BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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STANDARD OPERATING PROCEDURE:

FLUROSENCE MICROSCOPY (LEICA)

Figure 1. Leica fluorescuence microscope DM6000 in 381 CRL

Purpose of this Instrument: It is an important optical tool to monitor dynamic cellular processes in living

specimens or fixed sample slides.

Location: WVU – Chemistry Research Laboratory Building – Room: 381

Primary Staff Contact: Dr. Huiyuan Li (304) 293-0747

The Shared Research Facilities are operated for the benefit of all researchers. If you encounter any

problems with this piece of equipment, please contact the staff member listed above immediately. There is

never a penalty for asking questions. If the equipment is not behaving exactly the way it should, contact a

staff member.

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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1) INITIAL CHECK

This text is a guide to operating the Leica fluorescence microscope DM6000. A more detailed and thorough

account of these procedures can be found in the Leica DM6000 B Operating Manual.

1. Log in your “BNRF CRL Fl scope/plate reader” session on the FOM. (fom.wvu.edu/fom)

2. Check log sheet to see the notes of previous users. This will inform you about anything unusual that

occurred before.

3. Write down your name, date, start/end time, sample type, and etc on the log sheet

2) START-UP

1. Turn on the main power switch (Figure 2).

Figure 2: Microscope main power switch

2. Wait for one minute, or until the green light above the main power switch turns on.

3. Turn on the microscope, making sure the shutter open light is on.

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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Figure 3: Microscope power switch and shutter open light

3) OPERATION

4. Double click the program icon on the desktop (Figure 4). The instrument will perform an initial check

while the program loads.

Figure 4: Microscope program desktop icon

5. Place sample on the proper sample holder on the stage of microscope, with the sample side down

(Figure 5).

Shutter open light

Shutter switch

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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Figure 5. Place sample on sample stage.

6. Active “Acquire” tap on the top of the tool bar. And click on “Mic1” (Figure 6)

Figure 6: Microscope program

7. Use the low magnification lens (5 ×) and bright field (BF) first to find the area of interest and focus.

You can view the LIVE image window on the screen in the right side to do the adjustment. If the light

intensity is too high, you decease the light intensity by turn the light intensity wheel (Figure 7).

Light intensity adjustment

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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Figure 7. Microscope program

8. You also can use the Auto exposure to find the best light intensity (Figure 8).

Figure 8. Auto exposure

9. If the color of the entire picture is not correct (like yellowish), select an area in background of your

picture, right click and choose the “white balance”. The color of your picture will be automatically

adjusted (Figure 9).

Figure 9. White balance the image.

10. Then use the stage position control to move the sample stage and find the area of interest (X and Y

adjustment) (Figure 10).

Auto exposure

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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Figure 10. Sample stage X and Y adjustment.

11. On the right side of the microscope, there is adjustment nob. Click “Z coarse” first to do the basic

adjustment. Then click “Z fine” to do the fine focusing (Figure 11 and 12).

Figure 11. Sample stage Z direct adjustment

Height adjust nob

Fine adjustment switch

Coarse adjustment

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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Figure 12. Microscope screen shows the current height and adjustment mode (coarse).

12. After you get clear image of the area of interest, you can click “FLUO”, then choose correct filter for

your sample/dye(s) (Figure 13 and table 1)

Warning: Wear UV safe goggles at all times when using UV light. (ANSI 287+ UV proof

plastic).

Figure 13. Choose FLUO and lens filter for fluorescence imaging.

Choose filter

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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Table 1. Range of filter tubes installed.

Filter cube Excitation range Excitation filter Dichromatic

mirror

Suppression filter

A4 UV BP 360/40 400 BP470/40

L5 Blue BP 480/40 505 BP527/30

Y3 Green BP 545/30 565 BP 610/75

Y5 Red BP 620/60 660 BP 700/75

13. Click “Camera” bar on the top. You can adjust your image with exposure time, Gain, Saturation, and

Gamma based on your need (Figure 14).

Figure 14. Fluorescence exposure adjustment.

14. After image adjustment, click “Acquire Image” at the bottom of the control window to take photo

(Figure 14). And save the image in your folder. Some examples are shown below (Figure 15).

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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Figure 15. Examples of fluorescence images. (Slide information:

15. If you want to take overlay picture, select acquisition mode as “image overlay”, then click the “λ” on

the tool bar (Figure 16). Select the filter that you want to include. You still can make exposure

adjustment for each single filter. Then click the “Acquire overlay” to take the overlay picture. It will

save pictures with each single filter and an overlay picture. The example of overlay picture is shown

below (Figure 17).

Click and choose acquisition mode as “image overlay”

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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Figure 16. Choose acquisition mode.

Figure 17. An example of overlay image.

16. After image is saved, you can use the “Process” function to further modify your images, such as

color adjustment, adding scale bar, or image stitch. Please check the “Leica Application Suite LAS

V2.7, LAS user manual” for the image processing. You can find the manual book on the desk beside

the microscope.

3) HIGH MAGNIFICATION IMAGING

1. After focusing the microscope as described above, remove the microscope slide under the bright

field (BF) light and select the 63x objective on the computer program.

2. Apply a small drop of immersion oil with a sterile cotton swab to the microscope lens. Do not use too

much, only a very small amount to just barely cover the lens (Figure 18).

Figure 18: Applying immersion oil to microscope lens

3. Replace the sample on the top of the objective and be sure that the immersion oil and the sample

are touching so that the immersion oil seals the gap between the microscope slide and the lens

(Figure 19).

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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Figure 19: Slide placed on microscope with immersion oil

4. At this time the microscope can be changed from bright field to fluorescence and pictures can be

taken.

5. After a microscope slide has been covered on immersion oil it should not be viewed on other

objectives to minimalize the amount of immersion oil that gets on the other lenses.

6. When competed, clean the lens with lens paper (Figure 20 and 21) to remove the immersion oil. If it

is still very dirty it can be re-wiped with ethyl alcohol using cotton swab several times.

Figure 20: Lens paper

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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Figure 21: Cleaning microscope lens with lens paper

19) SHUT DOWN PROCEDURE

1. After finish the fluorescence imaging taking, turn the light back to bright field (BF).

2. Switch the objective to the lowest magnification.

3. Remove your sample.

4. Close the software.

5. Turn off the microscope.

6. Turn off the main powder.

BNRF SOP Updated on 10/29/2014 by Huiyuan Li

BioNano research Facility – WVU SRF

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EMERGENCY OPERATING PROCEDURES

If you have any questions, even if you are just slightly unsure, ASK someone who knows and can help.

There are no penalties for asking for help but there may be for not reporting damage to the equipment that

may delay or prevent others from working.

If, at any time, you need to contact someone for help, call or locate the following staff of the Shared

Research Facility (SRF):

Huiyuan Li Office: 381 CRL Phone: (304) 293-0747 Cell: (304) 906-5368

This microscope is protected by various safety devices. If none of the above-listed individuals is available,

the user must:

Turn the instrument OFF.

If possible, the user should stay with the microscope while trying to contact the above individuals. If it

becomes necessary to leave the microscope then the user should leave a large, legible note on both the

microscope and at least one of the above individuals’ offices, stating:

The problem (describe what happened and steps taken)

When it occurred (date and time)

User name and phone number

If a dangerous situation is evident (smoke, fire, sparks, etc), ONLY if it is safe to do so, the user

should turn OFF power to the entire microscope and notify the proper emergency personnel. In any

case, the user should leave the facility and contact emergency personnel as soon as possible from

a safe place.

Huiyuan Li Office: 381 CRL Phone: (304) 293-0747 Cell: (304) 906-5368

Marcela Redigolo Office: ESB G75D Phone: (304) 293-9973 Cell: (304) 680-3007