2024年8月26日发(作者：廉醉柳)
Author: Chaowei Shang
Aug 2021
Nikon TE300 Eclipse Brightfield
Microscope
User Guide
LSU Health Science Center-Shreveport
Research Core Facility
1
Author: Chaowei Shang
Aug 2021
User manual for Nikon Elements software
Equipment:
If the room lights are not on when you arrive to use the Nikon, check and confirm that no one is using
the Zeiss two-photon microscope before you turn on the light.
Check whether the floating table and the microscope is clean, if not, clean it.
Sign in on the sign in sheet; please use both your given name and your surname, your department, your
PI, and provide a telephone number at which you can be reached. If you do not use your lab telephone
as your main contact, please provide your mobile number.
Nikon TE300 Eclipse microscope
DS Fi2 color camera (For colored images)
2
Author: Chaowei Shang
Aug 2021
Turning on/preparing microscope
1. Turn on the surge protector by pressing its power button
2. Turn on the computer
1
2
Once the computer is on, please login to your own user account with the following user ID and
password:
User ID: lsumc-masterusername
The password is your personal school email password
Turn on the camera before you start the software.
• Turn on the Nikon Digital Sight DS-U3 unit, which is connected to the camera via a cable. The
green light will come on; once it stops blinking and remains a solid green, the camera is ready to
use.
Connecting cable
Digital Sight DS-U3 unit
DS Fi2 color camera
Open the NIS Elements software by double clicking on the icon .
A log-in dialogue box will appear after clicking on the software icon. Log in use:
User ID: nikonuser
3
Author: Chaowei Shang
Aug 2021
Password: corescopes
In the camera dialogue box, choose the DS-U3 color camera (Neglect the other options).
1. The Nikon lamp switch on the left side of the scope should be pushed in (ON). If not, push it in.
2. Choose the correct stage insert and use the nylon screws to fasten it in place. Extra screws are
in a white plastic box and the yellow screwdriver is for changing stage. You can find stage inserts
in the box labeled “Nikon Wide-field stage”.
3. The motorized stage joystick located to the right of the microscope has three speeds: 100%, 50%
and 25%, which can be changed by pressing the switch to the left of the joystick. The switch
continuously cycles through the speeds, so try different speeds to find the one you prefer.
2
1
Nikon lamp switch
3
Screws and screw
driver for changing
stage
Motorized stage joystick
and focus knob
Setting up for imaging (objective, plates, slides)
1. Lower the objective positions with the focus wheel. The lengths of the objectives are different,
and the taller objectives can easily hit the stage while you turn the objective turret.
4
Author: Chaowei Shang
Aug 2021
2. Place your sample securely in the stage tray. Remember to invert slides, coverslip side down.
3. The microscope objectives are engineered to work best with coverslips 0.17 mm in thickness,
usually referred to as number 1.5 or 1 ½ when ordering.
4. Plates and dishes should not be flipped. If possible, you may want to remove plastic lids, since
plastic can distort imaging.
Observing images through the eyepieces (light path: A)
1. When viewing your sample through the eyepieces, set the selector knob on the lower right side
of the scope to A, which directs 100% of the light to the eyepieces. B & D allow light to go to the
eyepieces and the camera at the same time; C directs 100% of the light to the camera.
Light path selector wheel
2. Rotate the objective turret manually to select an objective; please do not touch the objectives
as you rotate the turret, since those with correction collars can have the collars inadvertently
changed.
3. In the OC Panel of the software, click on the objective name corresponding to the one that you
have manually selected. Note: Clicking on objectives in the OC panel does not control the actual
turning of objective turret, but it tells the software which objective is currently in place and
allows the software to adjust the acquisition settings best for the selected objective. Although
you have to manually choose objectives, you still need to click on the corresponding objective
button in the software to ensure best imaging result. Scale bars won’t be in the correct um
format if no or wrong objective is selected.
[The objectives currently mounted on the turret comprise the following: 4x, 10x, 20x, 20x, 40x
dry, 40x oil. Please note that only ONE (40x oil) of these is an oil objective, requiring the use of
immersion fluid; the others are all air objectives, which do not require any immersion fluid, and
will in fact be damaged by oil, glycerin, or water. If needed, 60x (oil) or 100x (oil) objectives are
available to be checked out.]
5
Author: Chaowei Shang
Aug 2021
PLEASE use ONLY the Nikon oil in the brown plastic container at the microscope station with the
40X oil objective.
4. Turn on the light switch
5. Look through the eyepiece with transmitted light. Find and focus on your sample.
Observing live images on the computer and acquisition (light path: C)
1. To view samples in the software, manually set the selector knob on the lower right side of the
scope to C, which directs 100% of the light to the camera.
2. Click on the live button in the top tool bar at the top of the software.
3. A live view window will pop up.
Live view window
4. Use the fine focus knob to readjust the focus on screen, because the focus through eyepiece is
not exactly the same with the focus of the software.
5. Adjust the white balance of an image using the AWB button at the top tool bar of the live view
window.
6. Capture an image by clicking the camera button .
DS Fi2 (color) Camera Settings
6
Author: Chaowei Shang
Aug 2021
• Mode: Normal and Binning. Default Normal.
• Resolution: you will see Fast (Focus) and a drop down menu, and Quality (Capture) and another
drop down menu. Below those menus is a box called High Quality Capture that can be checked
or unchecked.
• Exposure: Uncheck auto Exposure to manually change exposure time.
• Analog Gain: software sets value
• AE Compensation: software sets value
• Color: Auto White button will add white balance to your image.
• Scene Mode >: no action
• Commands: no action
Saving an image (ND, TIFF, JPEG; folders; offline access)
When you are ready to save an image, go to File and choose Save As. You will have choice in which
format to save the image. Each user has a personal folder located in the Users Folder on the desktop.
Images can be saved there temporarily, but should be removed in good time. The easiest way is to use a
flash drive and the USB ports on computer.
Save your image as a .tif file from your original image. Keep in mind that TIFF format is required for
publication. JPEG images is not always accepted.
To save into a TIFF file. Go to File->Save as, then set the options as below:
7
Author: Chaowei Shang
Aug 2021
Shutting down the equipment
1. If you have used oil, make sure to clean the objective with the lens paper and cleaner
provided. Currently, this only applies to the 40x oil objective used for fluorescent imaging.
2. Clean your slides with cleaner and kimwipes.
3. Lower the objectives and turn the objective to 4X.
4. Log off and Shut Down the computer.
5. Turn off the DS Fi2 color camera by turning off the Nikon Digital Sight DS-U3 unit.
6. Turn OFF the surge protector under the table.
7. Replace the cover and remember to sign out on the login sheet.
Miscellaneous
Binning averages the grey values in pixels into various sizes of bundles, which makes the signal brighter
but reduces resolution. The software allows you to set the binning range for both cameras, with None
as the default. The 2x2 selection will produce a good size image for focusing. If you change the binning
size, you may need to redo the Auto Exposure. Using 2x2 binning, which averages the information in 4
pixels, increases your S/N ratio by a factor of 4. This is advantageous when you have a weak fluorescent
signal. The use of 2x2 binning also decreases image file size and time transfer rates for the camera.
Choose No Binning or the lowest value of binning for the best resolution.
Tips for Better Imaging
Coverslips: It is very important that your coverslip be glass, size 1 ½, 0.17 mm thick. Some of the
objectives have correction collars that adjust for differences in thickness, but some do not. The
correction collars may help if you are trying to image cells in plates or flasks. However, they may not be
in a position ideal for your samples.
Plastic: although in general images made through plastic ware are good, it may be worth the extra
money to buy optically corrected plastic.
Immersion medium objectives: If you are using the 40x, 60x, or 100x oil immersion objectives, please
use ONLY the Nikon immersion oil next to the microscope. Residues from different oil types on the lens
8
Author: Chaowei Shang
Aug 2021
can cause aberrations in the image. Kimwipes are fine for cleaning the oil off of slides (this can be
followed with lens cleaner sprayed onto a Kimwipe, then used to remove the last traces of oil). When
cleaning the objective lens, which is very sensitive, use the provided lens paper and draw it gently across
the objective, which allows it to soak up and remove the oil without scratching or damaging the lens.
For professional quality images, each component of the microscope system should be set to the
optimal position for your samples before you begin to collect images. This is especially important for
brightfield images that require Kohler illumination.
Equipment Specifications for Publication
Microscope: Nikon Eclipse TE300 inverted microscope with an epi-fluorescence attachment
Objectives: there are 6 objectives mounted on the turret; the rest are available for check out
Objective Imm Type WD (mm) Use with Where located?
4x/ 0.20 NA Dry Plan Apo WD 20 Microscope turret
10x/ 0.30 NA Dry Plan Fluor WD 16.0 Ph1 DLL Microscope turret
20x/ 0.45 NA Dry Plan Fluor ELWD WD 7.4 DIC L Microscope turret
20x/ 0.50 NA Dry Plan Fluor WD 2.1 Ph1 DLL Microscope turret
40x/ 0.60 NA Dry Plan Fluor ELWD WD 3.7-2.7 DIC M Microscope turret
40x/ 1.30 NA Oil Plan Fluor WD 0.2 DIC H Microscope turret
60x/ 1.40 NA Oil Plan Apo WD 0.21 DIC H Cabinet
100x/ 1.40 NA Oil Plan Apo WD 0.13 DIC H Cabinet
Condenser A Bright field
∞Ph1 Phase contrast
∞L DIC
∞M DIC
∞H DIC
Software: NIS Elements 4.50 (Windows 7, PC)
9
2024年8月26日发(作者：廉醉柳)
Author: Chaowei Shang
Aug 2021
Nikon TE300 Eclipse Brightfield
Microscope
User Guide
LSU Health Science Center-Shreveport
Research Core Facility
1
Author: Chaowei Shang
Aug 2021
User manual for Nikon Elements software
Equipment:
If the room lights are not on when you arrive to use the Nikon, check and confirm that no one is using
the Zeiss two-photon microscope before you turn on the light.
Check whether the floating table and the microscope is clean, if not, clean it.
Sign in on the sign in sheet; please use both your given name and your surname, your department, your
PI, and provide a telephone number at which you can be reached. If you do not use your lab telephone
as your main contact, please provide your mobile number.
Nikon TE300 Eclipse microscope
DS Fi2 color camera (For colored images)
2
Author: Chaowei Shang
Aug 2021
Turning on/preparing microscope
1. Turn on the surge protector by pressing its power button
2. Turn on the computer
1
2
Once the computer is on, please login to your own user account with the following user ID and
password:
User ID: lsumc-masterusername
The password is your personal school email password
Turn on the camera before you start the software.
• Turn on the Nikon Digital Sight DS-U3 unit, which is connected to the camera via a cable. The
green light will come on; once it stops blinking and remains a solid green, the camera is ready to
use.
Connecting cable
Digital Sight DS-U3 unit
DS Fi2 color camera
Open the NIS Elements software by double clicking on the icon .
A log-in dialogue box will appear after clicking on the software icon. Log in use:
User ID: nikonuser
3
Author: Chaowei Shang
Aug 2021
Password: corescopes
In the camera dialogue box, choose the DS-U3 color camera (Neglect the other options).
1. The Nikon lamp switch on the left side of the scope should be pushed in (ON). If not, push it in.
2. Choose the correct stage insert and use the nylon screws to fasten it in place. Extra screws are
in a white plastic box and the yellow screwdriver is for changing stage. You can find stage inserts
in the box labeled “Nikon Wide-field stage”.
3. The motorized stage joystick located to the right of the microscope has three speeds: 100%, 50%
and 25%, which can be changed by pressing the switch to the left of the joystick. The switch
continuously cycles through the speeds, so try different speeds to find the one you prefer.
2
1
Nikon lamp switch
3
Screws and screw
driver for changing
stage
Motorized stage joystick
and focus knob
Setting up for imaging (objective, plates, slides)
1. Lower the objective positions with the focus wheel. The lengths of the objectives are different,
and the taller objectives can easily hit the stage while you turn the objective turret.
4
Author: Chaowei Shang
Aug 2021
2. Place your sample securely in the stage tray. Remember to invert slides, coverslip side down.
3. The microscope objectives are engineered to work best with coverslips 0.17 mm in thickness,
usually referred to as number 1.5 or 1 ½ when ordering.
4. Plates and dishes should not be flipped. If possible, you may want to remove plastic lids, since
plastic can distort imaging.
Observing images through the eyepieces (light path: A)
1. When viewing your sample through the eyepieces, set the selector knob on the lower right side
of the scope to A, which directs 100% of the light to the eyepieces. B & D allow light to go to the
eyepieces and the camera at the same time; C directs 100% of the light to the camera.
Light path selector wheel
2. Rotate the objective turret manually to select an objective; please do not touch the objectives
as you rotate the turret, since those with correction collars can have the collars inadvertently
changed.
3. In the OC Panel of the software, click on the objective name corresponding to the one that you
have manually selected. Note: Clicking on objectives in the OC panel does not control the actual
turning of objective turret, but it tells the software which objective is currently in place and
allows the software to adjust the acquisition settings best for the selected objective. Although
you have to manually choose objectives, you still need to click on the corresponding objective
button in the software to ensure best imaging result. Scale bars won’t be in the correct um
format if no or wrong objective is selected.
[The objectives currently mounted on the turret comprise the following: 4x, 10x, 20x, 20x, 40x
dry, 40x oil. Please note that only ONE (40x oil) of these is an oil objective, requiring the use of
immersion fluid; the others are all air objectives, which do not require any immersion fluid, and
will in fact be damaged by oil, glycerin, or water. If needed, 60x (oil) or 100x (oil) objectives are
available to be checked out.]
5
Author: Chaowei Shang
Aug 2021
PLEASE use ONLY the Nikon oil in the brown plastic container at the microscope station with the
40X oil objective.
4. Turn on the light switch
5. Look through the eyepiece with transmitted light. Find and focus on your sample.
Observing live images on the computer and acquisition (light path: C)
1. To view samples in the software, manually set the selector knob on the lower right side of the
scope to C, which directs 100% of the light to the camera.
2. Click on the live button in the top tool bar at the top of the software.
3. A live view window will pop up.
Live view window
4. Use the fine focus knob to readjust the focus on screen, because the focus through eyepiece is
not exactly the same with the focus of the software.
5. Adjust the white balance of an image using the AWB button at the top tool bar of the live view
window.
6. Capture an image by clicking the camera button .
DS Fi2 (color) Camera Settings
6
Author: Chaowei Shang
Aug 2021
• Mode: Normal and Binning. Default Normal.
• Resolution: you will see Fast (Focus) and a drop down menu, and Quality (Capture) and another
drop down menu. Below those menus is a box called High Quality Capture that can be checked
or unchecked.
• Exposure: Uncheck auto Exposure to manually change exposure time.
• Analog Gain: software sets value
• AE Compensation: software sets value
• Color: Auto White button will add white balance to your image.
• Scene Mode >: no action
• Commands: no action
Saving an image (ND, TIFF, JPEG; folders; offline access)
When you are ready to save an image, go to File and choose Save As. You will have choice in which
format to save the image. Each user has a personal folder located in the Users Folder on the desktop.
Images can be saved there temporarily, but should be removed in good time. The easiest way is to use a
flash drive and the USB ports on computer.
Save your image as a .tif file from your original image. Keep in mind that TIFF format is required for
publication. JPEG images is not always accepted.
To save into a TIFF file. Go to File->Save as, then set the options as below:
7
Author: Chaowei Shang
Aug 2021
Shutting down the equipment
1. If you have used oil, make sure to clean the objective with the lens paper and cleaner
provided. Currently, this only applies to the 40x oil objective used for fluorescent imaging.
2. Clean your slides with cleaner and kimwipes.
3. Lower the objectives and turn the objective to 4X.
4. Log off and Shut Down the computer.
5. Turn off the DS Fi2 color camera by turning off the Nikon Digital Sight DS-U3 unit.
6. Turn OFF the surge protector under the table.
7. Replace the cover and remember to sign out on the login sheet.
Miscellaneous
Binning averages the grey values in pixels into various sizes of bundles, which makes the signal brighter
but reduces resolution. The software allows you to set the binning range for both cameras, with None
as the default. The 2x2 selection will produce a good size image for focusing. If you change the binning
size, you may need to redo the Auto Exposure. Using 2x2 binning, which averages the information in 4
pixels, increases your S/N ratio by a factor of 4. This is advantageous when you have a weak fluorescent
signal. The use of 2x2 binning also decreases image file size and time transfer rates for the camera.
Choose No Binning or the lowest value of binning for the best resolution.
Tips for Better Imaging
Coverslips: It is very important that your coverslip be glass, size 1 ½, 0.17 mm thick. Some of the
objectives have correction collars that adjust for differences in thickness, but some do not. The
correction collars may help if you are trying to image cells in plates or flasks. However, they may not be
in a position ideal for your samples.
Plastic: although in general images made through plastic ware are good, it may be worth the extra
money to buy optically corrected plastic.
Immersion medium objectives: If you are using the 40x, 60x, or 100x oil immersion objectives, please
use ONLY the Nikon immersion oil next to the microscope. Residues from different oil types on the lens
8
Author: Chaowei Shang
Aug 2021
can cause aberrations in the image. Kimwipes are fine for cleaning the oil off of slides (this can be
followed with lens cleaner sprayed onto a Kimwipe, then used to remove the last traces of oil). When
cleaning the objective lens, which is very sensitive, use the provided lens paper and draw it gently across
the objective, which allows it to soak up and remove the oil without scratching or damaging the lens.
For professional quality images, each component of the microscope system should be set to the
optimal position for your samples before you begin to collect images. This is especially important for
brightfield images that require Kohler illumination.
Equipment Specifications for Publication
Microscope: Nikon Eclipse TE300 inverted microscope with an epi-fluorescence attachment
Objectives: there are 6 objectives mounted on the turret; the rest are available for check out
Objective Imm Type WD (mm) Use with Where located?
4x/ 0.20 NA Dry Plan Apo WD 20 Microscope turret
10x/ 0.30 NA Dry Plan Fluor WD 16.0 Ph1 DLL Microscope turret
20x/ 0.45 NA Dry Plan Fluor ELWD WD 7.4 DIC L Microscope turret
20x/ 0.50 NA Dry Plan Fluor WD 2.1 Ph1 DLL Microscope turret
40x/ 0.60 NA Dry Plan Fluor ELWD WD 3.7-2.7 DIC M Microscope turret
40x/ 1.30 NA Oil Plan Fluor WD 0.2 DIC H Microscope turret
60x/ 1.40 NA Oil Plan Apo WD 0.21 DIC H Cabinet
100x/ 1.40 NA Oil Plan Apo WD 0.13 DIC H Cabinet
Condenser A Bright field
∞Ph1 Phase contrast
∞L DIC
∞M DIC
∞H DIC
Software: NIS Elements 4.50 (Windows 7, PC)
9