Lab Manual for 2D and 3D Confocal Microscopy€¦ · Grove Research Lab Lab Manual for 2D and 3D...

Grove Research Lab Lab Manual for 2D and 3D Confocal Microscopy

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Lab Manual for 2D and 3D

Confocal Microscopy

LSU’s Shared Instrumentation Facility provides the option to use the Leica TCS SP8 Confocal

microscope. To operate this instrument, follow the steps mentioned below sequentially:

1. Online log-in: To operate the Leica SP8 Confocal microscope, reserve the instrument using

LSU’s Shared Instrumentation Facility services website. For the first timers, sign up is required

on SIF web portal using MyLSU account credentials, after which you would be assigned an

iLab account and password. Without proper reservation, either online from home or using

the reservation console provided within the room, the instrument as well as the computer

system will not start.

2. Filling the log-in sheet: When you are done with the online reservation, write down all your

required details and the condition of the microscope on the log sheet folder provided by the

side of the microscope. Filling this sheet with your actual active time on this instrument can

also prevent you from extra charges (calculating online reservation hours) if you worked for

less time than expected.


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Please report any anomaly you find with the microscope or computer or anything that can be

a problem for you or the next user while using the microscope. Please report any defect you

find to help solve the problem as soon as possible. Also, write down the status of the mercury

lamp, whether on/off, and if ‘on’ already, write down the time mentioned below the

rotatable knob on the mercury lamp.


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3. Switching on the microscope and its console: After filling up the log-in sheet, remove cover

of the microscope and start switching on the instrument console one-by-one in the respective

sequence labeled on each switch/key. 1) Fluorescence Lamp, 2) Computer and Hardware, 3)

Confocal Scanner, 4) Lasers, and 5) Another laser line (looks fancy like key starting a Car). Now

wait for the Computer’s booting process to finish.


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4. TCS User log-in: Use TCS User password to log-in (currently: microscopy).

You can find the password written on a sticky note kept on the CPU.

5. Start Leica Application Suite X (LasX): Double-click on the LasX applet icon on the Desktop to

start the console.


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While starting, it will pause at Configuration check step. Confirm that the settings of

instrument are configuration: machine.xhw, excel: w, microscope: DMI8, resonance: off, and

knob setting at startup: off. Click ‘OK’ to proceed. If any of these values are different than

mentioned above, change them as described above.

6. Microscope stand initialize stage: Instrument console while starting up, asks “Microscope

Stand initialize stage DMI8 Stage?”, to which you respond “Yes”. You can now see Stage

initializing on itself by moving mechanically.


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In case of any error, the instrument will provide the cautions at this step and won’t start.

7. Instrument console settings: Go to “Configuration” tab on the Top left of screen and do the

following


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a. Set up Hardware: Change “Resolution Bit-depth” to 16 bits or according to your

needs, higher number of bits represent more number of pixels involved per unit

area of image captured. Thus, higher the resolution depth, the more detailed the

picture would be.


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b. Set up Objective lens: Select the appropriate objective lens for your work.


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c. Set up Laser configuration: Turn ‘On’ all the options 1) 405 Diode, 2) Argon, and

3) WLL (White Light Laser). Also, change the setting of “Argon laser” from Zero to

70%.

Now go to “Acquire” tab right by the side of “Configuration” to get back to the screen where

you can observe whatever is in the field of vision.


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8. Set microscope’s controller for Lenses and Lights: Go to the control panel at the bottom of

the microscope and Click on “Objective lens” and choose the appropriate objective lens as

selected in the console before. In this controller, while choosing the objective lens, be mindful

that the “drop” symbol means immersion needed. Options in the upper row provides Oil

immersion lenses and lower row provides Water immersion objective lenses.

9. Verification of Objective lens: Gently Lift the upper part of the microscope body and confirm

that the objective lens in place is the same one as mentioned in the controller below. Though

it is not advisable, you can also unscrew the lens and take it out to confirm if absolutely

required.


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10. Check lens settings: Check the lens spring for its proper working condition by pressing it down

3-4 times. This must be functional for proper absorbance of force that might be applied when

the mechanical stage moves to adjust its position on the lens cover. If the lens spring isn’t

functional, it may result in unexpected force applied and breakage of the cover glass during

adjustment.

Also, there is a rotatable ring on the neck of the lens. Rotate and adjust the ring for

appropriate cover glass thickness to be used. For using the cover glass of designated thickness

1 & ½ (make sure you use this thickness of cover glass only for best images), the ring should

be adjusted to 0.17. This setting will allow using 0.1 mm thick cover glass for the samples.


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Finally, verify that there is no scratch or residual immersion fluid from the last user on the tip

of the microscope. If there is, then use the specific lens paper provided to clean the lens. Do

not use the Kim wipe or any other paper for cleaning the lens.

Those may leave paper fibers on the lens.


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11. Ensure proper immersion liquids: In case you must use immersion liquid, make sure to use

De-ionized water for Water immersion lenses, and make sure to get “Type F immersion oil”

for the Oil immersion lenses. Make sure to retain the same service provider for Oil as for the

Microscope. In that case, we must use “Leica Type F immersion oil”.

12. Start setting up the sample: Apply immersion liquid on the Objective lens, use enough to let

it cover the entire surface of the lens, but not so much that it drops into the instrument. Using

too little may affect the refraction while moving the stage and the lens reaches dry surface.

Now place your sample on the slide with appropriate media/mounting liquid.


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Cover the sample with 1 & ½ cover glass and place the slide on the stage. Make sure to place

the slide upside down because the objective lens faces upward. Adjust the slide holder to fix

the slide in position.

13. Adjust slide position to make contact between lens and slide: To do so, there is a manual

controller towards the right side of the microscope, which is attached to the mechanical

controllers of the microscope and gradually moves the stage and objective lens position. It

has four small buttons designated as z-coarse, z-fine, xy-fast and xy-precise to move the stage

over z-axis fast & slow and xy-axes fast & slow, respectively.


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Dials designated for XY-axes have two parts, the upper dial moves Y-axis and the lower dial

moves X-axis. Moving Z-axis dials will adjust the lens position accordingly. Adjust it in a way

to touch the cover glass. Reposition the upper part of the Microscope back to its normal

position.

14. Transmission light and sample position adjustment: Press the icon of Colors on the control

panel within Microscope just above the Objective lens symbol. As you click the symbol, you

will see three categories, a) Transmitted, b) Incident, and c) Combined. Click DIC within

Transmitted category and the eye piece shutter will be switched on and light will be directed

through the eye pieces.

You can observe the sample through the eye piece and can adjust its position at this point.

This is one of the most crucial steps and once you are ready with your sample’s position to

be analyzed, reclose the Transmitted Light Shutter by clicking the TL-Shutter option on the

control panel.


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15. Adjustment of Incident Light properties: Click on the Incident Light category on the panel

and you will be given 4 choices of Filter Cubes to choose from i.e. a) DA/FI/TX, b) DAPI, c) GFP,

and d) DSRED. If you have the exact filter cube requirement as mentioned in the options it

would be great, but even if not, you can proceed with the closest available option that suits

your requirement.

When you are done with choosing your filter cube, switch on the Incident Light Shutter too

by clicking on the IL-Shutter icon.


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16. Laser settings: The first time, it is a must to set Laser in accordance with your requirements.

Once set, you can save settings as user defined laser settings and you won’t need to set it

back again. For instance, in this case we will use the White Light Laser. And then you will have

to choose the dye assistant closest to your fluorescent probe’s tag. For instance, set it

according to the Yellow Fluorescent Protein. On clicking the Dye assistant icon, you will be

asked to make a choice between PMT and HyD.


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These two choices are techniques to amplify signals using Photomultiplier Tube or Hybrid

Detectors. Let us choose HyD this time.

And now, make an appropriate choice of your Dye you are using within your sample. You may

need to enter the Excitation and Emission Spectra manually if you are not provided an

appropriate choice of dye.


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Now, select the spectrum of excitation and emission and switch on the HyD gating icon. A

part of this will be set automatically for emission spectra (511nm to 778nm). If you see above,

Laser power in use from the WLL is 1% of its power. Don’t use too much Laser power as it

may Photo bleach the samples.

17. Observe sample and adjust visual properties: Press Live icon at the bottom left of the screen

to start observing your sample.


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Now, you might be able to see the image very dimly.

At this step, adjusting SmartGain is important. Settings of SmartGain and Laser power usage

is crucial at this step. Since you cannot increase the Laser power too much to prevent

Photobleaching, increasing sensitivity towards photon signals can accomplish the task. For

that, we can use the SmartGain knob and adjust it according to our need and suitable image

quality. Since we are using HyD SmartGain, don’t increase SmartGain percentage over 100 to

avoid background signals.


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If you can’t see your image with 100% SmartGain, you can use the leverage of increasing Laser

power a bit because it had been 1% till now. Even in this case, you must not increase Laser

power more than 5% to avoid any chances of Photobleaching. Most Biological samples are

photobleached by the time laser power reaches 25%.


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18. Color selection and image capturing: By now, the sample appears in Gray shade.

To visualize colorful images/ fluorescent signals, double-click on the color symbol over HyD

option.

Select color (yellow for currently running situation) from the options and all the samples will

turn yellow.


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Set saturation color intensity as well so that you can identify the saturation level. This color

will be representing the highest intensity of fluorescent signals.

In case of using Chlamydomonas (since we are using it as a sample in this case), what we see

is mostly Chlorophyll because it has a wide range of excitation. Moreover, default excitation

and emission spectra for eYFP is suitable to excite chlorophyll. To resolve this, narrow down


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the emission spectra using HyD range of sensitivity for higher wavelength. Restrict longer

wavelength between 511nm-640nm and increase laser power to 5% and set SmartGain to

100%.

You might not be able to see anything at this point; for that purpose, click on TLD at the

bottom of all laser and sensor option. You will be able to see PMT (Photo Multiplier Tube)

option; since signal photons captured are for a very narrow range of wavelength, they might

not be detected by the sensors.


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PMT helps in amplifying signals, so switch it on. You will see two windows; increase the PMT

Gain using SmartGain knob below for the bottom screen. One thing to observe in SmartGain

for PMT is Voltage instead of HyD.

Increase it until you see the sample. To observe a region, turn on Zoom in option and select

the area to be observed known as ROI (Region of Interest).


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Go Live and select Overlap to superimpose the signals from different samples.

And you can observe both signals overlapped in a single screen. Adjust YFP signals using Laser

Power (adjusted to 10%) to see it more efficiently and adjust to lower down the PMT

SmartGain because it will Go up with increased Laser Power. Your Image is ready to be

Captured.

19. Capturing Image and format settings: Adjust Pixel size of image format according to your

need by choosing from the options provided in the window on the upper left screen. The,

higher the pixel size, the greater amount of time it will take to capture the image. With live


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samples, capturing high resolution may pose a problem of distorted images because if the

sample moves while scanning is on (because of its live status) it would create distortions.

Now go and save the image in E:\ Drive using Project option. Change its name for the sake of

good practice to not lose the file.

This is done for 2D image


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3D-imaging starts here

20. Set up 3D imaging properties: To start 3D-imaging we need to set the ‘acquisition mode’ for

xyz-axes. You will see various options including all the axes for 3 dimensions of space as well

as dimension ‘t’ for time. The latter option can be used for Time-lapse imaging to track the

path of protein movements within the cells. You may also use several wavelengths and time

at once to track movement of proteins excited by different wavelengths of light using Δ- and

γ-options in combination with axes options.


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21. Setting up Z-stack and step size: Switch on ‘Begin’ and ‘End’ options to enter coordinates you

want to cover horizontally with respect to the point of focus as ‘Zero’. Feeding the coordinate

data is important as we must keep at least 2.5µm of margin at both top and bottom edges.

Besides the total length of the Z-stack, we also need to set the number steps we want the 3D-

image to be captured in. Note: Number of steps will help the software to calculate step-size

for each stack. This step is pretty much automated, but you may need to adjust the speed of

stack capturing because a larger number of steps may take more time and thus proportionally

increases the chances of the 3D-image being distorted for live samples as they are moving.


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A smaller number of steps would result in larger step size and thus compiling the 3-

Dimentional feature at a low resolution.

22. Capture image and compile 3D image: Click “Start” to initiate image capturing and it will

capture the stack images one by one starting from the upper edge coordinate to the lower

edge coordinate.


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Once all stacks are captured, click “3D” on the side tab to compile the image in 3-dimensional

form and your image is ready to analyze.

23. Save project: As described for 2D image projects, save the 3D image using any name

convenient to you.


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24. Log-off and Sign out after switching off the complete system: You may now close the

application and shut down the computer system. Also, switch off each part of the instrument

in reverse order one by one, but not the laser system. Allow some time to let the laser cool

down (at least 10 minutes) and then switch it off as well. In case you know that another user

will use the microscope immediately after you, leave the laser on. Remember that Sign-up

sheet we filled in our signing in detail before starting the system? Now, fill in your log out

details in that sheet and write the current reading on the mercury laser source. Log-off from

the online web server as well and you are done with all the responsibilities towards the

Confocal microscope.

Lab Manual for 2D and 3D Confocal Microscopy€¦ · Grove Research Lab Lab Manual for 2D and 3D...

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