Nikon Ti-U Inverted Light Microscope (basic instructions)

Note: If there is a user after you, leave the Hg lamp on. Every time you turn on the Hg bulb keep it on for 4 hours, roughly 2+ hours off the lifetime of the bulb is removed every ignition. Runtime for bulb is calculated for at least 4 hr run time sessions.

1. Turn on dehumidifier 1 hr before use and close door to room.
2. Turn on dry N2 and begin cooling down Cryo Stage with LN2. Leave door open
3. Turn on all power switches - 8 in total. Make sure hub is the last unit turned on otherwise all the units will not be recognized by the system.
4. Make sure objectives lowered away from the cryostage.
5. Open Nikon Elements software and make sure all units recognized.
6. Insert test grid to obtain rough focal plan and test system.
7. Insert sample.

FAQ

How large is the CCD?

What objectives are available? Their resolution?

How large are the individual frames?

What happens if my grid is not flat?

You have several options:
1) take a Z-stack between a defined Z range
2) auto focus each image or every so often
3) use a focus surface - see How to deal with uneven grid?
4) manually take images in focus at select areas.

But each method has its drawbacks so it's a matter of choosing what you can live with.

How long does it take to capture the whole grid?

Test 1: Focus surface
Time: ~25min
1) Take a 4x image to use as a map.
2) Change to the 50X and manually select 2 dozen or so points randomly to manually find the correct Z-height.
3) After enough points are selected you can calculate a focus surface. Use those manually entered XYZ points to define the focus surface and define an area to take a large image. In the ND acquisition menu: the manually entered points will form a green line for z-height and calculate an orange line that it'll use to capture an image.
4) Take a large image in both BF and GFP using the focus surface.

This procedure took me about 25 minutes start to finish with steps 1-3 taking 5-7 min and step 4 taking the remainder of the time. The computer paused a couple times to save the data, but kept going. I took 180 images at 50x to stitch an area of 1.5mm x 1.5mm that covered ~80% of the grid. When taking so many images I saved all the individual files and have the program stitch separately from data acquisition. If it tries to do both it might crash unless I have a lot of memory.

There still are disadvantages: 1) it extrapolates the z-height between points, and 2) it assumes that the specimen remains at an absolute Z height. So if you knock the stage it will go to the predetermined height but not know it is out of focus. The problem I see is that within a field view the whole image may not be in focus. Also, this module was designed with a uniform surface in mind, such as a glass slide that is on an angle.

Test 2: Focus surface + auto focus
Time: ~35min
My second test was to use the surface focus and add what I was doing before, where it searches for focus in a range. I used the focus surface as the center and had it search +/- 7.5µm from that Z-height in 1.5 µm steps. This took about 35 minutes, but more of the image was in focus. I also put together the large image after it collected data. In both cases I used a 15% overlap between images.

• Set ALLOWTOPICVIEW =

Nikon Ti-U Inverted Light Microscope (basic instructions)

Note: If there is a user after you, leave the Hg lamp on. Every time you turn on the Hg bulb keep it on for 4 hours, roughly 2+ hours off the lifetime of the bulb is removed every ignition. Runtime for bulb is calculated for at least 4 hr run time sessions.

1. Turn on dehumidifier 1 hr before use and close door to room.
2. Turn on dry N2 and begin cooling down Cryo Stage with LN2. Leave door open
3. Turn on all power switches - 8 in total. Make sure hub is the last unit turned on otherwise all the units will not be recognized by the system.
4. Make sure objectives lowered away from the cryostage.
5. Open Nikon Elements software and make sure all units recognized.
6. Insert test grid to obtain rough focal plan and test system.
7. Insert sample.

FAQ

How large is the CCD?

What objectives are available? Their resolution?

What happens if my grid is not flat?

You have several options:
1) take a Z-stack between a defined Z range
2) auto focus each image or every so often
3) use a focus surface - see How to deal with uneven grid?
4) manually take images in focus at select areas.

But each method has its drawbacks so it's a matter of choosing what you can live with.

How long does it take to capture the whole grid?

Test 1: Focus surface
Time: ~25min
1) Take a 4x image to use as a map.
2) Change to the 50X and manually select 2 dozen or so points randomly to manually find the correct Z-height.
3) After enough points are selected you can calculate a focus surface. Use those manually entered XYZ points to define the focus surface and define an area to take a large image. In the ND acquisition menu: the manually entered points will form a green line for z-height and calculate an orange line that it'll use to capture an image.
4) Take a large image in both BF and GFP using the focus surface.

This procedure took me about 25 minutes start to finish with steps 1-3 taking 5-7 min and step 4 taking the remainder of the time. The computer paused a couple times to save the data, but kept going. I took 180 images at 50x to stitch an area of 1.5mm x 1.5mm that covered ~80% of the grid. When taking so many images I saved all the individual files and have the program stitch separately from data acquisition. If it tries to do both it might crash unless I have a lot of memory.

There still are disadvantages: 1) it extrapolates the z-height between points, and 2) it assumes that the specimen remains at an absolute Z height. So if you knock the stage it will go to the predetermined height but not know it is out of focus. The problem I see is that within a field view the whole image may not be in focus. Also, this module was designed with a uniform surface in mind, such as a glass slide that is on an angle.

Test 2: Focus surface + auto focus
Time: ~35min
My second test was to use the surface focus and add what I was doing before, where it searches for focus in a range. I used the focus surface as the center and had it search +/- 7.5µm from that Z-height in 1.5 µm steps. This took about 35 minutes, but more of the image was in focus. I also put together the large image after it collected data. In both cases I used a 15% overlap between images.

• Set ALLOWTOPICVIEW =

-- EdEng - 20 Sep 2012

FAQ

How large is the CCD?

What objectives are available? Their resolution?

What happens if my grid is not flat?

You have several options:
1) take a Z-stack between a defined Z range
2) auto focus each image or every so often

But each method has its drawbacks so it's a matter of choosing what you can live with.

How long does it take to capture the whole grid?

Test 1: Focus surface
Time: ~25min
1) Take a 4x image to use as a map.
2) Change to the 50X and manually select 2 dozen or so points randomly to manually find the correct Z-height.
3) After enough points are selected you can calculate a focus surface. Use those manually entered XYZ points to define the focus surface and define an area to take a large image. In the ND acquisition menu: the manually entered points will form a green line for z-height and calculate an orange line that it'll use to capture an image.
4) Take a large image in both BF and GFP using the focus surface.

This procedure took me about 25 minutes start to finish with steps 1-3 taking 5-7 min and step 4 taking the remainder of the time. The computer paused a couple times to save the data, but kept going. I took 180 images at 50x to stitch an area of 1.5mm x 1.5mm that covered ~80% of the grid. When taking so many images I saved all the individual files and have the program stitch separately from data acquisition. If it tries to do both it might crash unless I have a lot of memory.

There still are disadvantages: 1) it extrapolates the z-height between points, and 2) it assumes that the specimen remains at an absolute Z height. So if you knock the stage it will go to the predetermined height but not know it is out of focus. The problem I see is that within a field view the whole image may not be in focus. Also, this module was designed with a uniform surface in mind, such as a glass slide that is on an angle.

Test 2: Focus surface + auto focus
Time: ~35min
My second test was to use the surface focus and add what I was doing before, where it searches for focus in a range. I used the focus surface as the center and had it search +/- 7.5µm from that Z-height in 1.5 µm steps. This took about 35 minutes, but more of the image was in focus. I also put together the large image after it collected data. In both cases I used a 15% overlap between images.

• Set ALLOWTOPICVIEW =

-- EdEng - 20 Sep 2012

Nikon Ti-U Inverted Light Microscope

Note: If there is a user after you, leave the Hg lamp on. Every time you turn on the Hg bulb keep it on for 4 hours, roughly 2+ hours off the lifetime of the bulb is removed every ignition. Runtime for bulb is calculated for at least 4 hr run time sessions.

1. Turn on dehumidifier 1 hr before use and close door in room.
2. Begin cooling down Cryo Stage with LN2.
3. Turn on all power switches - 8 in total. Make sure hub is the last unit turned on otherwise all the units will not be recognized by the system.
4. Make sure objectives lowered away from the cryostage.
5. Open Nikon Elements software and make sure all units recognized.
6. Insert test grid to obtain rough focal plan and test system.
7. Insert sample.

FAQ

How large is the CCD?

What objectives are available? Their resolution?

What happens if my grid is not flat?

You have several options:
1) take a Z-stack between a defined Z range
2) auto focus each image or every so often
3) use a focus surface - which was the feature I just figured out
4) manually take images in focus at select areas.

But each method has its drawbacks so it's a matter of choosing what you can live with.

How long does it take to capture the whole grid?

Test 1: Focus surface
Time: ~25min
1) Take a 4x image to use as a map.
2) Change to the 50X and manually select 2 dozen or so points randomly to manually find the correct Z-height.
3) After enough points are selected you can calculate a focus surface. Use those manually entered XYZ points to define the focus surface and define an area to take a large image. In the ND acquisition menu: the manually entered points will form a green line for z-height and calculate an orange line that it'll use to capture an image.
4) Take a large image in both BF and GFP using the focus surface.

This procedure took me about 25 minutes start to finish with steps 1-3 taking 5-7 min and step 4 taking the remainder of the time. The computer paused a couple times to save the data, but kept going. I took 180 images at 50x to stitch an area of 1.5mm x 1.5mm that covered ~80% of the grid. When taking so many images I saved all the individual files and have the program stitch separately from data acquisition. If it tries to do both it might crash unless I have a lot of memory.

There still are disadvantages: 1) it extrapolates the z-height between points, and 2) it assumes that the specimen remains at an absolute Z height. So if you knock the stage it will go to the predetermined height but not know it is out of focus. The problem I see is that within a field view the whole image may not be in focus. Also, this module was designed with a uniform surface in mind, such as a glass slide that is on an angle.

Test 2: Focus surface + auto focus
Time: ~35min
My second test was to use the surface focus and add what I was doing before, where it searches for focus in a range. I used the focus surface as the center and had it search +/- 7.5µm from that Z-height in 1.5 µm steps. This took about 35 minutes, but more of the image was in focus. I also put together the large image after it collected data. In both cases I used a 15% overlap between images.

• Set ALLOWTOPICVIEW =

-- EdEng - 20 Sep 2012