Introduction to Light Introduction to Light Microscopy

L A B O R A T O R Y M A N U A L

Introduction to LightIntroduction to Light Microscopy

December 7th – 9th, 20109:00 AM-12:30 PM

Stewart Biology, Rm N2/2gy,

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McGill University Imaging Facility Introduction to Light Microscopy Course December 7-9, 2010 1

T A B L E O F C O N T E N T S

Lab Exercise 1: Fluorescence Labelling 2

Lab Exercise 2: Brightfield Microscopy 5

Lab Exercise 3: Contrasting Techniques 9

Lab Exercise 4: Resolution and Objectives 15

Lab Exercise 5: Fluorescence Microscopy 19

Lab Exercise 6: Wide Field Live Cell Imaging 24

Lab Exercise 7: CLSM Live Cell Imaging 27

Laboratory Exercises 6 and 7 Follow Up 33

Appendix Index 34

Appendix I: Fluorescence Labelling Reagents 35

Appendix II: Microscope Schematics

Axiovert 200M Transmitted Light Components 36

Axiovert 200M Fluorescence Components 37

Axioscope A1 38

Axioskop 2 Motorized 39

Axioskop 2 Manual Transmitted Light Components 40

Axioskop 2 Manual Fluorescence Components 41

AxioLab A1 42

Appendix III: Köhler Alignment 43

Appendix IV: DIC Alignment Protocol 45

Appendix V: Oil Immersion Lens Cleaning Protocol 47

Appendix VI: Live Cell Imaging Summary 48


Lab Exercise 1: Fluorescence Labelling Objective: To become familiar with techniques for fluorescently labelling cells. This lab will span over

three days and will involved two labelling protocols one for fixed cells and one for live cells.

A detailed list of reagents can be found in Appendix I.

Part 1: Fixing, fluorescent labelling and slide mounting of cells. You will be given four coverslips and

an IBIDI chambered slide with live CHO-K1 cells growing on them. Two coverslips with unlabelled

cells, two coverslips and the IBIDI slide with cells expressing paxillin-EGFP. Rinse the cells three

times with 1 mL of phosphate buffered saline without calcium or magnesium (PBS-).

1. Aspirate the solution off of the coverslips and out of the chambers on the IBIDI slide.

2. Apply 1 mL* of 4% paraformaldehyde (PFA) in PBS- to each sample and leave for 10 minutes.

3. Wash the cells three times with 1 mL* of PBS-.

4. Permeabilize the cells with 1 mL* of 0.2% Triton-X-100 detergent in PBS- for 3 minutes.

5. Wash the cells three times with 1 mL* of PBS-.

6. Block non-specific binding for 10 minutes in 1 mL* of 5% bovine serum albumin (BSA) in PBS-.

7. First Labelling Step: Make sure to dry the area around the coverslip very well before apply the

labelling reagents.

Coverslip 1: Unlabelled cells.

Control for cellular autofluorescence. Apply 200 μL* of PBS- with 2% BSA.

15 minutes

at RT

Coverslip 2: Paxillin-EGFP cells labeled for

the nucleus and actin filaments. Apply 200 μL* of DAPI/phalloidin-

Alexa555 in PBS- with 2% BSA.

15 minutes

at RT

Coverslip 3: Paxillin-EGFP cells will be

labeled for nucleus and microtubules. *Note: Two day protocol.

Apply 200 μL* of tubulin primary

antibody in PBS- with 2% BSA.

Overnight at 4oC

Coverslip 4: Unlabelled cells. Control for non-specific binding of 2o antibody.

Apply 200 μL* of PBS- with 2% BSA. Overnight

at 4oC *When labelling with the IBIDI chamber slides you only need 100 μL of solution.


8. Leave coverslips 3 and 4 at 4oC overnight. The labelling will be finished on Day Two.

9. Wash coverslips 1 and 2 three times with 1 mL* of PBS- and they are ready to mount.

Mounting the Coverslips:

a. Wipe two microscope slides with a KimwipeTM moistened with ethanol (EtOH).

b. Label the slides for each sample.

c. Use the wooden end of a cotton swab handle to place a small drop of cytoseal 60 in

the centre of the coverslip.

d. Remove as much liquid as possible from coverslip 1. Lift the corner of the coverslip

with a needle tip and grab a corner using fine tipped tweezers.

e. Tilt the slide and dry any excess liquid at the corner of the coverslip on a KimwipeTM.

f. Invert the slide and place it gently at a 45o angle onto the drop of mounting medium.

g. Use the cotton end of a cotton tip applicator (QTip) to gently press down on the

coverslip and displace any air bubbles in the mounting media to the edges of the

coverslip.

h. Leave the coverslip covered with foil overnight so the mounting medium can cure.

i. The samples will be ready for visualization on Day Two.


10. Second Labelling Step (Day 2):

a. Wash coverslips 3 and 4 three times with 1 mL of PBS-.

b. Apply 200 μL of tubulin secondary antibody in PBS- with 2% BSA for 45 minutes.

c. Wash the coverslips three times with 1 mL of PBS-.

d. Mount the coverslips as in Step 9. Samples will be ready for visualization on Day 3.

Part 2: Live paxillin-EGFP expressing CHO-K1 cells will be labelled with nuclear and mitochondrial dyes.

1. Remove the DMEM medium form the cells.

2. Apply 1 mL of DMEM containing MitoTracker RedCMX-Ros and Hoechst 33342.

3. Place the cells at 37oC for 10 minutes.

4. Rinse once with 1 mL of DMEM culture media.

5. Place in 1 mL of fresh culture media and take to the microscope.


Lab Exercise 2: Brightfield Microscopy

Objective: To become familiar with the transmitted light path components, Köhler Illumination

alignment of the microscope, the shape of the cone of light produced by the

condenser and how the manipulation of various components within the optical train

of the microscope change that cone of light.

Part 1: Know your microscope. When using any new microscopy platform it is important to

familiarize yourself with the components and operations of the system.

Identify the type of microscope stand you are using and find the appropriate schematic

for the system in Appendix II. Following from the light source to the specimen to the

eyepiece or detector identify the components in the light path.

Part 2: Köhler Alignment. In this section of the lab exercise you will learn how to align the

condenser for proper Köhler Illumination of the microscope.

1) Choose a 10x or 20x objective lens and rotate it into the transmitted light path.

2) Place a stained kidney slide on the microscope stage. Which way should the coverslip face?

_________________________________________________________________________

_________________________________________________________________________

3) Focus on the sample and carefully observe it, if you are using a microscope equipped with a

camera take a snapshot of the sample.

4) Perform Köhler Illumination alignment using the protocol in Appendix III.

5) Observe the specimen again and note any differences in the image. Again if you are using a

microscope equipped with a camera take a snapshot of the sample.

6) Compare the two images. What differences do you see? Why?

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________


Part 3: Contrast with stained tissues. Take a look at the three kidney tissue slides. One is

unstained and unmounted, one is unstained and mounted and one is stained with H&E

(hematoxylin and eosin – proteins appear pink and DNA purple) and mounted.

1) Hold the slides up to the light and look at them by eye.

a) Which slide shows the most contrast? ________________________________________

b) Which slide shows the least contrast? ________________________________________

c) Why?__________________________________________________________________

______________________________________________________________________

d) What is generating the contrast in the unstained tissue?__________________________

2) Place the stained tissue on the microscope (make sure you are aligned for Köhler

illumination). Identify some of the features of the tissue. You should see purple nuclei and

cross sections of tubules in the kidney tissue.

_________________________________________________________________________

_________________________________________________________________________

3) Place the unstained tissue on the microscope and see if you can identify the same

structures you see in the stained tissue. Is the tissue architecture more evident in the

stained or unstained slice?____________________________________________________

Challenge: Can you identify the blood cells in the stained kidney section?

How about in the unstained section?


Part 4: Visualization of the transmitted light cone focused by the condenser. The

transmitted light beam and how it changes shape when the field and condenser

apertures are adjusted will be observed using a Lucite cylinder.

1) Make sure the microscope has been aligned for Köhler illumination.

2) Remove the slide used for alignment from the stage.

3) Move the microscope objective turret to an empty position.

4) Place the Lucite cylinder on the stage and make sure it is centered with respect to the top

lens of the condenser.

5) Close the condenser aperture.

6) Sketch the cone of light propagating from the condenser in the Lucite cylinder shown below.

7) Increase the numerical aperture of the condenser by opening the aperture diaphragm.

a. What happens to the cone of light? Sketch below.

b. How does opening up the condenser aperture affect the cone of light?

______________________________________________________________________

______________________________________________________________________


c. Should you use the condenser aperture diaphragm to adjust light?

______________________________________________________________________

______________________________________________________________________

d. What other ways are there to reduce light intensity?

______________________________________________________________________

______________________________________________________________________

e. Do you foresee any problems with using your proposed methods?

______________________________________________________________________

______________________________________________________________________

8) Close the field diaphragm.

a. What happens to the cone of light?__________________________________________

b. Why is controlling the diameter of the illuminating light path important?

______________________________________________________________________

______________________________________________________________________

9) Place a phase annulus into the light path. What happens with the cone of light?

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________

10) Remove the Lucite cylinder, put the 10x objective lens in place and adjust the microscope

for Köhler illumination.

11) Opaque samples are not amenable to transmitted light techniques. For this reason,

materials such as silicon wafers and thick bone sections are observed in reflection. In this

configuration, the objective also serves as the condenser. Can you think of a way to

visualize the cone of light produced and accepted by an objective?

_________________________________________________________________________

_________________________________________________________________________


Lab Exercise 3: Contrasting Techniques

Objective: To become familiar with various contrasting techniques in order to visualize cellular

structures using brightfield, phase contrast, darkfield and DIC.

Part 1: Phase Contrast Microscopy

1) Have one of the laboratory staff members take a phase objective off of the microscope so

you can look through the lens. What do you see? __________________________________

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________

2) Check the labels on the objective casings to see what types of phase lenses you have

(every company has its own labelling scheme). The laboratory staff member can then screw

the lens back into the opening in the objective turret.

3) With the phase lens in place, take the eyepieces out and look down to see the back aperture

of the lens. What do you see? _________________________________________________

_________________________________________________________________________

_________________________________________________________________________

4) Rotate the condenser turret to place the corresponding phase annulus into the transmitted

light path (you may need help from a laboratory staff member). Look down the eyepiece

tubes again. What do you see? Is the phase annulus centered?

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________


5) Turn the condenser turret and see how the other phase annuli look at the aperture at the

back focal plane of the objective.

a. Do all the annuli line up with the phase plate in the objective? Why or why not?

______________________________________________________________________

______________________________________________________________________

6) Return to the correct phase annulus and replace the eyepieces.

7) Put the unstained kidney sections with mounting media on the microscope.

a. What features are visible with phase contrast that are not visible with brightfield?

______________________________________________________________________

______________________________________________________________________

______________________________________________________________________

b. Do all the features in the image correspond to physical structures on the sample? Why

or why not? ____________________________________________________________

______________________________________________________________________

______________________________________________________________________

8) Place the stained kidney section on the microscope without the phase annulus in place. Can

you see the same features? __________________________________________________

_________________________________________________________________________

_________________________________________________________________________

9) Would you recommend phase contrast for observing these samples? __________________

_________________________________________________________________________

_________________________________________________________________________


Part 2: Darkfield Microscopy.

1) Put a 10x or 20x lens in place and put a pollen grain slide on the microscope and align the

microscope for Köhler illumination as described in Appendix III.

2) Put the darkfield annulus into place.

3) If your microscope is not equipped with darkfield components insert a phase annulus for a

high NA objective lens.

4) Remove the eyepieces and look down the eyepiece tubes at the back aperture of the lens.

What do you see?___________________________________________________________

_________________________________________________________________________

5) Put the eyepieces back in place and look at the pollen grain slide. Do you need to increase

the intensity of the transmitted light lamp? Why or why not? _________________________

_________________________________________________________________________

6) Open both the field and condenser aperture diaphragms completely before proceeding any

further. What does opening up the diaphragms accomplish?

_________________________________________________________________________

_________________________________________________________________________

7) How do the brightfield, phase contrast, and darkfield images compare? Try some other

samples._________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________


Part 3: Differential Interference Contrast (DIC) Microscopy.

1) Examine the objectives to determine whether DIC lenses are available on the microscope

(labels vary according to manufacturer, but “DIC” usually appears).

2) List the components of a typical DIC light path starting from the source to the eyepiece or

detector. __________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________

3) What is meant by crossed polarizers? __________________________________________

_________________________________________________________________________

_________________________________________________________________________

4) Align the DIC components of the microscope using the protocol in Appendix IV.

5) Look at the unstained kidney sample without the polarizers or the prisms in the light path. If

you are using a camera based system take an image of the kidney sample.

6) Now look at the unstained kidney sample with the polarizers and prisms in the light path. If

you are using a camera based system take an image in DIC.

7) What differences do you see between brightfield and DIC? Are there features you can see in

DIC that are not visible in brightfield or vice versus? _______________________________

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________


8) Repeat step 2 with the stained kidney section. If you have a camera take some images.

9) Do you see any difference between stained and unstained samples in the DIC images?

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________

10) Is it helpful to have a stained specimen when imaging with DIC?

_________________________________________________________________________

_________________________________________________________________________

11) Focus along the z-axis through the kidney section with DIC and brightfield. Do you notice

any differences? Can you see 3D structures in brightfield? In DIC?

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________


Part 4: Comparison of transmitted light techniques.

1) Compare brightfield, phase contrast, darkfield and DIC microscopy. In particular, what are

the distinctive features of each technique? _______________________________________

_________________________________________________________________________

_________________________________________________________________________

2) Which types of samples are ideal for:

a) Brightfield?_____________________________________________________________

______________________________________________________________________

______________________________________________________________________

b) Phase Contrast?_________________________________________________________

______________________________________________________________________

______________________________________________________________________

c) Darkfield?______________________________________________________________

______________________________________________________________________

______________________________________________________________________

d) DIC? __________________________________________________________________

______________________________________________________________________

______________________________________________________________________


Lab Exercise 4: Resolution and Objectives

Objective: To become familiar with diffraction patterns, how they are collected by objective

lenses, and how this relates to numerical aperture. Learn how to distinguish lower

quality and higher quality lenses. Explore optimal sampling frequencies.

Part 1: Visualization of the diffraction pattern from a diffraction grating.

1) Place a stained sample on the microscope and, using a 20x lens, align the

microscope for Köhler Illumination (Appendix III).

2) Remove the sample and place a diffraction grating slide on the stage.

3) Turn on the transmitted light lamp.

4) Close down the field diaphragm and open the condenser aperture diaphragm.

5) Move the condenser wheel to an open position (no phase annulus or DIC prism).

6) Remove the eyepieces from the microscope and look down the eyepiece tubes.

a. What do you see? ______________________________________________

b. Can you see separation of the blue and red light? _____________________

c. What happens if you turn the diffraction grating? ______________________

_____________________________________________________________

d. Move higher magnification lenses in place. What do you see? ____________

_____________________________________________________________

e. Place two diffraction grating slides at 90o to one another. What do you see?

________________________________________________________________

________________________________________________________________


Part 2: Distinguish different qualities of lenses. Your microscope is equipped with two or

three different lenses of the same magnification.

1) Using your stained kidney slide determine the identity of each of the two or three lenses

on your microscope system. Record the lens on your system in the table below.

Magnification and Type Numerical Aperture Lens Identity (A, B, or C)

2) Now place a fluorescently stained sample on the microscope. Do you still think you have

properly identified the objectives? ___________________________________________

3) Why or Why not?________________________________________________________

4) Is it easier to use the fluorescently stained slide or the visibly stained slide to determine

the quality of the lenses or are they similar? ___________________________________

5) If you find one sample type easier than the other why is that?______________________

______________________________________________________________________

______________________________________________________________________

______________________________________________________________________


Part 3: Determine the optimal lateral sampling frequency.

1) Choose a 40x or higher magnification lens.

2) Place a fluorescent sample on the stage. 3) Make sure the camera is set to full resolution (i.e. no camera binning). 4) Take an image of the fluorescent slide. Choose a stain with fine structures like actin

or mitochondria to make it easier to see resolution differences. 5) Put the camera to a 2x2 binning setting. Take another image of the sample. 6) Put the camera to a 3x3 binning setting. Take another image of the sample. 7) Compare the three images.

a. Which image is brightest? (Hint: You may need to look at the intensity histogram

to determine this.) _________________________________________________

________________________________________________________________

b. Which image provides the highest resolution? (Hint: You may need to zoom in to

see the difference). ________________________________________________

________________________________________________________________

c. Which image provides the lowest resolution?_____________________________

________________________________________________________________

d. Save your files and compare their sizes. Why are the sizes different? ________

________________________________________________________________

________________________________________________________________


Part 4: Determine the optimal axial sampling frequency. Note: Requires a motorized z-focus drive.

1) Choose a 20x or 40x objective lens.

2) Place a fluorescent sample on the stage.

3) Note the z-focus value at the top and the bottom of your sample.

4) Take several z-stacks through your sample with 2 μm, 1 μm and 0.5 μm spacing

between image sections.

5) Using the orthogonal viewer along the z-axis compare the axial resolution of the

three image stacks.

a. Which image provides the greatest amount of detail?______________________

b. Which image looks the thinnest? Why? _________________________________

________________________________________________________________

c. Zoom in to see the pixel resolution.

6) Save the files and compare your file sizes.

a. Which file is biggest? _______________________________________________

b. Why? __________________________________________________________

________________________________________________________________

________________________________________________________________


Lab Exercise 5: Fluorescence Microscopy

Objectives: To become familiar with the fluorescence light path and how to collect fluorescence

images. To learn how to detect and minimize photo-bleaching. Look at field

uniformity. Live cell fluorescence imaging.

Part 1: Microscope components, light path and basic operation.

1) Identify the components in the fluorescence light path for your microscope. See Appendix II.

a. What is your fluorescence light source? ______________________________________

______________________________________________________________________

b. Are there neutral density (ND) filters or other controls for lamp intensity?_____________

______________________________________________________________________

c. Are there UV or IR or other filters in the light path? ______________________________

______________________________________________________________________

d. Do you have control over the aperture diaphragm and the field diaphragm? __________

e. How do you control which filter cubes are in light path? __________________________

______________________________________________________________________

f. What filter cubes are in place? _____________________________________________

______________________________________________________________________

g. What detector(s) is(are) available on the microscope? ___________________________

______________________________________________________________________

2) What colours of fluorophores can be imaged on your microscope?

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________


Part 2: Collect fluorescence image data.

1) Put a 10x or 20x lens in place and collect a few multi-colour images of a fluorescent sample.

The laboratory staff will help you with the software settings.

2) What is your sample? _______________________________________________________

_________________________________________________________________________

_________________________________________________________________________

3) What are your cells labelled with? ______________________________________________

_________________________________________________________________________

_________________________________________________________________________

4) What settings do you need to use for your imaging?

a. Exposure time___________________________________________________________

b. Camera gain____________________________________________________________

c. Pixel binning____________________________________________________________

d. Lamp power ____________________________________________________________

e. Other settings __________________________________________________________

5) Take your sample off of the microscope and put a high NA oil immersion lens in place. Have

the laboratory staff review with you how to put some oil on the lens.

6) Place your sample with the coverslip facing the lens and bring the objective into contact with

the oil. Use the fine focus to focus on your sample.

7) Now collect a few multi-colour images of a fluorescent sample using the same settings as

above (Step 4). How do you images look? Why? __________________________________

_________________________________________________________________________


8) Before the lab, calculate the optimal sampling frequency for your sample using the Nyquist-

Shannon Theorem that states that sampling should be at least 2.3x the resolution. The

resolution is defined as Resolution = 0.61 * wavelength/ NA. Assume you have a 1.4 NA

lens and that green light is coming from your sample at 520 nm. Assume that the axial (z-

axis) resolution should be ~3x the lateral resolution in x and y.

What is the optimal sampling frequency?

x-axis resolution = __________________ x-axis sampling = __________________

y-axis resolution = __________________ y-axis sampling = __________________

z-axis resolution = __________________ z-axis sampling = __________________

9) How can you achieve optimal sampling on your microscope? Are you able to achieve

optimal sampling in the end? __________________________________________________

_________________________________________________________________________

_________________________________________________________________________

10) What filters did you use for your image acquisition? Are these the ideal filters for sensitivity

or for separating the dyes well?

Part 3: Fluorescence photo-bleaching.

1) Using the highest magnification dry objective lens available focus on your fluorescent

sample. Make sure to use minimal excitation light – ND filters in place or low lamp power.

2) Close the fluorescence field diaphragm to about two-thirds of the field of view.

3) Use the highest lamp intensity possible, no neutral density filters, fully open aperture

diaphragm. Leave the shutter open for 2-3 minutes.

4) Reduce the incident light with ND filters and/or lamp intensity controls.


5) Reopen the field diaphragm and look at the sample by eye to determine if bleaching has

occurred. If it did, can you think of ways to minimize bleaching? ______________________

_________________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________

Note: If your system does not have a field diaphragm then simply move the sample partially

out of the field of view and see if you can see the edge of the bleached region.

Part 4: Evaluate the quality of fluorescence illumination in the field of view.

1) Choose the highest magnification dry lens available on your system.

2) Place a plastic slide on the microscope stage.

3) Focus on the sample and observe it with the filter set matching the color of the plastic.

4) Acquire a picture. Use the software to evaluate the intensity variation across the image. An

intensity profile going horizontally across the image can be compared to one going vertically

to one going diagonally. You can also use a rainbow look up table (LUT) to see slight

intensity changes across the field of view.

5) What would be the consequence of such intensity variation on quantitative microscopy?

_________________________________________________________________________

_________________________________________________________________________

6) Try another colour of plastic slide with another filter cube. Do you see the same pattern in the field illumination? Why or why not? __________________________________________

_________________________________________________________________________

7) If needed what can be done to improve on the field illumination uniformity? _____________ __________________________________________________________________________________________________________________________________________________


Part 5: Live cell fluorescence. Onion skin labelled with DiO will give off a green fluorescent

emission. Vesicles in the onion cells will be visibly moving within the tissue.

1) A freshly cut onion skin sample will be provided.

For future reference the sample can be prepared in the following way:

a) Put a drop of DiOC6(3) solution (provided at the working concentration) on a clean

coverslip. Take care not to break the coverslip.

b) Take a piece of freshly cut onion skin of about 5 mm x 5 mm and put the ripped side on

the drop of DiOC6(3).

c) Place the sample on a microscope slide and press down gently.

2) Use the highest magnification dry lens available. Focus on the onion membrane using

brightfield imaging.

3) Choose an area on the sample that is larger than the field of view, and homogeneous.

4) Close the transmitted light path, and switch to fluorescence using the appropriate filter.

DiOC6(3) excitation peak is 484 nm, and its emission peak is 501 nm. Use an ND filter or

decrease the excitation intensity to minimize photobleaching.

5) Can you see the dynamics of the onion cells by eye?

6) If you have a camera based system you can take a short time lapse movie at 5 second

intervals. Are you properly sampling the dynamics at this rate? ______________________

_________________________________________________________________________

_________________________________________________________________________

7) If not try rates that are slower or faster so you can properly see the cellular dynamics.


Lab Exercise 6: Wide Field Live Cell Imaging

Objective: To become familiar with the conditions for maintaining live cells on the microscope.

Perform live cell time lapse imaging on samples prepared in Lab Exercise 1.

Part 1: Live Cell Environment.

1) Look at the live cell incubation system on your microscope if you have one.

2) Identify the components that regulate the temperature. _______________________

___________________________________________________________________

___________________________________________________________________

3) Is 5% CO2 provided to the cells? If so how is it regulated? _____________________

___________________________________________________________________

___________________________________________________________________

4) Is the CO2 humidified when going into the system? If so how? ________________________

_________________________________________________________________________

_________________________________________________________________________

Part 2: Live Cell Imaging.

1) Visually examine your live cell sample from Lab Exercise 1.

2) How is the culture media different from “regular” culture media? ______________________

_________________________________________________________________________

_________________________________________________________________________


3) Why do you think this media is used? ___________________________________________

_________________________________________________________________________

_________________________________________________________________________

4) Put the 63x 1.4 NA oil objective lens (or a similar one) in place. Have the laboratory staff

show you how to put a small drop of oil on the lens.

5) Place your sample on the microscope and locate your cells with brightfield contrast. Make

sure the microscope is aligned for Köhler illumination (see Appendix III if necessary).

6) Use the neutral density filters in the fluorescent light path and/or the attenuation settings on

the lamp power controller to adjust the intensity of the illumination. The live cells are

expressing a green fluorescent protein (GFP) so put the FITC or GFP fluorescent cube into

place. Open the fluorescence light shutter and observe the cells.

7) What is the lowest illumination power you can use and still see the cells? You can turn off

the lights or the computer monitor to have better contrast if you like. ___________________

_________________________________________________________________________

8) Is it good to use a low lamp power? Why or Why Not? ______________________________

_________________________________________________________________________

9) Repeat step 7 with the fluorescence cubes for the blue Hoechst stain and the Red

MitoTracker stain.

10) Set the illumination to a minimal value, bin the camera 2x2 and determine a “good”

exposure time for your three fluorophores. What exposure times did you set? ___________

_________________________________________________________________________

_________________________________________________________________________


11) Take a 5-10 minute time-lapse movie of your cells with 30-60 seconds delay between

frames. Watch your movie.

12) Find another area of your sample and collect another movie using a much higher lamp

power. How does the intensity of your GFP change over time compared to the lower lamp

power? __________________________________________________________________

13) Are the cells as dynamic when you use more light? ________________________________

_________________________________________________________________________

14) Do you see the mitochondria slow down and start to break up and form large aggregates?

_________________________________________________________________________

15) If yes, what is happening to the cells? ___________________________________________

_________________________________________________________________________

WITH THE ASSISTANCE OF THE LABORATORY STAFF CLEAN

THE OIL IMMERSION LENS ACCORDING TO APPENDIX V.

16) If time allows and samples are available try live cell imaging of live brine shrimp. Are the

shrimp sensitive to the light? __________________________________________________

_________________________________________________________________________

17) Try fluorescence and brightfield and various powers of lamp intensity.


Lab Exercise 7: CLSM Live Cell Imaging

Objective: To become familiar with the conditions of maintaining live cells on the microscope

platform. Perform live cell time lapse imaging on a confocal microscope. Explore

sampling conditions for confocal laser scanning microscopy (CLSM).

Part 1: Live Cell Environment.

1) Look at the live cell incubation system on your microscope if you have one.

2) Identify the components that regulate the temperature. _____________________________

_________________________________________________________________________

_________________________________________________________________________

3) Is 5% CO2 provided to the cells? If so how is it regulated? ___________________________

_________________________________________________________________________

_________________________________________________________________________

4) Is the CO2 humidified when going into the system? If so how? ________________________

_________________________________________________________________________

_________________________________________________________________________


Part 2: Live Cell Imaging. 1) Visually examine your live cell sample from Lab Exercise 1.

2) How is the culture media different from “regular” culture media? _______________

___________________________________________________________________

___________________________________________________________________

3) Why do you think this different media is used? ______________________________

___________________________________________________________________

___________________________________________________________________

4) Put the 63x 1.4 NA oil objective lens (or a similar one) in place. Have the laboratory staff

show you how to put a small drop of oil on the lens.

5) Place your sample on the microscope and locate your cells with brightfield contrast. Make

sure the microscope is aligned for Köhler illumination (see Appendix III if necessary).

6) Use the neutral density filters in the fluorescent light path and/or the attenuation settings on

the lamp power controller to adjust the intensity of the illumination. The live cells are

expressing a green fluorescent protein (GFP) so put the FITC or GFP fluorescent cube into

place. Open the fluorescence light shutter and observe the cells.

7) What is the lowest illumination power you can use and still see the cells? You can turn off

the lights or the computer monitor to have better contrast if you like. ___________________

_________________________________________________________________________

8) Is it good to use a low lamp power? Why or Why Not? ______________________________

_________________________________________________________________________

9) Repeat step 7 with the fluorescence cubes for the blue Hoechst stain and the Red

MitoTracker stain.

10) Examine your sample and choose a field of view for imaging.

Note: In general with CLSM you do not need to look at your cells with the fluorescence lamp. You should find your cells only with brightfield and then optimize the fluorescence settings within the confocal software. So steps 6-9 can be omitted.


11) Using the confocal software and the help of the laboratory staff collect and save an image of

the green fluorescence (paxillin-EGFP) with the following settings:

1024x1024 pixels Zoom factor = 2 488 nm laser power = 2-3%

PMT gain = 800 Pinhole = 2 Airy units Scan speed = 5

12) How does the image of the cells look? __________________________________________

_________________________________________________________________________

13) Go to zoom 10, put the PMT gain to 600 and put the 488 nm laser line power to 20% and

take 10 consecutive images. You can use the continuous feature for this.

11) Go back to zoom 2, PMT gain of 800 and collect and save a new image. How does this

image compare to the one you took in step 11? ___________________________________

_________________________________________________________________________

12) With the help of the laboratory staff set up the instrument settings for three colour imaging of

the Hoechst, paxillin-EGFP and MitoTracker Red using sequential scanning (one image at a

time will be collected). If you have the option use sequential line scanning. The laboratory

staff can explain to you what this means.

13) Collect and save the three images of the fluorescent dyes in your cells.

14) Collect and save another set of images but this time use the simultaneous settings collecting

all three images at once.

15) Compare the two image sets. What do you see and why? ___________________________

_________________________________________________________________________

16) Is it better to use sequential or simultaneous imaging? Why? ________________________

_________________________________________________________________________


17) Can you think of a situation when you could benefit from using simultaneous imaging?

_________________________________________________________________________

_________________________________________________________________________

18) With the help of the laboratory staff take a short 5-10 minute time-lapse movie of your cells

at 30-60 second intervals. If you like you can use the ROI tool to crop out just a few cells to

speed up the acquisition and make the image files smaller. Watch your movie. What do you

see? _____________________________________________________________________

_________________________________________________________________________

19) Find the top and bottom of your sample and collect a z-stack of images with 0.5 μm between

image slices. How long does it take to collect one image stack? ______________________

20) Would this be practical for time-lapse imaging? ___________________________________

21) Name at least three ways you could speed up the acquisition. ________________________

_________________________________________________________________________

_________________________________________________________________________

Part 3: Confocal instrument settings.

1) Find a fresh field of view. Collect and save an image of the MitoTracker Red using the same

settings as in Part 2, step 11 except use the 543 nm laser line at 30% power.

2) Collect and save two additional images with all the settings the same except with the pinhole

set to 1.5 and then 1 Airy unit. Is there any difference between the images? _____________

_________________________________________________________________________


3) Are there any features you can see with the smaller pinhole that you cannot see at 2 Airy

units? ____________________________________________________________________

_________________________________________________________________________

_________________________________________________________________________

4) Put the pinhole back to 2 Airy units. Take images with the PMT gain at 800, 750 and 700.

Compare the images. As you decrease the gain what happens to the signal? What happens

to the noise? ______________________________________________________________

_________________________________________________________________________

5) Find a new field of view. Keep the PMT gain at 700. Take images with the laser power at

20%, 40%, 60%. Compare the images. What happens to the signal? __________________

_________________________________________________________________________

6) What happens to the noise? __________________________________________________

_________________________________________________________________________

7) Are there features you cannot see at 20% power that you can see at 60% power? ________

_________________________________________________________________________

_________________________________________________________________________

8) Put the PMT gain back up to 800. Collect and save an image with 1024x1024 pixels with a

zoom factor of 2. Collect and save a second image of the same field of view with 512x512

pixels with a zoom factor of 1. Displaying the images 1:1 how do they compare? _________

_________________________________________________________________________

_________________________________________________________________________


9) Digitally zoom in on the images. Now how do they compare? ________________________

_________________________________________________________________________

10) Take a small z-stack of 10 images using the settings in step 1 with 1 μm spacing between

slices. At 2 Airy units what is the thickness of your z-slice? __________________________

_________________________________________________________________________

11) According to Nyquist theory, is the 1µm spacing thin enough to accurately sample your z-

slices? (Refer to the equations in Lab Exercise 4 if needed). _________________________

_________________________________________________________________________

_________________________________________________________________________

12) Take another z-stack of 40 images with 0.25 μm spacing between slices. According to

Nyquist theory, is the 0.25 μm spacing thin enough to accurately sample your z-slices?

_________________________________________________________________________

_________________________________________________________________________

13) Compare the two stacks of images with different z resolution with the orthogonal viewer.

Digitally zoom in to see the resolution differences. How do these z-stacks compare?

_________________________________________________________________________

_________________________________________________________________________

WITH THE ASSISTANCE OF THE LABORTORY STAFF CLEAN

THE OIL IMMERSION LENS ACCORDING TO APPENDIX V.


Laboratory Exercises 6 and 7 Follow Up

Platform Comparison between wide-field and confocal laser scanning microscopy

1) Which system has the most straightforward software? WF CLSM

2) Which system produces images the fastest? WF CLSM

3) Are the cells more dynamic on one platform? YES NO

a. If so which one? WF CLSM

4) Does one system give better image quality over the other? WF CLSM

Any other comments about the two platforms?

____________________________________________________________________________

____________________________________________________________________________

____________________________________________________________________________

____________________________________________________________________________

____________________________________________________________________________


Appendix Index

Appendix I: Fluorescence Labelling Reagents 35

Appendix II: Microscope Schematics

Axiovert 200M Transmitted Light Components 36

Axiovert 200M Fluorescence Components 37

Axioscope A1 38

Axioskop 2 Motorized 39

Axioskop 2 Manual Transmitted Light Components 40

Axioskop 2 Manual Fluorescence Components 41

AxioLab A1 42

Appendix III: Köhler Alignment 43

Appendix IV: DIC Alignment Protocol 45

Appendix V: Oil Immersion Lens Cleaning Protocol 47

Appendix VI: Live Cell Imaging Summary 48


Appendix I: Fluorescence Labelling Reagents Product Company Catalogue # Notes

DMEM Invitrogen 11885-084 low glucose, L-glutamine, 110 mg/ml sodium

pyruvate, pyridoxine hydrochloride

DMEM phenol red free

Invitrogen 11054-020 Same as above, but no phenol red and you

need to add L-glutamine.

Non-essential amino acids

Invitrogen 11140-050 Add 5 mL of stock to DMEM

Penicillin-Streptomycin (Pen/Strep)

Invitrogen 15140-122 Add 5 mL of stock to DMEM

L-Glutamine Invitrogen 25030-081 Add 5 mL of stock to DMEM

Fetal Bovine Serum (FBS) Invitrogen 26140-079 Add 50 mL to DMEM

Geneticin (G418) Invitrogen 11811-031 Add to DMEM for stable GFP expressing

cells. 2.5 mL of 100 mg/mL stock/bottle for 0.5 mg/mL.

PBS- Invitrogen 70011-044 10X solution ph 7.4

Paraformaldehyde (PFA) Polysciences 50-00-0 Specify 16%, dilute 1:4 for 4%. Always work under Fume Hood.

Coverslips Fisher 12-544A Any form but thickness #1.5

Triton-X-100 Fisher BP151-100 Very viscous, use % v/v

Bovine serum albumin (BSA)

Jackson Immuno Research

001-000-162 IgG-free, Protease-free.

Drop on top of liquid (PBS), dissolve by gravity. Avoid shaking. Use % w/v.

Phalloidin-Alexa555 Invitrogen A34055 Use at a 1:1000 dilution.

DAPI (dilactate) Invitrogen D3571 5 mg/ml stock, 1:5000 dilution (1 μg/ml)

Can be added during 2o Antibody or phalloidin labelling steps.

1o Mouse monoclonal anti-alpha tubulin

Sigma T9026 Use at a 1:200 dilution.

Cytoseal 60 Fisher 23-244-256 To get a nice small drop use the end

of a wooden cotton tip applicator. Tubulin 2o antibody

AlexaFlour546 goat anti-mouse

Invitrogen A11003 1:2000 dilution of molecular probes stock

MitoTracker Red CMX-Ros

Invitrogen M7512 Make a 1 mM stock. Use at 2-100 nM.

Hoechst 33342 Sigma-Aldrich 14533 2.5 μg/mL

1:400 dilution of 1 mg/mL stock


Appendix II: Microscope Schematics Axiovert 200M


Axiovert 200M

Fluorescence Components


Axioscope A1


Axioskop 2 - Motorized


Axioskop 2 – Manual

1) Transmitted light intensity adjustment. 2) Course and fine focus adjustments. 3) Condenser turret. 4) Field aperture. 5) Condenser aperture adjustment. 6) Objective lens turret.


Axioskop 2 – Manual Fluorescence Light Path

1) Fluorescence filter turret. 2) HBO fluorescence lamp. 3) Aperture Diaphragm. 4) Field Diaphragm. 5) ND Filter Slider. 6) Fluorescence Shutter.


AxioLab A1


Appendix III: Köhler Alignment Köhler alignment should be performed every time you change your sample or the microscope objective. With proper alignment, transmitted light imaging will require lower intensity illumination light and the images will show better contrast. Note the double image and the poor colour reproduction due to mis-alignment in the images below.

1. Choose the desired objective lens. It is often best to start with 10x and then fine tune the alignment on higher magnification lens.

2. Place the sample on the microscope and focus on it.

3. Close the Field Diaphragm by

moving the lever towards the back of the microscope.

4. Move the Condenser Focus Knob so that the field diaphragm is in focus in the eyepieces.

5. Use the centering screws on the condenser to position the field diaphragm in the centre of the field of view.

Misaligned Aligned


6. Open the field diaphragm so that it just touches the edges of the field of view.

7. Use the centering screws to fine tune the

alignment so that the field diaphragm in the centre of the field of view.

8. Open up the field diaphragm until it is just slightly larger than the field of view.

9. For high magnification high resolution lenses (NA higher than 0.7) make sure the field aperture is set to the maximum open position (NA = 0.55).

10. For high NA objective lenses (NA > 0.7) set the condenser aperture to the maximum setting

of NA = 0.55. For lower NA lenses set the aperture to ~80% of the NA for the objective lens. For example for a 0.5 NA lens the condenser aperture is set to 0.4. You can remove the eyepieces and look down the oculars to make sure the aperture is filling ~80% of the back aperture of the objective lens.

***** Köhler alignment should be performed EVERY TIME you change your sample or the microscope objective. *****


Appendix IV: DIC Alignment Protocol

1. Choose the desired objective.

2. Place you sample on the stage and focus on the specimen

using a DIC equipped objective. DIC capability will be indicated on the objective casing. A DIC objective will also have an objective-side prism mounted into the turret directly underneath the objective.

3. Follow the Köhler Illumination procedure to make sure that the condenser is properly aligned.

4. Swing out the condenser side polarizer.

5. Use the Insert Selector Keys on the condenser to select brightfield mode (BF

on the LED display). 6. Slide out the objective-side prism.

7. Slide out the analyzer.

8. Remove one or both of the eyepieces so

that you can see the back focal plane of the objective.


9. Insert the polarizer and analyzer into the beam path. The image of the specimen should appear black and a Maltese cross should appear in the back focal plane.

10. Insert the objective-side prism into the objective turret. You should see a brightfield image of the specimen in the image plane and a 45° angled black interference fringe in the back focal plane.

11. Adjust the prism translator on the prism to center the

interference fringe in the back focal plane.

12. Use the Insert Selector Keys to move the correct

condenser-side prism into the beam path. The appropriate condenser DIC prism should be indicated on the objective lens and on the back side of the objective lens prism. Typically DIC III for 63x and 100x (NA 1.4) and DIC II for 10x-40x.

13. The specimen should appear very dark and highly contrasted. A Maltese cross should re-appear in the back focal plane of the objective.

14. Slightly adjust the objective-side prism translator until you see the Maltese cross split apart and a grey image of your specimen with nice contrast. Depending on the direction you turn the prism translator the sample will appear to be coming out of the sample or going into it.

I M P O R TA N T .

Widefield The analyzer underneath the objective should be in place.

The polarizer on the excitation lamp side above the condenser

should be set to 0o.

LSM Confocal

The analyzer underneath the objective should NOT be in place.

The polarizer on the excitation lamp side above the condenser

should be set at 90o.


Appendix V: Oil Immersion Lens Cleaning Protocol Materials required:

• Lens paper • Lens cleaner • De-ionized water

1. Obtain a sheet of lens paper and fold it into a rectangle as shown. Only touch the edges of paper or the oil from your fingers could transfer to the wiping surface.

2. Holding the lens paper wipe the excess oil from the objective three times by dragging the lens paper from the back of the stand towards you. Never put any pressure with your finger near the centre of the lens. Move to a fresh surface of the lens paper with each wipe.

3. Discard the used lens paper.

4. Add lens cleaning solution to a fresh piece of folded lens paper and repeat steps 2 and 3.

5. Add de-ionized water to a fresh piece of folded lens paper repeat steps 2 and 3.

6. Obtain a fresh dry piece of lens paper and repeat steps 2 and 3.


Appendix VI: Live Cell Imaging Check List

1) What will I grow my cells in?

a. Plastic is fine if you have long working distance lenses and/or low magnification.

b. Glass bottom dishes, specialized plates, coverslip holder for high resolutoin.

2) Is my media buffered?

3) Can I provide 5% CO2? 4) How can I minimize the fluorescence lamp intensity?

a. ND filters b. Low power settings (if availalbe) c. UV filters d. IR filters e. Turn off the room lights f. Dark adjust your eyes

5) How can I minimize the light exposure to my cells?

a. Avoid blue dyes

b. Minimum number of dyes

c. High NA lenses.

d. Widefield

i. Reduce exposure time ii. Bin the camera

e. Confocal

i. Large pixels ii. Fast scanning iii. High PMT voltage iv. Open the pinhole (~ 2Airy units)

Microscopy Imaging Analysis

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miacellavie.com

OUR VISION

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EQUIPMENTPlanning and consulting on appropriatetype of microscopy technique, matchingthe needs of the project. Well maintained,tested, calibrated and operated equipmentto perform at maximum potential.

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OPERATORTrained and educated operator to ensurecompetent use and protection of equipment.Operator knowledgeable and aware of benefitsand limits of sample, system, software andprocessing/analysis methods.

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McGill Life SciencesComplex Imaging Facility

www.lifesciencescomplex.mcgill.ca/imaging

IFmaging

acilityIF

maging

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Image & distinguish multiple dyes of similar colour. To distinguishing specific fluorescence from autofluorescence.

T E C H N I Q U E S1) Spectra Imaging (Zeiss‐LSM 510 Meta)2) Total Internal Reflection Fluorescence (TIRF)

(Olympus) IX‐81)3) Fluorescence Correlation Spectroscopy (FCS)

(Zeiss‐ Confocor2)4) Laser Micro‐dissection, Catapult & Capture (Zeiss‐PALM)5) FRET and FRAP and Photoactivation (Zeiss‐LSM 510 Meta)6) High Content Screening (HCS) Automated Imaging

S P E C T R A L I M A G I N G

Removal of auto-fluorescence contributions.

Separate Similarly Coloured Dyes

AlexaFluor® 568 – Actin SYTOX® Orange – Invitrogen Fluorescence

Autofluorescence & GFP GFP

Autofluorescence OverlayEmission Spectra

Image within ~100 nm of the sample coverslip. Adhesion, extracellular matrix (ECM), endocytosis and exocytosis.

TIRFWidefield

6) High Content Screening (HCS) Automated Imaging (MDS –ImageXpress Micro)

7) 3D Image Deconvolution (AutoQuant, Zeiss Axiovision )8) Image Analysis Workstations (MetaMorph and Imaris)9) Live Cell Imaging (LSM, TIRF, Widefield) T I R F M I C R O S C O P Y

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Real-time estimation of chelatable iron in the mitochondria

gNucleus, Mitotracker® Red CMXRos ‐Mitochondria

gSpectra Viewer Sample courtesy of Mr. Yu Lu, Richard Roy Lab, McGill University

C. Elegans ‐ Autofluorescence, Spinal‐defect 2‐EGFP, (SPD‐2) in centrosomeof germline cells.

Paxillin-EGFPTime-lapse of Cell Division

Green = Alexa 488 TfSheftel, A.D., A.S. Zhang, C. Brown, O.S. Shirihai, and P. Ponka. 2007. Blood. 110:125-32.

Red = RDA – iron sensitive mitochondrial dye

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html

Widefield

Restorative deconvolution of widefield and confocal image stacks to improve resolution and signal‐to‐noise.

0 min 15 min 30 min25 min 40 min 55 min

F R E T Primary Hippocampal neurons

F R A P (Fluorescence Recovery After Photobleaching)Paxillin-EGFP

32 cnt A U T O M A T E D

I M A G I N G

Reagent Titration

D E C O N V O L U T I O N

Wide-fieldContrasted Wide-field Deconvolution

Wide-field Deconvolution

1

Automated imaging and analysis of cells on multi‐well plates. Analysis for counting, cell

cycle, mitotic index, translocation, etc.

Fluorescence CorrelationSpectroscopy (FCS)

Raichu-WT RacFRET signal goes upwhen Rac is active.

Zhang et al., J. Neuroscience 25:3379, 2005

Control ∆F508-GFP CFTR-GFP

DAPI

Anti‐GFP

DAPI

Actin

3D Iso-surfaces – Imaris Software

WGA

1) 96 or 384 well plate imaging.2) Image on glass or plastic.3) Up to 5 colours at once.4) Fully Automated.5) Chemical Library or RNAi Screening.

Mitochondria

ConfocalRaw Data

Deconvolved Data

Histone‐GFP

Bates et al., Biophysical J., 91:1046 2006.

Focal Volume CFTR‐EGFP

Measure molecular (i.e. protein, lipid) dynamics in the cell.

Ms. Jacynthe LalibertéMicroscopy Technician

Dr. Dongmei ZuoMicroscopy Specialist

Mr. Aleks SpurmanisMicroscopy Specialist

Dr. Claire M. BrownDirector

S T A F FCFTRDye in Solution

Time (ms)

G(τ)

Time (ms)

G(τ)

D = 33 ± 0.07 μm2s‐1 D = 0.17 ± 0.05 μm2s‐1

Introduction to Light Introduction to Light Microscopy

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