at some pond water. What do we see? Why is this significant? How does the microscope help us study these items? What is the utility of the the concepts of magnification, FOV and DOF when we use microscopes to study biological samples?

# The Scientific Method

The Scientific Method is the process for systematically investigating phenomena to better understand occurrences. Scientific inquiry is based on observation. Observations are based on sensory evidence, measurements and definitions pertaining to the subject or phenomenon. We refer to this type of information acquisition as empirical. In science, we use experimentation to modify variables associated with a phenomenon to identify correlation and causation. In this case, empirical is practically equivalent to experimental.

## Not an Educated Guess

One of the great failures in scientific education is the reduction of the concept of a hypothesis to an educated guess. A hypothesis is actually a proposed explanation for a phenomenon based on previous observations, evidence or experiments. These proposed explanations are often formulated in such a way that one can test them to acquire further observations that may agree or disagree with the underlying assumptions of the hypothesis. While this suggests a role for educated, there isn’t any guessing involved. The Scientific Method continues on the ideas of observation and empiricism to formulate hypotheses that can be experimentally demonstrated. In short, we use inductive reasoning to combine isolated facts into a cohesive sum and for devising predictions about the phenomenon.

Beginning with observations and previous knowledge, we can formulate a characterization for certain phenomena and develop a testable idea for inquiry –the hypothesis. The crux of hypothesis testing lies in the ability to gather new evidence or observations while controlling for external variables that may obfuscate correlation and causation of the phenomenon. The systematic collection of new data comes from experimentation. As we gain new information about the phenomenon, we can further support or reject the original hypothesis. The testing of our hypothesis, therefore relies on deductive reasoning (cause/effect & if/then).

## Random Speculation? I have a theory about that!

In the vernacular, people often see something happening (any phenomenon) and will remark “I’ve got a theory about that.” When they say this, it means they have a loose speculation regarding association. Sometimes it’s completely baseless and will lack any rigor. In science, we use the word theory to indicate a joining of multiple, well-supported hypotheses. Here, the definition is more stringent as it pulls from multiple tested and refined hypotheses that result in the same observed phenomenon. When a theory is accepted by an overwhelming number of people in a field, we refer to it as a principle.

## Testing Speculation

Even speculation can be tested when posing the speculation as a testable idea or hypothesis.

In 1991, a young male hawk made a home in Central Park. Because of his physical appearance he was called Pale Male. He is of some notoriety because he was the first hawk to return to nest in Manhattan in about 100 years. His nest is built on a 5th Avenue building overlooking the Park.

Since the early 1990s, Pale Male and his numerous mates have generated about 25 offspring and at least 7 nesting pairs of hawks make their home in Manhattan.

Erick is a bird watcher and he says to the passers-by, “He’s the father of all the other hawks in Manhattan.”

Erick indicates the following pieces of evidence for this assumption:

1. No other hawks made their “homes” in Manhattan until after his arrival –temporal correlation
2. Many of the other hawks, like Pale Male, build nests on buildings instead of trees (implying his origin in their culture)
3. Some have physical attributes similar to Pale Male (sometimes coloration or behavior) —anthropomorphisms

While these observations and associations are parsimonious, there is a great degree of spuriousness in the assertions.

How do we think about this issue in a scientific way? Do we have enough evidence or observations to test, refute or accept Erick’s assertions? Let’s add a new piece of information to the list. While adults nest and stay close to their nest year-round, the offspring are forced out of the territory and have a large dispersal. Even with this new piece of information, we cannot reject or accept the original hypothesis, but we can state that the claim is less likely to be authentic. How do we use this new evidence to revise and refine our hypothesis? How can we possibly test the assertions more definitively? (Think what Jerry or Maury would do)

# Ecosystems and Populations in New York City

## Biology: The study of life

Organisms are any contiguous living things that are characterized by their ability to respond to environmental (external/internal) stimuli, grow and reproduce. They are composed of chemical elements and governed by the principles of physics and chemistry. Organisms display an inherent synergy where a greater organization of chemistry defines the underlying properties yet appear as a synthesis of more than an assemblage of individual parts. Life, in this context, maintains a complex order and counters entropy.

## How do organisms live?

Living things use energy to actively regulate their internal environments in response to internal, external or environmental stimuli. We call this regulation homeostasis. Active regulation can refer to energy being used to counter environmental changes (maintenance of body temperature in mammals and birds) or behavioral adaptations (a lizard basking in the sun to stay warm).

Energy is the physical capacity to do work. Biological systems are governed by a complex series of chemical reactions and energy is stored/released in the form of chemicals. Chemicals within biological systems cycle as they are transformed from one compound to another, but energy flows within the system. The set of chemical reactions that occur within organisms is referred to as metabolism. Metabolism consists of two types of reaction: building larger chemical compounds from simpler parts (anabolic) or reduction of chemicals to simpler forms (catabolic).

## Ecosystems

The air, land and water on Earth where organisms exist is referred to as the biosphere. The biosphere contains populations (members of a given species within a given area) and communities (collections of interacting populations). We call the physical environment where various populations interact within a community an ecosystem. Ecosystems are comprised of all the organisms, as well as the abiotic physical components of the environment. In short, the biosphere is the sum total of all ecosystems on the planet.

Ecosystems are characterized by chemical cycling and energy flow. Energy is used to do work and is lost through inefficient transfers. Chemicals are mostly locked into ecosystems and cycle from one form to another through various anabolic and catabolic processes. The biosphere receives a constant input of energy from the Sun. Organisms that are capable of producing complex molecules from simpler ones using the light energy (phototrophs) or energy from inorganic compounds (chemoautotrophs) are referred to as autotrophs (self feeders). Heterotrophs are organisms that cannot self feed and must ingest or absorb organic material to acquire energy and raw material.

## Looking outside

Nature and ecosystems can be found even in New York City! Follow the list items and video below.

• Look around and think about the various organisms
• Identify autotrophs and heterotrophs
• What are the energy sources and how is the energy flowing?
• How are chemicals cycling in this system?

## Humans and ecosystems

Humans abound in the city. Humans depend on healthy ecosystems for:

• food
• medicines
• raw materials

But Humans also have a large impact on ecosystems. Much of the wildlife we associate with NYC are foreign invasive species that were introduced by people. These invasive species change the dynamics of local ecosystems. Some examples include:

• rock doves (pigeons)
• house sparrows

Identify the invasive pest species in the video below:

Here, the invasive species is a food item. Ordinarily, we relate this species with disease. Why does this pest species flourish in a place like NYC? How do we counter control this pest species in a way that is not detrimental to other species within the ecosystem?

# Success at Learning Science

Science is a systematic organization of knowledge about the universe. Our method of science is built upon testing predictions and explanations of phenomena. The organization of this knowledge comes with a large vocabulary. To succeed at learning science, we first need to master the language behind it. What we hear in class must be reinforced by reading the text and associated assignments. Mastering a language comes from practice and reinforcement. We can’t wait until the day before the exam to learn the language since the assessments in science are often about concepts. Those who believe that science is about the memorization of facts will not succeed at learning science.

The material presented on Openlab is meant to provide real examples and multimedia for a better understanding of the concepts at hand. But to really excel, the underlying language or lexicon must be mastered. Take an active role in learning by reading ahead of classes. Utilize the concepts from lecture and reinforce them in practice in the lab component of the class. If the ideas are difficult to understand in my words or the book’s, look to other sources for examples. You will find that early entries in Openlab will contain multiple links to other sources. Learn to identify what you don’t understand and seek out ways to learn on your own.

## Why learn science?

Science and Biology is all around us. Laboratory science is a general education requirement. We learn science even if we have no interest in it because it is the basis for all that is around us. Most importantly, science changes the way we think. It challenges us to become more analytical and understand mechanisms.

In a New York Times exercise, Neil deGrasse Tyson provided an eloquent response to what he would do differently as President of the country that actually explains why we learn science.

When you’re scientifically literate, the world looks different. Science provides a particular way of questioning what you see and hear. When empowered by this state of mind, objective realities matter. These are the truths on which good governance should be based and which exist outside of particular belief systems.

Our government doesn’t work — not because we have dysfunctional politicians, but because we have dysfunctional voters. As a scientist and educator, my goal, wouldn’t be to lead a dysfunctional electorate, but to bring an objective reality to the electorate so it could choose the right leaders in the first place.

In short, we learn science to become conscientious members of society.