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REMOTE LAB ACTIVITY

SUBJECT SEMESTER: ________________

TITLE OF LAB: Membrane Osmosis

Lab format: This lab is a remote lab activity.

Relationship to theory (if appropriate): In this lab you will be examining the underlying processes of transport/osmosis.

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 on-line period, and answer questions in analysis sections after your on-line period. Your instructor will let you know if you are required to complete any optional exercises in this lab.

Remote Resources: Primary - Microscope.

CONTENTS FOR THIS NANSLO LAB ACTIVITY:

Learning Objectives......................................................................................................... 2Background Information ................................................................................................ 2 - 6Equipment ...................................................................................................................... 7Preparing to Use the Remote Web-based Science Lab (RWSL) ..................................... 7Experimental Procedure – Exercise 1: Brownian Motion .............................................. 7 - 8Experimental Procedure – Exercise 2: Osmosis in Plant Cells ........................................ 8 - 9Experimental Procedure – Exercise 3: Osmosis in Animal Cells ..................................... 9 - 10Preparing for this NANSLO Lab Activity .......................................................................... 11 - 25

1 | P a g e L a s t U p d a t e d A p r i l 1 9 , 2 0 1 4

LEARNING OBJECTIVES:

With respect to the membrane transport process of osmosis:

1. State what type of material is moved in each process.2. Give examples of the osmotic process in the human body.3. Describe the effects of hypertonic, isotonic, and hypotonic conditions on cells.4. Explain the effect of temperature on Brownian motion.5. Demonstrate the process of osmosis and, given appropriate information, predict the outcomes

of the demonstration on the cell.6. Describe the mechanism of osmosis.7. Relate tonicity to solute concentration.8. Describe the effects of water gain or loss in animal or plant cells.

BACKGROUND INFORMATION:

This laboratory activity will focus on the movement of molecules with respect to living cells. Specifically, we will look at how molecules move based on the inherent energy of Brownian motion. Then we will use that understanding to explore the process of osmosis in plant and animal cells.

In a cell, molecules move across a semipermeable membrane or in the cytosol. Before we look at how the cell regulates molecular movement we need to review some basic principles of molecular vibrational movement. Molecules are in constant state of movement. Even in a solid state, the molecules exhibit vibrational movement as they move against each other in position. Molecules in a liquid are farther apart than in a solid (for most molecules), but not as far apart as in a gas. As molecules move from a solid to a liquid to a gas, they increase their motion. As molecules move from a solid to liquid state, they gain the ability to move with respect to each other both in orientation and position. In the liquid state, molecules are in constant motion with particles frequently colliding with each other. This type of motion is called Brownian movement. It can be defined as any of various physical phenomena in which some quantity is constantly undergoing small, random fluctuations. (Encyclopedia Britannica. http://www.britannica.com/EBchecked/topic/81815/Brownian-motion).

In the gaseous state, molecules are much farther apart and the number of collisions between them is greatly decreased compared to the liquid state. In Figure 1 you can see the arrangement of the molecules in three of the states of matter. The arrows in Figure 1 show how molecules move between the three states.


http://www.britannica.com/EBchecked/topic/81815/Brownian-motion)

Figure 1: States of Matter

This understanding of basic molecular movement can now be applied to molecular movement within a cell.

The plasma membrane, common to all cells, is a unique structure that delineates the cell’s boundaries. It has the ability to regulate the entry and exit of molecules into and out of the cell. The plasma membrane is composed of phospholipids and proteins arranged in a phospholipid bilayer. The specific type of lipid molecule used in the cell wall is the phospholipid. This is a type of lipid molecule that has a phosphate group attached to the glycerol subunit. The glycerol molecule is called the head and it is a polar molecule. This polarity is important because the head group is hydrophilic or “water loving”. The fatty acid tails are non-polar and hydrophobic or “water fearing”. This molecule structure is what causes the formation of the lipid bilayer (see Figure 2). The fatty acid tails in the phosoplipids interact with each other to exclude water and form the core of the bilayer while the head group interacts with the water both on the internal side of the membrane (cytosol or cytoplasm) and the external side (extracellular fluid).


Figure 2: Phospholipid Structure

The glycerol molecules on the internal and external surfaces protect the fatty acid tails from coming into contact with the water.

The plasma membrane layer also contains protein molecules. These molecules have a variety of functions: transport, identification, attachment, metabolic functions and signal transduction. The plasma membrane’s composition of both phospholipids and proteins has led to the Fluid Mosaic Model (see Figure 3 below). This model was first proposed in 1972 by S.J Singer and G. Nicolson to describe the structure of a living membrane. Scientists use the term “fluid” to describe the plasma membrane because of its hydrophobic integral components such as the lipids and membrane proteins that have the ability to move laterally (sideways) within the membrane. This movement or potential for movement gives it a “fluid” quality. The term mosaic is used to describe something that is made up of many different parts. Within the membrane there are many different macromolecules. In Figure 3 you can see there are cholesterol molecules, sugar side chains and many different types of proteins. To date there have been more than 50 membrane proteins identified. (Campbell Biology 9th ed.2011)

Figure 3: Cell Membrane Structure


Passive Transport

Passive transport does not require the input of additional energy. It takes place solely based on the inherent molecular movement of Brownian motion. We have listed several types of passive transport below:

Diffusion – This is the net movement (the movement in one direction minus the movement in the opposite direction) is dictated by relative concentrations of molecules. Molecules move from a place of higher concentration to a place of lower concentration. Equilibrium is when there is no longer a net movement in one direction; the number of molecules moving in one direction equals the number of molecules moving in the other. Concentration gradient is the difference in the concentration of the molecules over a distance.

Osmosis – This is the net movement (diffusion) of water molecules through a selectively permeable membrane with respect to the solute concentration in the water. Selectively permeable means that the membrane will allow certain molecules to pass across it and will prevent others from doing so.

Osmolarity can be defined as the measure of osmotic pressure of a solution. In other words, it is the measure of the amount of solute in the solution. Osmolarity, therefore, is the movement of water from one side of the cell membrane to the other. Tonicity, on the other hand, refers to relative concentration of solute particles inside a cell with respect to concentration outside the cell. The result of tonicity is the cell’s ability to gain or lose water based in part on the concentration of the non-penetrating solutes in the solution surrounding the cell.

The following terms are used to describe the tonicity of a solution and the impacts of the solutions on a cell. It is important to note that concentration is typically given for the non-penetrating solute not the solvent. This means that in order to determine which direction the water molecules will move you need to be able to calculate the % of water. For example, in a 5% Glucose solution there is 95% water. In Figure 4 shown below, you can see the three tonic solutions and how the movement of the free water across a plasma membrane changes as a result of the concentration of the solutes. As we use these terms isotonic, hypertonic and hypotonic, we are referring the solution not the cell.

Isotonic – iso means same -a solution that contains a concentration of water and dissolved materials (solutes) which equal that of the internal environment of the cell. In this solution, there is no net movement. In an animal cell, this results in a stable cell. See Figure 4A.

Hypotonic – hypo means less - a solution that has a lesser amount of non-penetrating solutes outside of the cell. Based on a concentration gradient, the net movement of water is into the cells. As a result, the cells swell. In a plant cell, as a result of a cell wall, the result is turgor. In an animal, cell without a cell wall, the cell will swell and burst. This is called lysis. See Figure 4B.

Hypertonic – hyper means more - a solution that has a greater amount of non-penetrating solutes outside of the cell. Based on a concentration gradient the net movement of water is out of the cells. As a result, the cells shrink. This is called crenation. See Figure 4C.


Figure 4: Tonic Solutions and the Effect on Cells

Dialysis- If osmosis is the movement of water then dialysis is the movement of ions. It follows the same rules as other forms of passive transport. This type of membrane is used in dialysis for persons with poor kidney function. In dialysis, ions move from a higher concentration (in the blood) to a lower concentration (in the water on the other side of the membrane).

Facilitated Diffusion – when molecules move across a membrane by flowing through a protein channel or by combining with specific carrier proteins, the rate of diffusion is increased. It is still moving from a high concentration to a low concentration.


EQUIPMENT:

Paper Pencil/pen Slides

o India Inko Anachariso Red Blood Cell

Computer (access to remote laboratory)

PREPARING TO USE THE REMOTE WEB-BASED SCIENCE LAB (RWSL):

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.

EXPERIMENTAL PROCEDURE:

Once you have logged on to the microscope, you will perform the following laboratory procedures:

EXERCISE 1: Brownian Motion

This exercise will introduce you to the random motion of particles caused by their inherent energy. We know that this random movement (Brownian motion) is based on the kinetic energy of the molecules and that the movement of the molecules is effected by temperature.

Pre-Lab Questions:

1. Do you think you will see any movement of the ink particles? What do you think is causing the movement and will temperature alter the movement?

2. Hypothesis/Prediction – Set this up as an - if…..then…… statement. For example: If heat is applied to particles in random motion then observable differences will be seen in the movement at the different temperature. This example is meant to be very general. Your job is to use your answer to question #1 and make it into a more specific if-then statement based on your understanding prior to observing the slides.

Procedure:

3. Request an India ink slide from the lab technician. The slide has been kept in the freezer at _____ 0C.

4. Begin observing your slide at 10X and increase magnification to 40x.5. Describe the motion that you see. Include in your description a statement about the speed the

molecules are moving.6. Continue to observe the movement for a minute. As you watch the slide, it will warm to Room

temp in the lab is ~ 250C. The light used on the microscope is an LED lights so there will be little


https://cheo.pbworks.com/w/page/69376066/Membrane%20Osmosis%20-%20NANSLO%20Lab%20Activity#microscopeinstructions

to no impact on the slide temperature from this light. Describe or estimate the change in the speed of the molecules.

7. Once you are ready request that the lab technician hold the slide over a hot plate very briefly (this will heat very quickly) and replace it on the microscope. The approximate temperature of the hot plate on a low setting is ~ 700 C.

8. Observe and describe the motion and speed of the molecules.

Analysis:

9. Is there any directionality to the molecular movement? Explain your answer.10. Think about the effect of temperature on molecular movement in which temperature do you

think there would be more molecular interactions? Use your observations to support your answer.

11. Make a claim about what you learned and back it up with the data or evidence you gathered. You may have more than one claim and evidence statement.

12. Think back to the initial hypothesis/prediction you made. Was your prediction correct? Write a statement that uses your data to either support or reject your hypothesis.

EXERCISE 2: Osmosis in Plant Cells

Osmosis is the diffusion of water through a selectively or semipermeable membrane. It is caused by the unequal distribution of solutes on the two sides of the membrane as a result the water is “drawn” towards the side of the membrane with the higher solute concentration. In a plant cell, the vacuole draws water in from the cytoplasm as the result of a hypotonic environment. As this happens, the central vacuole pushes out and increases pressure between the cytoplasm and the cell membrane. The cell wall acts as a strong barrier containing the pressurized cytoplasm. This is known as turgor and is responsible for plant cells being turgid or “full”.

Pre-Lab Question:

1. You are starting with a plant cell that is in a turgid state. Describe what you think you will see in a turgid plant cell? What do you think will happen if the cell is exposed to a hypertonic solution?

2. Hypothesis/Prediction – Set this up as an - if…..then…… statement. For example: If a cell is exposed to a hypertonic solution then observable differences will be seen in the movement of the cells fluid. This example is meant to be very general. Your job is to use your answer to question # 1 and make it into a more specific if then statement based on your understanding prior to observing the slides.

Procedure:

3. Request the lab technician make a slide of a leaf of Anacharis and place it on the microscope.4. Begin observing your slide at 10X and increase magnification to 40x.5. Describe and take a picture of what you see to include in your lab submission.6. While watching the screen ask the lab technician to add a drop of a 20% salt solution.7. Observe the process for a few minutes. Then describe and take a picture of the Anacharis as its

cells are exposed to the salt solution.


Analysis:

8. What tonicity condition was the solution to the cell was in at the beginning of the experiment and at the end of the experiment?

9. Was the cell ever at an isotonic condition? Explain your answer.10. What is the role of the cell wall with regard to a plants ability to control its internal turgor

pressure?11. Many organelles such as the chloroplasts and mitochondrion have internal membrane systems.

What is the explanation for the fact that many cells and their membrane systems have many convolutions? How does this relate to transport of molecules?

12. Based on your understanding of the impact of temperature on molecular movement, predict the effect of temperature on osmotic movement.

13. Think about what you learned in the first exercise on Brownian motion. Use this understanding to explain how the molecules are moving in this activity.

14. Make a claim about what you learned and back it up with the data or evidence you gathered. You may have more than one claim and evidence statement.

15. Did the data support your hypothesis? Write a statement that uses your data to either support or reject your hypothesis.

EXERCISE 3: Osmosis in Animal Cells

In cells that do not have a cell wall, i.e. animal cells, the impacts of tonicity can impact the function of cells in the body. If the balance is disturbed, the cells will swell or shrink. The tonicity of the extracellular fluid is regulated at all times by controlling its relative amounts of solutes and water.

Pre-Lab Questions:

1. You are starting with an anucleated mammalian red blood cell that is in an isotonic solution. Describe what you think you will see in an animal cell in an isotonic solution? What do you think will happen if the cell is exposed to a hypertonic solution? A hypotonic solution?

2. Hypothesis/Prediction – Set this up as an - if…..then…… statement. For example: If a cell is exposed to a hypertonic solution then observable differences will be seen in the movement of the cells fluid. This example is meant to be very general. Your job is to use your answer to question # 1 and make it into a more specific if then statement based on your understanding prior to observing the slides.

Procedure:

3. Select a slide of the blood and place it on the microscope.4. Begin observing your slide at 10X and increase magnification to 60x.5. Describe and take a picture of what you see to include in your lab submission.6. The field of view at 60X is ____________. With a ruler, measure the size of the isotonic red

blood cell. Calculate the size of the red blood cell with the following ratio.

Captured Image Measured Actual Image (X)Measured diameter of captured image Given field of View


Record the size of the RBC ____________________________________________7. Select a slide of the blood that has been in a hypotonic solution. For this activity, the

concentration of the solution is _________________. 8. Begin observing your slide at 10X and increase magnification to 60X. 9. Observe the slide. Then describe and take a picture of the blood cells as they are exposed to the

hypotonic solution. 10. With a ruler, measure the diameter of the image you have captured. Record that here

______________. Now measure the size of the isotonic red blood cell. Calculate the size of the red blood cell with the following ratio.


Record the size of the RBC ____________________________________________

11. Select a slide of the blood that has been in hypertonic solution. For this activity, the concentration of the solution is ______________________.

12. Begin observing your slide at 10X and increase magnification to 60X.13. Observe the slide. Then describe and take a picture of the blood cells as they are exposed to the

hypertonic solution.14. With a ruler, measure the diameter of the image you have captured. Record that here

____________. Now measure the size of the isotonic red blood cell. Calculate the size of the red blood cell with the following ratio.


Record the size of the RBC ____________________________________________

Analysis:

15. What was the tonicity condition of the blood cell for the first slide at the beginning of the experiment and at the end of the experiment?

16. Research what conditions could result in crenation of blood cells in a human.17. In what tonicity condition were the blood cells on the second slide in at the beginning of the

experiment and at the end of the experiment?18. As the cells reacted to the distilled water what happened to them? Explain what caused this to

happen.19. Research what conditions could result in lysis of blood cells in a human.20. Why are concentration gradients such as we saw in the tonicity of the plant and animal cells so

important to transport in living things?21. Make a claim about what you learned and back it up with the data or evidence you gathered.

You may have more than one claim and evidence statement.22. Did the data support your hypothesis? Write a statement that uses your data to either support

or reject your hypothesis.


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 - https://www.youtube.com/watch?

feature=player_embedded&v=m7w9ssIgVdw. 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.


http://www.wiche.edu/info/nanslo/creative_science/Using_the_Microscope_for_a_NANSLO_RWSL_Lab_Activity_for_NANSLO_labs.pdf

http://www.wiche.edu/nanslo/lab-tutorials

http://www.wiche.edu/nanslo/scheduling-software

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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