NANSLO REMOTE LAB ACTIVITY

 

SUBJECT SEMESTER: XXXXXXXXXXXXXXXXXXXXX

TYPE OF LAB:  XXXXXXXXXXXXXXXXXXXX

 

 

 

 

 

 

 

TITLE OF LAB:  CELL DISEASES

 

Diseased Cell NANSLO Lab Activity in Word Format last updated April 30, 2014.

 

Lab format: This lab is a remote lab activity: Microscopy.

 

Relationship to theory:  In this lab you will learn identify the underlying cellular changes that lead to several human diseases.

 

Instructions for Instructors: This protocol is written under an open source CC BY license. You may use the procedure as is or modify as necessary for your class.  Be sure to let your students know if they should complete optional exercises in this lab procedure as lab technicians will not know if you want your students to complete optional exercise.

Instructions for Students: Read the complete laboratory procedure before coming to lab.  Under the experimental sections, complete all pre-lab materials before logging on to the remote lab, complete data collection sections during your online period, and answer questions in analysis sections after your online period.  Your instructor will let you know if you are required to complete any optional exercises in this lab.

 

Remote Resources:  Primary - Microscope, Secondary – Diseased Cell slide set.

 

CONTENTS FOR CELL DISEASES LAB:

 

Learning Objectives

Background Information

Equipment

Experimental Procedure

Pre-Lab Exercise 1:  Skin Composite and Skin Cancer Slides Observations

Exercise 1:  Skin Composite and Skin Cancer Slides Observations

Pre-Lab Exercise 2:  Normal Red Blood Cells and Sickled Cells Slide Observations

Exercise 2:  Normal Red Blood Cells and Sickled Cells Slide Observations

Pre-Lab Exercise 3:  Normal and Cancerous Breast Tissue

Exercise 3:  Normal and Cancerous Breast Tissue

Pre-Lab Exercise 4:  Normal and Cancerous Spleen

Exercise 4:  Normal and Cancerous Spleen

IF CARDIAC IS USED IN ADDITION TO OR IN PLACE OF SPLEEN ADD

Pre-Lab Exercise 4A:  Cardiac Muscle and Myocardial Infarction

Exercise 4A:  Cardiac Muscle and Myocardial Infarction

Pre-Lab Exercise 5:  Cirrhosis of the Liver

Exercise 5:  Cirrhosis of the Liver

Summary Questions

Preparing for this NANSLO Lab Activity

 

LEARNING OBJECTIVES:

 

After completing this laboratory experiment, you should be able to do the following:

1. Identify the differences between normal skin tissue layers and skin cancer.
2. Compare the shape of normal red blood cells and sickled red blood cells. 
3. Identify the cellular differences between normal breast tissue and breast cancer. 
4. Identify the cellular differences between a normal spleen and a spleen suffering the effects of leukemia. 
   IF CARDIAC IS USED IN ADDITION TO OR IN PLACE OF SPLEEN ADD - Use the following objective
   Identify and describe the difference between healthy cardiac muscle and cardiac muscle suffering the effects of myocardial infarction.
5. Identify the cellular changes that occur in the liver due to cirrhosis.

 

BACKGROUND INFORMATION:

 

Cells inside organisms undergo a variety of changes as part of adaptations due to growth, stress, and aging.  During these adaptations, cells are trying to maintain homeostasis to prevent diseases. The human body can be affected by several stimuli such as:

 

• Physical agents (mechanical trauma, temperature variation).
• Chemical agents (radiotherapy, glucose, or lipid accumulation).
• Infectious microorganisms (toxins produced by bacteria or viruses interfering with normal cell metabolism).
• Hypoxia: lack of oxygen supply to cells (it could be detrimental for the brain).
• Genetic factors leading to abnormal cellular metabolism or malformation.
• Nutritional imbalances (more frequent in children).
• Hypersensitivities and allergic reactions

 

However, some of the changes could cause irreversible effects leading to pathological changes that could either be obvious or difficult to detect. Many of the common diseases that affect humans are caused by disruptions of the basic mechanisms of the cell. Medical professionals and scientists rely on histopathology, the microscopic study of tissue, to accurately diagnose diseases of the human body. During this laboratory activity, you will be viewing and comparing different types of cells and tissues to identify the differences between diseased and normal tissues at the cellular level.  We will be looking at cells from several different tissues in the body including the skin, internal organs, and the blood.  Each tissue type is described in detail below.

Skin

 

Our skin is the largest organ. It has several functions:  serving as a protective barrier; regulating our body temperature; producing sweat; and other functions. Although most diseases affecting the skin originate in the layers of the skin (Figure 1), such abnormalities are also important factors in the diagnosis of a variety of internal diseases. There is some truth in the belief that the skin mirrors a person’s internal health. Often, the visibility and accessibility of skin make it the first organ of the body to show detectable signs of underlying disease.

 

Figure 1:  Normal human skin tissue specimen

 

Normal human skin is composed of three layers: epidermis; dermis; and hypodermis.  The epidermis is the outermost layer.  The epidermis and dermis together make up the cutis.  The hypodermis makes up the subcutis and contains structures like the hair follicles and sweat glands.

 

Abnormalities of the skin frequently suggest metabolic, malignant, and glandular diseases. Skin cancer is the most common form of cancer in the United States. The two most common types of skin cancer—basal cell and squamous cell carcinomas—are highly curable. However, melanoma, the third most common skin cancer, is more dangerous. About 65%–90% of melanomas are caused by exposure to ultraviolet (UV) light  (http://phil.cdc.gov/phil/details.asp.)

 

UV light is not the only cause of skin cancer.  Kaposi sarcoma (Figure 2) is a malignant tumor of the lymphatic endothelium caused by the Human herpesvirus 8 (HHV8), i.e., Kaposi’s sarcoma-associated herpesvirus (KSHV) and arises from a cancer of the lymphatic endothelial lining. It is characterized by bluish-red cutaneous nodules. Kaposi’s sarcoma is thought of as an opportunistic infection affecting patients whose immune systems have been compromised, as in the case of patients with HIV/AIDS (http://phil.cdc.gov/phil/details.asp.)

 

Figure 2: Human skin biopsy specimen due to Kaposi's sarcoma

 

Above is a photomicrograph depicting the histopathologic changes seen in human skin biopsy specimen due to Kaposi’s sarcoma. Of importance is the appearance of the dermal layer which contains a cellular infiltrate and a proliferation of vascular elements.

 

Like other tissues, skin is afflicted by all types of pathological changes, including hereditary, inflammatory, benign and malignant (neoplastic), endocrine, hormonal, traumatic, and degenerative processes. Emotions affect the health of the skin as well. The reaction of the skin to certain diseases and disorders differs from that of other tissues in many ways. For example, extensive inflammation of the skin may affect metabolism within other organs and systems of the body, causing anemia, circulatory collapse, disorders of body temperature, and disturbance of water and electrolyte balance in the blood (http://phil.cdc.gov/phil/details.asp.)

 

Internal Organs

 

The internal organs of a body are complex structures consisting of a combination of parenchymal tissue and stromal tissue. The cells of the parenchyma are specialized to carry out the function of the organ, while the stromal cells are involved in support (e.g. connective tissue and blood vessels).

For example, the microscopic anatomy of the liver reveals a uniform structure of clusters of cells called lobules where the vital functions of the liver are carried out. Each lobule, measuring about one millimeter in diameter, consists of numerous cords of rectangular liver cells,or hepatocytes, that radiate from central veins, or terminal hepatic venules, toward a thin layer of connective tissue that separates the lobule from other neighboring lobules.

Hepatocytes occupy about 80 percent of the volume of the liver, and their cytoplasm (the area surrounding the nucleus) contains many mitochondria which provide the energy needed for the many synthetic and metabolic functions of the liver cell. The cytoplasm also contains a series of long tubules called the endoplasmic reticulum which provides many enzymes essential to liver function. Some of the membranes of the endoplasmic reticulum appear granular, or rough, owing to the presence of ribosomes which are responsible for forming specific polypeptide (protein) chains after having had the amino group removed (deamination) and having been converted into glucose through a process called gluconeogenesis. The ammonia released from gluconeogenesis is converted to urea in the hepatocyte by way of the urea cycle. The nonribosomal, or smooth, endoplasmic reticulum is where cytochromes (combinations of heme from hemoglobin with various proteins), and certain enzymes undertake the important hepatic functions of drug and hormonal metabolism and also cholesterol synthesis. Hepatocytes also conjugate with carbohydrate components of bilirubin and other fat-soluble metabolic and foreign compounds and thereby are made soluble in water. Bilirubin is the product of hemoglobin metabolism that is formed in the bone marrow and the lymphatic tissue and is carried to the liver after becoming bound to plasma albumin. It is released at the hepatocytic sinusoidal membrane and is transported to the smooth endoplasmic reticulum where it is conjugated with one or two molecules of glucuronic acid and thereby becomes soluble in water and excretable in bile. The Golgi apparatus, a series of tubular structures between the endoplasmic reticulum and the canaliculus, acts as a transport station for newly made proteins and other hepatocytic products before they are conveyed to other parts of the cell or out of the cell entirely. Lysosomes, another important cytoplasmic constituent, are responsible for the intracellular storage of pigments, such as iron or copper, and for the digestion of certain contents, such as glycogen or foreign particles. The nucleus of the hepatocyte guides replication of the cell and transmits genetic material in the form of messenger ribonucleic acid (mRNA) from deoxyribonucleic acid (DNA) to organelles located in the cytoplasm (http://phil.cdc.gov/phil/details.asp.).

The parenchymal cells of certain organs, such as the liver, the heart, and the kidneys, are responsible for the metabolism and elimination of excess fat.  If the fat droplets are not properly metabolized, they end up accumulating in the endoplasm reticulum and the Golgi apparatus of the cells (Figure 3). This accumulation could be irreversible, leading to more serious health problems, and may even lead to death.

 

Figure 3: Alcoholic Cirrhosis Liver

 

Above is an image of a liver with alcoholic cirrhosis. The yellow color indicates the accumulation of fat inside the hepatocytes interfering with normal activity of these cells due to excessive alcohol consumption.

 

Reference:

Encyclopedia Britannica

http://www.britannica.com/EBchecked/topic/1081754/human-digestive-system/242929/Microscopic-anatomy#ref212928

 

Blood and the Circulatory System

 

The body’s circulatory system is composed of several parts:  the blood vessels; the blood; heart; and the lungs. The main function of the circulatory system is to transport food and oxygen to the body’s cells and remove waste products and CO₂.  The structure of the blood vessels, both large and small, is closely associated with their function (Figure 4). All nutrient materials and waste products exchanged between the organs and the blood must traverse perivascular spaces in the walls of the vessels that are occupied by connective tissue. One of the important functions of the connective-tissue cells is to maintain conditions in the extracellular spaces that favor this exchange (http://www.britannica.com/EBchecked/topic/36874/artery#md-media-strip-tab-image-content.)

 

Figure 4:  Artery: transverse section of an artery

 

Blood vessels are composed of multiple layers of tissue.  Each of these layers provides specific functions to the vessel.  Connective tissue makes up the outer surface of the vessels and controls transport into and out of the vessel.  The elastic membrane and elastic fibre give the vessel its flexibility while the involuntary muscles help to maintain blood pressure and the endothelium forms the inner surface of the vessel.

 

Any variation in the diameter of the lumen of the blood vessels will have an impact on the blood flow, nutrients supply, blood pressure, and the overall function of the cardiovascular system.

 

The blood vessels provide the pathway through which the blood circulates. The blood is composed four different types of cells: red blood cells (erythrocytes); white blood cells (leukocytes); and platelets (fragment of giant cells also known as thrombocytes).  Each one of these cells is responsible for specific functions inside our organs. Any variation of the number, shape, or size of these cells will have an impact on our health. The function of these cells is:

 

• Red blood (erythrocytes) cells function in the transport of oxygen and carbon dioxide to and from the cells.
• White blood (leukocytes) cells are involved in the immunity.
• Platelets are involved in hemostasis (stopping the bleeding).

 

ADD IF CARDIAC OBJECTIVE IS USED

 

Cardiac Muscle

 

The heart is the pump that keeps blood circulating throughout the body and thereby transports nutrients, breakdown products, antibodies, hormones, and gases to and from the tissues. The heart consists mostly of muscle.  The myocardial cells (collectively termed the myocardium) are arranged in ways that set it apart from other types of muscle. The outstanding characteristics of the action of the heart are its contractility, which is the basis for its pumping action, and the rhythmicity of the contraction.

 

Heart muscle differs from its counterpart, skeletal muscle, in that it exhibits rhythmic contractions. The amount of blood pumped by the heart per minute (the cardiac output) varies to meet the metabolic needs of the peripheral tissues (muscle, kidney, brain, skin, liver, heart, and gastrointestinal tract.)  The cardiac output is determined by the contractile force developed by the muscle cells of the heart (myocytes) as well as by the frequency at which they are activated (rhythmicity.)  The factors affecting the frequency and force of heart muscle contraction are critical in determining the normal pumping performance of the heart, and its response to changes in demand.

 

The heart is a network of highly branched cardiac cells 110 μm in length and 15 μm in width which are connected end to end by intercalated disks. The cells are organized into layers of myocardial tissue that are wrapped around the chambers of the heart. The contraction of the individual heart cells produces force and shortening in these bands of muscle with a resultant decrease in the heart chamber size and the consequent ejection of the blood into the pulmonary and systemic vessels.

Your heart muscle needs oxygen to survive. A heart attack occurs when the blood flow that brings oxygen to the heart muscle is severely reduced or cut off completely.   View an animation of blood flow. This happens because coronary arteries that supply the heart muscle with blood flow can slowly become narrow from a buildup of fat, cholesterol, and other substances that together are called plaque. This slow process is known as atherosclerosis.  In a heart artery, when plaque breaks, a blood clot forms around the plaque. This blood clot can block the blood flow through the heart muscle. When the heart muscle is starved for oxygen and nutrients, it is called ischemia. When damage or death of part of the heart muscle occurs as a result of ischemia, it is called a heart attack or myocardial infarction (MI). About every 34 seconds, someone in the United States has a myocardial infarction (heart attack).

 

END OF TEXT IF CARDIAC OBJECTIVE IS USED

 

Reference:

Encyclopedia Britannica

http://www.britannica.com/EBchecked/topic/132995/connective-tissue 

 

EQUIPMENT:

 

• Paper
• Pencil/pen
• Slides
• Skin Cancer, Section
• Adult Skin, Unpigmented, ection
• Human Sickle Cell Anemia, Smear
• Human Blood, Smear
• MISSING SLIDES USED FOR SPLEEN
• Mycardial Infarction, Section
• Cardiac Muscle, Section
• Breast Carcinoma, Section
• Mammary Gland, Inactive, Section
• Cirrhosis, Section
• Liver, Section
• Computer with Internet access (for the remote laboratory and for data analysis)

 

EXPERIMENTAL PROCEDURE:

 

Once you have logged on to the remote lab system, you will perform the following laboratory procedures.  See Preparing for the Microscope NANSLO Lab Activity below.

PRE-LAB EXERCISE 1: Skin Composite and Skin Cancer Slides Observations

 

In this exercise, you will use the microscope to distinguish between the three layers of human skin.  In this exercise you will identify the skin layers on images you have taken, and you will measure and compare the relative size and shape of cancerous skin cells with healthy skin cells.

 

Pre-Lab Questions:

 

1. Using what you know about cancer, do you think the size and shape of the cancerous skin cells will be bigger, smaller, or the same as the ones of the healthy skin?
   
   
2. Rewrite your answer to question one in the form of an If … Then … hypotheses.

 

EXERCISE 1:  Skin Composite and Skin Cancer Slides Observations

 

Data Collection:

 

3. Select the composite skin slide (Slide Cassette 4:  TBA)  from the slide loader. Using the 10X objective, identify the skin sample and bring it in to focus.
   
   
4. Carefully work your way through all the objectives focusing with each one until you reach the 60X objective and capture an image.  Insert your image of normal skin in the space below.
   
   
5. Select the skin cancer slide (Slide Cassette 4:  TBA) from the slide loader. Using the 10X objective, identify the skin sample and bring it in to focus.
   
   
6. Carefully work your way through all the objectives focusing with each one until you reach the 60X objective and capture an image.  Insert your image of skin cancer in the space below.

 

Analysis:

 

7. Using the image you took in question 4 of this exercise, label the different layers of the skin and the 3 different parts of a single cell (nucleus, cytoplasm and cell membrane) from either the epidermis or the dermis. Insert your labeled image below.
   
   
8. Using the image you took in question 6 of this exercise, label the different layers of the skin and the 3 different parts of a single cell (nucleus, cytoplasm and cell membrane) from either the epidermis or the dermis. Insert your labeled image below.  Describe the shape of the cell and its nucleus.
   
   
9. Based on your observation, describe the difference in the shape and number of cell layers between normal skin and skin cancer.
   
   
10. Next we are going to measure the size of normal and cancerous cells.  To determine the size of the cells, we are going to use the ratio method.  In order to do this, you will need one piece of information which is the width of your field of view.  On our microscopes, the field of view at 60X is 205µm.
    
    
11. Now, if we use the image in figure 5, we can see that the total width of the field of view is 13.6 cm or 136 mm (Image A).  The cell (Gray) is 3.7 cm or 37mm (Image B).
    
    
                                                            Figure 5:  Measurements
    
    
12. Now, if we divide 37mm/136mm = 0.272, which we multiply by the total length of the field of view.  So 0.272 * 205µm = 55.77 µm rounded for significant figures, we get a cell size of 56µm.
    
    
13. Measure the difference in the size of the healthy skin and compare that to the size of the cancerous skin.
    
    
14. Are your results in correlation with what you have predicted earlier?
    
    
15. Rewrite your hypothesis in light of the new information you collected in this exercise.
    
     

PRE-LAB EXERCISE 2: Normal Red Blood Cells and Sickeled Cells Slide Observations

 

The function of red blood cells is to transport oxygen to cells and carbon dioxide away from them. The shape and size of the red blood cell is important for its function.  In this lab exercise, you will measure and compare the relative size and shape of normal red blood cells with the sickled celled ones.

 

Pre-lab Questions:

 

1. Do you predict the size of normal red blood cells to be smaller, bigger, or the same as the sickled celled one?
   
   
2. Rewrite your answer to question one in the form of an If … Then … hypotheses.

 

EXERCISE 2:  Normal Red Blood Cells and Sickeled Cells Slide Observations

 

Data Collection:

 

3. Select the human blood slide (Slide Cassette 4: TBA) from the slide loader. Using the 10X objective, identify the blood cells and bring them into focus.
   
   
4. Carefully work your way through all the objectives focusing with each one until you reach the 60X objective and capture an image.  Insert your image of normal red blood cells below.
   
   
5. Select the sickle cell anemia slide (Slide Cassette 4:  TBA) from the slide loader. Using the 10X objective, identify the blood cells and bring them in to focus.
   
   
6. Carefully work your way through all the objectives focusing with each one until you reach the 60X objective and capture an image.  Insert your image of sickled cells below.

 

Analysis:

 

7. Using the method from exercise 1, determine the length of the normal and sickle red blood cells.
8. Based on your observation, describe the difference in the shape of normal red blood cells versus sickled cells.
9. Are your results in correlation with what you have predicted earlier?
10. Rewrite your hypothesis to take into account the new information you have learned in this exercise.
11. What is the impact of sickle cell anemia on oxygen transport?

 

PRE-LAB EXERCISE 3: Normal and Cancerous Breast Tissue

 

As we mentioned in experiment 1, there are several forms of cancer. The most common form of cancer in women is breast cancer. Rather than being a cancer of the epithelial tissue, breast cancer is a cancer of the mesenchymal tissue. In this exercise, you will compare and identify the differences between normal and cancerous breast tissue.

Pre-Lab Questions:

 

1.  Do you think the cancerous breast cells will be bigger, smaller, or the same as the healthy breast cells?
2. Rewrite your answer to question one in the form of an If … Then … hypotheses.

 

EXERCISE 3: Normal and Cancerous Breast Tissue

 

Data Collection:

 

3. Select the mammary gland slide from the slide loader. Using the 10X objective, identify the cells and bring them into focus.
4. Carefully work your way through all the objectives focusing with each one until you reach the 60X objective and capture an image. Insert your image of normal breast tissue below.
5. Select the breast carcinoma slide from the slide loader. Using the 10X objective, identify the carcinoma cells and bring them in to focus.
6. Carefully work your way through all the objectives focusing with each one until you reach the 60X objective and capture an image. Insert your image of the breast carcinoma below.

Analysis:

 

7. Using the method form exercise 1, measure and compare the size of the cells from the mammary gland with breast carcinoma cells.
8. Are your results in correlation with what you have predicted earlier?
9. Rewrite your hypothesis to take into to account the new information you learned in this exercise.

 

PRE-LAB EXERCISE 4:  Normal and Cancerous Spleen

 

Leukemia is often thought of as a cancer of the blood which it is.  However, leukemia is more accurately described as cancer of the white blood cells.  The white blood cells play a large role in the immune response, which also involves many other tissues and organs.  In this exercise, we are going to look at the effect of leukemia on the spleen.

 

Pre-lab Questions:

 

1. What differences do you expect to see in the tissue samples taken from a normal spleen versus a tissue sample taken from a leukemia infected spleen?
   
   
2. Write a hypothesis in the IF … THEN … format that predicts what effect you expect to see with respect to fat and lumen size in an Atherosclerosis blood vessel.

 

EXERCISE 4:  Normal and Cancerous Spleen

 

Data Collection:

 

3. Select the spleen slide from the slide loader. Using the 10X objective, identify the cells and bring them into focus.
   
   
4. Carefully work your way through all the objectives, focusing with each one until you reach the 60X objective and capture an image. Insert your image of the normal spleen below.
   
   
5. Select the leukemia spleen slide from the slide loader. Using the 10X objective, identify the cells and bring them into focus.
   
   
6. Carefully work your way through all the objectives focusing with each one until you reach the 60X objective and capture an image. Insert your image of the leukemia spleen below

 

Analysis:

 

7. Using the method from exercise 1, measure and compare the size of the cells in the normal spleen versus the cells from the leukemia spleen.
   
   
8. What is the difference in the frequency of white blood cells in the normal spleen versus the leukemia spleen?
   
   
9. Are your results in correction with what you have predicted earlier?
   
   
10. Rewrite your hypothesis to take into account the new information you learned in this exercise.

 

ADD IF CARDIAC IS USED IN ADDITION TO OR AS A REPLACEMENT FOR SPLEEN

 

PRE-LAB EXERCISE 4A: Cardiac Muscle and Myocardial Infarction

 

One of the leading causes of death in the developed world is heart disease.  A specific type of heart disease is a Myocardial Infarction, commonly referred to as a heart attack, which is caused by the loss of oxygen to part of the heart.  It is often common for a heart attack to lead to the death of a small amount of cardiac muscle which will be replaced by scare tissue.

 

Pre-Lab Questions:

 

1. What do you predict the differences between the normal cardiac muscle and the cardiac muscle suffering from myocardial infarction will be?
2. Rewrite your answer to question #1 in the form of an If ... Then ... hypotheses.

 

EXERCISE 4A:  Cardiac Muscle and Myocardial Infarction

 

Data Collection:

 

3. Select the cardiac muscle slide (Slide Cassette 4:  TBA) from the slide loader.  Using the 10X objective, identify the cells and bring them into focus.
4. Carefully work your way through all the objectives focusing with each one until you reach the 60X objective and capture an image.  Insert your image of cardiac muscle below.
5. Select the myocardial infarction slide (Slide Cassette 4: TBA) from the slide loader.  Using the 10X objective, identify the cells and bring them into focus.
6. Carefully work your way through all objectives focusing with each one until you reach the 60X objective and capture an image.  Insert your image of the myocardial infarction muscle below.

 

Analysis:  

 

7. Based on your observations, describe the difference you see between the two cardiac muscles in the samples.
8. Are your results in correlation with what you have predicted earlier?
9. Rewrite your hypothesis to take into account the new information you learned in this exercise.

 

END IF CARDIAC IS USED IN ADDITION TO OR AS A REPLACEMENT FOR SPLEEN

 

PRE-LAB EXERCISE 5: Cirrhosis of the Liver

 

At a first pass, the liver seems to be a relatively simple organ.  Its appearance is basically uniform and only interrupted by blood vessels.  However, what the liver may lack in appearance it makes up for in metabolic complexity.  As we mentioned in the introduction, the liver is responsible for the metabolism (removal) of excess fat.  When this process is disrupted, fat will accumulate in the liver cells and lead to problems and eventually disease. One thing that will lead to a disruption of fat metabolism is excessive drinking (cirrhosis of the liver).  In this lab, you will examine normal and diseased liver cells.

 

Pre-lab Questions:

 

1. What defects do you think you will see in the cells from the cirrhosis liver?
2. Write a hypothesis in the IF … THEN … format that predicts what effect you expect to see with respect to a cirrhosis liver.

 

EXERCISE 5: Cirrhosis of the Liver

 

Data Collection:

 

3. Select the liver slide (Slide Cassette 4:  TBA) from the slide loader. Using the 10X objective, identify the cells and bring them into focus.
4. Carefully work your way through all objectives focusing with each one until you reach the 60X objective and capture an image. Insert your image of the liver tissue below.
5. Select the cirrhosis slide (Slide Cassette 4:  TBA) from the slide loader.  Using the 10X objective, identify the cells and bring them into focus.
6. Carefully work your way through all objectives focusing with each one until you reach the 60X objective and capture an image. Insert your image of the cirrhotic liver tissue below.

 

Analysis:

 

7. Based on your observation, describe the difference you see between the two liver samples.
8. Are your results in correlation with what you have predicted earlier?
9. Rewrite your hypothesis to take into account the new information you learned in this exercise.

 

SUMMARY QUESTIONS:

 

1. Explain the difference between etiology and pathogenesis.
2. Provide 2 examples of etiologic factors that may cause diseases in human.
3. Discuss the purpose of cellular adaptation in our body.
4. Provide an example of cellular adaptation and its cause(s).
5. Discuss the difference between reversible and irreversible cell injury and provide an example for each.
6. What is the definition of anemia? List three major causes of anemia.
7. Explain the rationale for each of the following manifestations of anemia: pale skin, increased heart rate, dizziness and fainting.
8. How does blood loss lead to anemia? Does all blood loss lead to anemia?
9. What is jaundice and why does it occur in some forms of anemia and not others?
10. Why do red blood cells need iron?
11. Define leukemia?
12. What is the difference between acute and chronic leukemia? Discuss the predominant cell type and the onset / course of the disease for each type of leukemia.
13. Discuss the role of vitamin K in blood coagulation? 
14. What effect does liver disease have on coagulation?
15. Provide an example of a specific type of intracellular accumulation and its impact on the cell’s metabolism and the overall health.
16. Discuss the role of diet in the development of atherosclerosis and its impact on the overall function of the cardiovascular system.
17. IF CARDIAC IS USED:  Describe the changes that occur in cardiac muscle that has suffered myocardial infarction.  How is the damaged tissue different than healthy cardiac tissue?

 

PREPARING FOR THE MICROSCOPE NANSLO LAB ACTIVITY

 

Read and understand the information below before you proceed with the lab!

  

Scheduling an Appointment Using the NANSLO Scheduling System

 

Your instructor has reserved a block of time through the NANSLO Scheduling System for you to complete this activity. For more information on how to set up a time to access this NANSLO lab activity, see www.wiche.edu/nanslo/scheduling-software.  

 

Students Accessing a NANSLO Lab Activity for the First Time

 

You must install software on your computer before accessing a NANSLO lab activity for the first time. Use this link to access instructions on how to install this software based on the NANSLO lab listed below that you will use to access your lab activity – www.wiche.edu/nanslo/lab-tutorials.

 

1. NANSLO Colorado Node -- all Colorado colleges.
2. NANSLO Montana Node -- Great Falls College Montana State University, Flathead Valley Community College, Lake Area Technical Institute, and Laramie County Community College.
3. NANSLO British Columbia Node -- Kodiak College.

 

Using the Microscope for a NANSLO Remote Web-based Science Lab Activity

 

We've provided you with three ways to learn how to use the microscope for this NANSLO lab activity:

 

1. Read these instructions.
2. Watch this short video. 
3. Print off these instructions to read (PDF version of the instructions.)

 

NOTE:   The conference number in this video tutorial is an example.  See “Communicating with Your Lab Partners” below to determine the toll free number and pin to use for your NANSLO lab activity.

 

MICROSCOPE RWSL LAB INTERFACE INSTRUCTIONS

 

The Remote Web-based Science Lab (RWSL) microscope is a high quality digital microscope located at the NANSLO Node.  Using a web interface as shown below, you can control every function of the microscope just as if you were sitting in front of it. 

 

The equipment control software shown below is written using the LabVIEW software from National Instruments. The user interface is presented as a LabVIEW control panel which will be referred to as the lab interface for the remainder of the document.

Figure 1:  Remote Web-based Science Lab (RWSL) Microscope Lab Interface

 

COMMUNICATING WITH YOUR LAB PARTNERS

 

As soon as you have accessed this lab interface, call into the toll free conference number shown on the control panel to communicate with your lab partners and with the Lab Technicians.  Use the PIN code noted to join your lab partners.  Only one person can be in control of the equipment at any one time so talking together on a conference line helps to coordinate control of the equipment and creates a more collaborative environment for you and your lab partners.

 

GAINING CONTROL OF THE MICROSCOPE

 

Right click anywhere in the grey area of the lab interface and choose “Request Control of VI” from the dialogue box that appears when multiple students are using the microscope at the same time,.  After you request control, you may have to wait a short time before you actually receive control and are able to use the features on this lab interface.

 

 

Figure 2:  Selecting "Request Control of VI"

 

RELEASING CONTROL OF THE MICROSCOPE

 

To release control of the microscope so that another student can use it, right click anywhere in the grey area of the lab interface and choose "Release Control of VI" from the dialogue box that appears. 

 

 

Figure 3:  Selecting "Release Control of VI" 

 

MICROSCOPE CONTROLS

 

 The Stage Controls allow you to adjust the visual of the specimen that has been placed on the stage of the microscope, select lenses with various magnifications, and select whether or not the condenser lens is in the light beam.  Below are more specific instructions on using these controls.  When using the arrows on this lab interface, click and hold the arrow until the desired effect is achieved or click and wait to view the result before clicking again. Quick clicks on the arrows may cause the system to lock up.

 

Figure 4:  Microscope Controls - Stage, Objective & Condenser

 

Stage Controls: Using the left and right and up and down arrows found to the right of the microscope image in the Stage Control area, moves the microscope stage which holds the specimen.  These arrows allow you to precisely control the position of the specimen on the stage. 

 

1. Use the "Right" and "Left" arrows to move the Stage so that you can view the specimen from left to right.
2. Use the "Backward" and "Forward" arrows to move the Stage so that you can view the top, middle or bottom of the specimen.
3. Use the "Up" and "Down" arrows to move the stage closer or farther away from the objective lens to bring a specimen into focus.   BE CAREFUL!  Don't move the stage too close to the lens.

 

When selecting the button between the "Up" and "Down" arrows, you can toggle between “Coarse” and “Fine” focus.  When the button is dark green and “Coarse/Fine” is displayed to the right of the button, the microscope is in “Coarse” focus.  When the button is bright green and “Fine” is displayed, the microscope is in “Fine” focus.   Typically, you will start with coarse focus which moves the stage in large increments and then use fine focus to complete your final focusing as it moves the stage in smaller increments.  There is no difference between the course and fine focus when using the 60X objective

 

NOTE:  When you click on these arrows, the specimen appears to move in the opposite direction.  Since the objective stays fixed, the image moves in the opposite direction of the stage.  This is how these controls work on most microscopes so the "feel" of the microscope is preserved over the web.

 

 

Figure 5:  Right/Left & Backward/Forward Stage Controls

Figure 6:  Up/Down Stage Controls &       Coarse/Fine Focus Control

                        

Objective:  A microscope mounts an objective lens very close to the object to be viewed.  Depending on need, different lenses with different power will be used on the microscope. This microscope feature multiple objectives, each with different power, mounted on a rotating turret.  The larger the magnification numbers the greater the magnification.  For example, if a specimen is viewed through a 40X objective lens, the magnifier in that lens displays the specimen 40 times larger than an equivalent view as seen by the unaided eye.  Remember that the ocular or other lenses also add to the magnification.

 

This microscope has five lenses – 4X, 10X, 20X, 40X, and 60X.  Use the arrows below the objective lens box that indicates the magnification of the current objective lens to move to a higher or lower magnification lens.  If you have activated the “Picture-in-Picture” Preset 2 (see below) you will be able to see the objective lens move when you select a new magnification.

 

Condenser:  The condenser controls whether or not the condenser lens is in the light beam. You want to have the condenser OUT for the 4x objective but IN for all the others.

 

SELECTING A CASSETTE AND LOADING SLIDES ONTO THE STAGE

 

There are two tabs on the lab interface.  When you first access the lab interface, the "Microscope" tab is displayed by default.  Click on the Slide Loader tab at the top of the screen to access the controls for the Slide Loader robot.  There can be up to four cassettes available on the Slide Loader.  These cassettes are used to store slides, and each can hold up to 50 slides.  The cassettes available to you are dependent on the lab activity to be completed.  Once a cassette has been selected, you will use the drop-down list to select your slides.

 

Figure 7:  Select the Slide Loader Tab to select a cassette and slides.

 

 

EXAMPLE OF HOW TO LOAD SLIDES

 

In this example, we have selected Cassette #1. Using the drop-down menu, we have selected "1:  Colored Threads Whole Mount."  Then, we selected the "Load" button.  A message indicates that the slide is loading.  Using the picture-in-picture camera, you can watch this happening.  The robotics selects the slide and places it on the microscope stage.

 

Figure 8:  Selecting the slide
"1: Colored Threads Whole Mount" from Cassette #1

 

Notice that when a slide is actually on the microscope (or when it is being loaded or unloaded), the cassette controls are greyed out so you cannot load a second slide until the first is removed.  Once the slide is on the microscope stage, it will be listed in the "Current Slide on Stage" box.  The only thing that the Slide Loader robot can do is return it to the cassette when the "Return Slide to Cassette" button is selected. 

 

Figure 9:  "LOADING SLIDE ... PLEASE WAIT" is displayed
in the "Current Slide on Stage" window

 

Select the "Microscope" tab to perform the NANSLO lab activity.  Once you are finished with the slide, select the "Slide Loader" tab and select "Return Slide to Cassette" button.  Once the slide is returned to the cassette, the Slide Loader controls are again available to select another slide from the cassette.

 

ENHANCING THE MICROSCOPE IMAGE

 

The digital camera mounted on the microscope has a camera control unit that is equipped with a series of image processing functions that enable you to quickly and easily correct imaging problems that arise from low or high contrast, poor focus, insufficient or uneven illumination, sample shading or discoloration and noise.  The most common reason for uneven elimination is a light source that does not completely fill the field of view on lower magnifications.  The White Balance should be used only if the image appears to be brown or gray, and you think you might need to adjust it (although it won't hurt anything to click this button).

A choice of color modes can be selected in the Microscope Image area and are used to display the image in different color palettes in order to highlight certain features.  The default setting is "Normal."

 

 

Figure 10:  Microscope Image Special Effects and Other Image Controls for Camera

 

Here is a description of each option:

1. In the “Normal” mode, the sample is displayed in its true  colors.
2. In the “Negative” mode, the sample is displayed in a color-inverted form, where red, green, and blue values are converted into their complementary colors. The technique is useful in situations when color inversion can be of benefit in exposing subtle details or in quantitative analysis of samples.
3. In the “Blue Black” mode, the black portions of a grayscale negative sample are displayed in blue. This mode is often useful to reveal details in samples having a high degree of contrast.  The “Blue Black” filter can aid you in examining a wide spectrum of difficult samples.
4. In the “Black & White” mode, a grayscale image of the sample is displayed.
5. In the “Sepia” mode, a brown scale (black and white) image of the sample is displayed. Although typically this filter is of little utility, it can be employed to alter image color characteristics to improve the visualization of sample detail.
6. At times, the sample may have an unacceptable color quality.  Use “White Balance” calibration to remove the color cast.  This process is often referred to as white balancing.
7. Auto Exposure is on automatically.  You do not need to do anything with Auto Exposure unless you are adjusting the luminance.  If you are doing so, you should turn off Auto Exposure by clicking on the button.  The green light is now off.  Now adjust the luminance.  See explanation below.

 

Reference:  http://www.microscopyu.com/articles/digitalimaging/dn100/correctingimages.html

 

Auto Exposure is normally turned on, but you can turn it off if you want to play around with the brightness of the light source and not have the microscope camera automatically adjust it.  It is usually best, though, to leave it turned on. 

 

When you turn off the Auto Exposure, the button turns dark green.  Some new controls appear that let you turn the LED off or on, and also adjust the intensity of the light source.  The intensity of the light source can be increased or decreased manually with the dial that now appears next to the Objective control when Auto Exposure is turned off.

 

Figure 11:  Additional controls available when Auto Exposure is turned off

 

 

CAPTURING AND SAVING A MICROSCOPE IMAGE

 

When the “Capture Image” button is pressed, a high-resolution image of what is currently in the field of view of the objective is captured.  While the image is being captured, the button will be illuminated bright green.  The capture is complete when the light turns off.  Be patient as this may take several seconds to complete.

 After the Capture Image light turns off, select the “View Captured Image” tab on the bottom of this control panel to view the image.

 

Figure 12:  Click the capture image button (#1), wait till the green light goes off,
and then select the View Captured Image tab (#2)

 

After opening this image through the View Captured Image tab, you will need to take a snapshot of it and save it to your computer.  There are several ways to do this, depending on your operating system.

WINDOWS:

1.  Pressing the two keys ALT and Print Screen simultaneously will copy the active window into your computer clipboard.  Then you can past it into a document.
2. Windows 7 and above has a Snipping Tool program under Programs/Accessories which can capture selected areas of the screen. 
3. Right click on it and select "Copy" from the menu presented.  After right clicking and selecting Copy, just open a document and right click and select Paste.  You can either paste it directly into your lab report document or into another one for safe keeping until you use it later.  You can use drawing tools in your word processing editor to annotate this image so you can show your instructor that you know what you were suppose to be looking for!

 

Figure 13:  Right click and select Copy to paste the image into a document

 

MAC:

1. Press these three keys simultaneously – .  This will change your cursor icon into a little cross. 
2. Now press the spacebar, and the icon becomes a camera.  Click in the image window you want to take a snapshot of, and it will save the image to a file on your desktop.

 

There are lots of free screenshot utilities you can also use to capture this image.

 

If you are familiar with saving a document to your computer, you also can select “Save Image As” from the pop-up menu,  give the image a name and then select a location on your computer where you want this image to be saved for future use. 

 

MICROSCOPE IMAGE VIEW WINDOW

 

The Image View Window displays the real-time video feed from the digital camera “looking through” the microscope. 

 

Figure 14:  Image View Window

 

PICTURE-IN-PICTURE CONTROLS - CAMERA PRESET POSITIONS AND PAN-TILT-ZOOM CONTROLS

 

When you click on the "Picture-in-Picture" button, it turns bright green.  A second real-time video feed from another digital camera appears in the Image View Window.  The controls shown in Figure 15 are all operational when the Picture-in-Picture feature is selected. 

 

Figure 15:  Picture-in-Picture Image Controls

 

CAMERA PRESETS

 

There are six camera preset positions.

 

Figure 16:  Picture-in-picture Camera Preset 1 and 6 - Displays the microscope, microscope camera,
and a camera control unit projecting the sample on the Stage. 

 

 

 

 

 

 

 

 

 

 

Figure 17:  Picture-in-picture Camera Preset 2:  Displays a closeup of the objective lens.

 

 

 

 

 

 

 

 

 

 

 

Figure 18:  Picture-in-picture Camera Preset 3 - Displays a closeup of the camera control unit
projecting the sample on the Stage.

 

 

 

 

 

 

 

 

 

 

 

Figure 19:  Picture-in-picture Camera Preset 4 - Displays the microscope eye piece and
the camera mounted to the microscope.

 

 

 

 

 

 

 

 

 

 

Figure 20:  Picture-in-picture Camera Preset 5 - Displays the Condenser Lens underneath the Stage that focuses the light on the sample.  The Condenser Lens controls the width of the beam.  In some instances you will want a tighter beam while in other cases you will want a broader beam to control the image quality.  This setting has been optimized for you.

 

 

 

 

 

 

 

 

PAN, TILT, ZOOM CONTROLS FOR PICTURE-IN-PICTURE

 

For each camera preset view, additional camera options are available. 

1. Use the up and down arrows to tilt the camera up or down. 
2. Use the right and left arrows to pan right or left.
3. Use the left "Zoom OUT" arrow and right "Zoom IN" arrow to zoom out and in.

Figure 21:  Picture-in-picture Camera - Example of "Zoom In" capability

 

 

For more information about NANSLO, visit www.wiche.edu/nanslo.

 

 

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Creative Commons Attribution 3.0 United States License 3  

 

 

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