REMOTE LAB ACTIVITY

SUBJECT SEMESTER: ________________

TITLE OF LAB: Histology - Connective Tissue

Lab format: This lab is a remote lab activity.

Relationship to theory (if appropriate): In this lab you will learn the underlying principles behind the histological study of tissues.

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 exercises.

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 – Histology slide set.

CONTENTS FOR THIS NANSLO LAB ACTIVITY:

Learning Objectives................................................................................................. 2Background Information ........................................................................................ 2 - 5Equipment .............................................................................................................. 5Experimental Procedure ......................................................................................... 5Pre-lab Exercise 1: Areolar Connective Tissue ....................................................... 5Exercise 1: Areolar Connective Tissue.................................................................... 5 - 6Pre-lab Exercise 2: Andispose Connective Tissue .................................................. 6Exercise 2: Andispose Connective Tissue ............................................................... 6Pre-lab Exercise 3: Reticular Connective Tissue .................................................... 7Exercise 3: Reticular Connective Tissue ................................................................ 7Pre-lab Exercise 4: Dense Connective Tissue ......................................................... 7Exercise 4: Dense Connective Tissue ..................................................................... 7 – 8

Pre-lab Exercise 5: Ground Substance Based Connective Tissue ........................... 8Exercise 5: Ground Substance Based Connective Tissue ....................................... 8

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Summary Questions................................................................................................ 9Preparing to Use the Remote Web-based Science Lab (RWSL) .............................. 10 - 24

LEARNING OBJECTIVES:

Overview of histology and tissue types

1. Define the term histology.2. List four major tissue types.3. Contrast the general features of the four major tissue types.

Microscopic anatomy, location, and functional roles of epithelial tissue

1. Classify the different types of connective tissue based on distinguishing structural characteristics.

2. Describe locations in the body where each type of connective tissue can be found.3. Describe the functions of each type of connective tissue in the human body and

correlate functions with structure for each tissue type.4. Identify the different types of connective tissue using proper microscope technique.

BACKGROUND INFORMATION:

A living organism is composed of a variety of cells of different sizes, shapes, structures and specialized functions. Cells of similar type are usually organized into groups. A group of cells with similar size, shape, structure and function form a tissue. There are four general classes of tissues. These classes are epithelial, connective, muscle, and neuronal. In this lab, we will use histology to examine several connective tissues.

Histology1 is the branch of biology concerned with the composition and structure of plant and animal tissues in relation to their specialized functions. The terms histology and microscopic anatomy are sometimes used interchangeably, but a fine distinction can be drawn between the two studies. The fundamental aim of histology is to determine how tissues are organized at all structural levels from cells and intercellular substances to organs.

Connective tissues maintain the form of the body and its organs and provide cohesion and internal support. The connective tissues include several types of fibrous tissue that vary only in their density and cellularity as well as the more specialized and recognizable variants — bone, ligaments, tendons, cartilage, and adipose (fat) tissue.

Some organs are suspended from the wall of a body cavity by thin sheets of connective tissue called mesenteries. Others are embedded in adipose tissue, a form of connective tissue in which the cells are specialized for the synthesis and storage of energy-rich reserves of fat or lipid. The entire body is supported from within by a skeleton composed of bone, a type of connective tissue endowed with great resistance to stress owing to its highly ordered laminated structure and to its hardness, which results from deposition of mineral salts in its fibers and amorphous matrix. The individual bones of the skeleton are held firmly together by ligaments, and muscles are attached to bone by tendons, both of which are examples of dense connective tissue in which many fiber bundles are associated in parallel array to

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provide great tensile strength. At joints, the articular surfaces of the bones are covered with cartilage, a connective tissue with an abundant intercellular substance that gives it a firm consistency well adapted to permitting smooth gliding movements between the apposed surfaces. The synovial membrane which lines the margins of the joint cavity and lubricates and nourishes the joint surfaces is also a form of connective tissue.

Components of the connective tissue: All forms of connective tissue are composed of (1) extracellular fibers, (2) an amorphous matrix called ground substance, and (3) cells (stationary and migrating. We will discuss each component below. The proportions of these components vary from one part of the body to another depending on the local structural requirements. The anatomical classification of the various types of connective tissue is based largely upon the relative abundance and arrangement of these components.

Extracellular fibers: The fibrous component of connective tissue fall into three kinds: collagenous, elastic, and reticular. The collagenous fibers are the most abundant and are composed of the protein collagen which is of commercial and medical interest. The behavior of collagen varies tremendously based on how it is treated. Leather is the collagen-dense dermis of animal skins preserved and toughened by the process called tanning. Fresh collagen dissolves in hot water and produces gelatin. Under appropriate conditions, collagen can be brought into solution without chemical change. The fundamental units in such solutions are slender tropocollagen. Collagen appears to be secreted in this form by the connective-tissue cells called fibroblasts, and the tropocollagen molecules assemble extracellularly to form striated collagen fibrils.

Elastic fibers are composed of the protein elastin and differ from collagenous fibers in dimension, pattern, and chemical composition. They do not have uniform subunits comparable to the unit fibrils of collagen. They present a variable appearance in electron micrographs. Sometimes they appear to have an amorphous core surrounded by minute fibrils, while in other sites they appear to consist exclusively of dense amorphous material. Whether there are in fact two components or whether these are differing forms of the same substance is not yet clear. At the light-microscope level, the fibers vary in diameter and often branch and reunite to form extensive networks in loose connective tissue. When present in high concentration, they impart a yellow color to the tissue. In elastic ligaments, the fibers are very coarse and are arranged in parallel bundles. In the walls of arteries, elastin is present in the form of sheets or membranes perforated by openings of varying size. As their name implies, elastic fibers are highly distensible and, when broken, recoil like rubber bands. Changes in this property and diminution in their numbers are thought to be, in part, responsible for the loss of elasticity of the skin and of the blood-vessel walls in old age.

Reticular fibers are distinguished by their tendency to form fine-meshed networks around cells and cell groups. They are small fascicles of typical collagen fibrils interwoven to form a network.

Ground substance: The amorphous ground substance of connective tissue is a transparent material with the properties of a viscous solution or a highly hydrated thin gel. Its principal constituents are large carbohydrate molecules or complexes of proteins and carbohydrates. All substances passing to and from cells must pass through the ground substance. Variations in its composition and viscosity may therefore have an important influence on the exchange of materials between tissue cells and the blood. Its physical consistency also constitutes a barrier to the spread of particulates introduced into the tissues. While all types of connective tissue have at least a small amount of ground substance, two types consist predominately of it.

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Cartilages are a form of connective tissue in which the ground substance is abundant and of a firmly gelated consistency that endows this tissue with unusual rigidity and resistance to compression. The cells of cartilage, called chondrocytes, are isolated in small lacunae within the matrix. Although cartilage is a vascular (without blood supply), gaseous metabolites and nutrients can diffuse through the aqueous phase of the gel-like matrix to reach the cells.

Bones like cartilage consist of cells, fibers, and ground substance, but, in addition, the extracellular components are impregnated with minute crystals of calcium phosphate. The mineralization of the matrix is responsible for the hardness of bone. Because the hardness of bone the tissue does not diffuse nutrients rapidly, bone tissue is vascularized. It also provides a large reserve of calcium that can be drawn upon to meet unusual needs for this element elsewhere in the body. The structural organization of bone is adapted to give maximal strength for its weight-bearing function with minimum weight. The cells in bone are called osteocytes.

Cells of connective tissue include two types that are relatively stationary—fibroblasts and adipose cells—and several types of motile migrating cells—mast cells, macrophages, monocytes, lymphocytes, and eosinophils. All the cells of connective tissue develop during embryonic life from the mesenchyme, a network of primitive stellate cells that have the potential to differentiate along several different lines depending upon local conditions.

Stationary cells: The ubiquitous fibroblasts are the principal active cells of connective tissue, occurring as long spindle-shaped cells stretched along bundles of collagen fibrils. Their function is to secrete collagen and constituents of the ground substance and to maintain these extracellular tissue components.

Adipose, or fat, cells are connective-tissue cells that are specialized for the synthesis and storage of reserve nutrients. They receive glucose and fatty acids from the blood and convert them to lipid, which accumulates in the body of the cell as a large oil droplet. This distends the cell and imposes upon it a spherical form. The nucleus is displaced to the periphery, and other metabolically active constituents of the cell are confined to a thin rim of cytoplasm around the large central droplet of lipid. Adipose cells may occur in small numbers anywhere in connective tissue, but they tend to develop preferentially along the course of small blood vessels where they accumulate in such large numbers that they become the predominant cellular element.

Migrating cells: In addition to the relatively fixed cell types described above, there are free cells that reside in the interstices of loose connective tissue. These vary in their abundance and are free to migrate through the extracellular spaces. (Since you mentioned these above, you need to define or discuss them here: mast cells, macrophages, monocytes, lymphocytes, and eosinophils.)

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References:

1. "histology." Encyclopedia Britannica. http://www.britannica.com/EBchecked/topic/267172/histology (22 April, 2014)

EQUIPMENT:

Paper Pencil/pen Slides

o Areolar tissue slideo Adipose tissue slideo Lymph node slideo Tendon white fibrous connective tissue slideo Skin pigmented slideo Hyaline cartilage slideo Compact bone slide

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.

During the study of each type of human tissue, you are required to photograph and label a representative sample of each tissue type. You should begin your microscopic examination of the tissue slide using the 4X objective to give you a perspective of the entire structure. This is very important, since sometimes more than one tissue type is often present on each slide. All observations should be made using the 40X or 60X objective lens (whichever gives you the best results.)

PRE-LAB EXERCISE 1: Areolar Connective Tissue

Pre-Lab Questions:

1. Based on your course research and pre-lab readings what characteristics do you expect to see in Areolar connective tissue?

EXERCISE 1: Areolar Connective Tissue

Data Collection:

2. Select the areolar tissue slide from the microscope interface. Using the 10X objective, locate the tissue sample and bring it into focus.

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3. Carefully work your way through all the objectives focusing with each one until you reach the 40X or 60X objective and capture an image of the areolar connective tissue. Insert your image below.

Analysis (can be completed offline):

4. Using your image from Exercise 1, question 3, label the 3 different parts of a cell (nucleus, cytoplasm and cell membrane) and the different types of fibers.

5. Based on your observation, describe the function of the areolar connective tissue in the human body and correlate its function with its structure.

6. Where do you find the areolar connective tissue inside the human body?

PRE-LAB EXERCISE 2: Andipose Connective Tissue

Pre-Lab Questions:

1. Based on your course research and pre-lab readings what characteristics do you expect to see in adipose connective tissue?

EXERCISE 2: Andipose Connective Tissue

Data Collection:

2. Select the adipose tissue slide from the microscope interface. Using the 10X objective, locate the tissue sample and bring it into focus.

3. Carefully work your way through all the objectives, focusing with each one, until you reach the 40X or 60X objective and capture an image of the adipose tissue. Insert your image below.

Analysis (can be completed offline:)

4. Using your image from Exercise 2, question 2, Label the 3 different parts of a cell (nucleus, cytoplasm and cell membrane) and the different types of fibers.

5. Based on your observation, describe the function of the adipose tissue in the human body and correlate its function with its structure.

6. Based on your observation, what is a particular difference you have noticed between the adipose tissue and the areolar tissue?

7. Where do you find the adipose tissue inside the human body?

PRE-LAB EXERCISE 3: Reticular Connective Tissue

Pre-Lab Questions:

1. Based on your course research and pre-lab readings, what characteristics do you expect to see in reticular connective tissue?

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EXERCISE 3: Reticular Connective Tissue

Data Collection:

2. Select the lymph node or the spleen slide from the microscope interface. Using the 10X objective, locate the tissue sample and bring it into focus.

3. Carefully work your way through all the objectives, focusing with each one until you reach the 40X or 60X objective and capture an image of the reticular connective tissue. Insert your image below.

Analysis (can be completed offline:)

4. Using your image from Exercise 3, question 2, label the 3 different parts of a cell (nucleus, cytoplasm and cell membrane) and the different types of fibers

5. Based on your observation, describe the function of the reticular tissue in the human body and correlate its function with its structure.

6. Where do you find the reticular connective tissue inside the human body?

PRE-LAB EXERCISE 4: Dense Connective Tissue

Pre-Lab Questions:

1. Based on your course research and pre-lab readings, what characteristics do you expect to see in dense regular connective tissue?

2. Describe the differences you expect to see between dense regular and irregular connective tissue.

EXERCISE 4: Dense Connective Tissue

Data Collection:

3. Select the tendon white fibrous connective tissue slide from the microscope interface. Using the 10X objective, locate the tissue sample and bring it into focus.

4. Carefully work your way through all the objectives, focusing with each one until you reach the 40X objective and capture an image of the dense regular connective tissue. Insert your image below.

5. Select the skin pigmented slide from the microscope interface. Using the 10X objective, locate the tissue sample and bring it into focus.

6. Carefully work your way through all the objectives, focusing with each one until you reach the 40X or 60X objective and capture an image of the dense irregular connective tissue. Insert your images below.

Analysis (can be completed offline):

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7. Using your image from Exercise 4, question 4, label the 3 different parts of a cell (nucleus, cytoplasm and cell membrane) and the different types of fibers.

8. Based on your observation, describe the function of the dense regular connective tissue in the human body and correlate its function with its structure.

9. Where do you find the dense regular tissue inside the human body?10. Using your image from Exercise 4, question 6, label the 3 different parts of a cell (nucleus,

cytoplasm and cell membrane) and the different types of fibers.11. Based on your observation, describe the function of the dense irregular connective tissue in the

human body and correlate its function with its structure.12. Where do you find the dense irregular tissue inside the human body?

PRE-LAB EXERCISE 5: Ground Substance Based Connective Tissue

Pre-Lab Question:

1. Based on your course research and pre-lab readings what are the similarities and differences you expect to see between the cartilage and bone?

EXERCISE 5: Ground Substance Based Connective Tissue

Data Collection:

2. Select the hyaline cartilage 2 from the microscope interface. Using the 10X objective, locate the tissue sample and bring it into focus.

3. Carefully work your way through all the objectives, focusing with each one, until you reach the 40X or 60X objective and capture an image of the hyaline cartilage. Insert your image below.

4. Select the bone dry ground human 1 slide from the microscope interface. Using the 10X objective, locate the tissue sample and bring it into focus.

5. Carefully work your way through all the objectives, focusing with each one, until you reach the 40X or 60X objective and capture an image of the compact bone. Insert your image below.

Analysis (can be completed offline):

6. Using your image from Exercise 5, question 2, label the 3 different parts of a cell (nucleus, cytoplasm and cell membrane) and the ground substance.

7. Describe the function of the cartilage in the human body and correlate its function with its structure.

8. Where do you find the hyaline cartilage inside the human body?9. Using your image from Exercise 5, question 4, label the 3 different parts of a cell (nucleus,

cytoplasm and cell membrane) and the different types of fibers.10. Based on your observation, describe the organization of the compact bone.11. What is the function of the compact bone in the human body and correlate its function with its

structure.12. Where do you find the compact bone inside the human body?

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SUMMARY QUESTIONS:

Answer the following questions regarding the connective tissue.

1. Where is the nucleus of an adipocyte located? Why?2. Where are the functional differences between dense irregular and dense regular connective

tissue? How do these differences help each to perform their unique function?3. Based on your observation, what is a particular difference you have noticed between the dense

irregular and the dense regular connective tissues?4. Based on your observation, what is a particular difference you have noticed between the hyaline

cartilage and the compact bone?5. Discuss the impact of diet and hormones on the onset of osteoporosis.

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PREPARING FOR THIS 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, seewww.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 - http://www.youtube.com/watch?v=m7w9ssIgVdw3. 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.

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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.

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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"

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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

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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.

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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

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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."

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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

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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.

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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!

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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.

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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.

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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.

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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.

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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

This product was funded by a grant awarded by the U.S. Department of Labor’s Employment and Training Administration. The product was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor. The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.

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