How to scan microscopic cuts - Epson 4990

[Marc Heusser]

I try to scan microscopic cuts hoping to reach at least 100x
magnification (9600 dpi scan -> 72 dpi screen).
I tried it on a Mac using Epson Scan, set at 9600 dpi, using the
transparency mode. I set the glass slides directly on the scanning glass
(both upside down and upright).
When I inspect the images, they seem to be much lower resolution (say
4800 dpi).
How do I best do that? Any experience?

TIA

Marc

 

[CSM1]

I try to scan microscopic cuts hoping to reach at least 100x
magnification (9600 dpi scan -> 72 dpi screen).
I tried it on a Mac using Epson Scan, set at 9600 dpi, using the
transparency mode. I set the glass slides directly on the scanning glass
(both upside down and upright).
When I inspect the images, they seem to be much lower resolution (say
4800 dpi).
How do I best do that? Any experience?

TIA

Marc

No flatbed scanner has enough resolution to scan a microscope slide. Not to
magnify 100X.

You need a microscope with a digital camera attached.

 

[-]

I set the glass slides directly on the scanning glass (both upside down

and upright). When I inspect the images, they seem to be much lower
resolution (say
4800 dpi).

If you look at your film holders, you will see they don't place the image to
be scanned directly on the glass. They suspend the film slightly off the
glass. If you are scanning a mounted film slide, the slide mount keeps the
film slightly of the glass. From your description, it sounds like your
image is right on the glass as opposed to in a mount. You need to make sure
your image is also slightly off the glass if you want the best resolution.

There is no use scanning over 4800 ppi. Anything over 4800 ppi is
interpolated resolution. In other words, it is just "made up" pixel
information based on the software's best guess. Many people feel there
isn't any real resolution gained over 2400 ppi.

Doug

 

[Marc Heusser]

I set the glass slides directly on the scanning glass (both upside down
and upright). When I inspect the images, they seem to be much lower
resolution (say
4800 dpi).

If you look at your film holders, you will see they don't place the image to
be scanned directly on the glass. They suspend the film slightly off the
glass. If you are scanning a mounted film slide, the slide mount keeps the
film slightly of the glass. From your description, it sounds like your
image is right on the glass as opposed to in a mount. You need to make sure
your image is also slightly off the glass if you want the best resolution.[/QUOTE]

Actually the microscope slides have a thick glass, and a thin one. I
used both configurations, the thick one below gave the better scan. The
actual tissue was therefore some 0.8 mm above the glass plate of the
scanner. Maybe it needs more? I'll try to fix them int the film holders
somehow. Would you know where the actual plane of focus lies?

There is no use scanning over 4800 ppi. Anything over 4800 ppi is
interpolated resolution. In other words, it is just "made up" pixel
information based on the software's best guess. Many people feel there
isn't any real resolution gained over 2400 ppi.

Epson claims a 4800x9600 optical resolution - it should be either 4800
or 9600 that gives the best resolution. 2400 ppi definitely gives a
worse scan. Epson claims an interpolated resolution of 12800 is possible.

No flatbed scanner has enough resolution to scan a microscope slide. Not to
magnify 100X.

You need a microscope with a digital camera attached.

I'd certainly get a better picture this way - I do not have a
photographic adapter though for my microscope (an 20y old Olympus CH2,
goes up to 600x)

But IF the scanner actuall gives the above mentioned 4800x9600
resolution, when looking at the picture at 72 dpi screen resolution I
should get a magnification of 67 to 134, ie roughly 100x. Unless the
picture is not as sharp as given by the scanner's specs. And the scanner
does scan in the transmissive mode.

Thank you both

Marc

 

[-]

actual tissue was therefore some 0.8 mm above the glass plate of the

scanner. Maybe it needs more? I'll try to fix them int the film holders
somehow. Would you know where the actual plane of focus lies?

Theoretically, it is supposed to be 1 mm but with these scanners the true
optimum scanner height quite often varies.

Epson claims a 4800x9600 optical resolution - it should be either 4800
or 9600 that gives the best resolution. 2400 ppi definitely gives a
worse scan. Epson claims an interpolated resolution of 12800 is possible.

The key word is "claims." You find many posts on the internet
discussing this. Most people find that true optical resolution maxxes out
at somewhere around 2400 for the 4180 and even it successor, the 4490. They
are good scanners for their price but you just have to keep things in
perspective. Microtek and Canon flatbeds have the same "issue."

Doug

 

[CSM1]

If you look at your film holders, you will see they don't place the image to
be scanned directly on the glass. They suspend the film slightly off the
glass. If you are scanning a mounted film slide, the slide mount keeps the
film slightly of the glass. From your description, it sounds like your
image is right on the glass as opposed to in a mount. You need to make sure
your image is also slightly off the glass if you want the best

resolution.

Actually the microscope slides have a thick glass, and a thin one. I
used both configurations, the thick one below gave the better scan. The
actual tissue was therefore some 0.8 mm above the glass plate of the
scanner. Maybe it needs more? I'll try to fix them int the film holders
somehow. Would you know where the actual plane of focus lies?

There is no use scanning over 4800 ppi. Anything over 4800 ppi is
interpolated resolution. In other words, it is just "made up" pixel
information based on the software's best guess. Many people feel there
isn't any real resolution gained over 2400 ppi.

Epson claims a 4800x9600 optical resolution - it should be either 4800
or 9600 that gives the best resolution. 2400 ppi definitely gives a
worse scan. Epson claims an interpolated resolution of 12800 is possible.

No flatbed scanner has enough resolution to scan a microscope slide. Not to
magnify 100X.

You need a microscope with a digital camera attached.

I'd certainly get a better picture this way - I do not have a
photographic adapter though for my microscope (an 20y old Olympus CH2,
goes up to 600x)

But IF the scanner actuall gives the above mentioned 4800x9600
resolution, when looking at the picture at 72 dpi screen resolution I
should get a magnification of 67 to 134, ie roughly 100x. Unless the
picture is not as sharp as given by the scanner's specs. And the scanner
does scan in the transmissive mode.

Thank you both

Marc
[/QUOTE]


This company has lens adapters for microscopes to various Digital Cameras.
You have to choose the one for your camera and microscope.
http://www.zarfenterprises.com/

 

[Don]

Actually the microscope slides have a thick glass, and a thin one. I
used both configurations, the thick one below gave the better scan. The
actual tissue was therefore some 0.8 mm above the glass plate of the
scanner. Maybe it needs more? I'll try to fix them int the film holders
somehow. Would you know where the actual plane of focus lies?

I don't know if getting a new scanner is an option but a film scanner
would considerably improve the result and simplify the workflow.

For example, Nikon scanners have a special holder/adapter for glass
microscope slides. Virtually all Nikon scanners can use this optional
adapter which has to be purchased separately.

Reading the Nikon manual there are actually two such adapters:

FH-8G1 - for high end 9000 ED and 8000 ED scanners.
FH-G1 - for all other Nikon scanners i.e. 5000 ED, 4000 ED, COOLSCAN V
ED and COOLSCAN IV ED.

For more check the relevant Nikon site in your region:
http://www.nikonusa.com/
http://www.europe-nikon.com/support
http://www.nikon-asia.com/

Don.

 

[George E. Cawthon]

If you look at your film holders, you will see they don't place the image to
be scanned directly on the glass. They suspend the film slightly off the
glass. If you are scanning a mounted film slide, the slide mount keeps the
film slightly of the glass. From your description, it sounds like your
image is right on the glass as opposed to in a mount. You need to make sure
your image is also slightly off the glass if you want the best resolution.

Actually the microscope slides have a thick glass, and a thin one. I
used both configurations, the thick one below gave the better scan. The
actual tissue was therefore some 0.8 mm above the glass plate of the
scanner. Maybe it needs more? I'll try to fix them int the film holders
somehow. Would you know where the actual plane of focus lies?

There is no use scanning over 4800 ppi. Anything over 4800 ppi is
interpolated resolution. In other words, it is just "made up" pixel
information based on the software's best guess. Many people feel there
isn't any real resolution gained over 2400 ppi.

Epson claims a 4800x9600 optical resolution - it should be either 4800
or 9600 that gives the best resolution. 2400 ppi definitely gives a
worse scan. Epson claims an interpolated resolution of 12800 is possible.

No flatbed scanner has enough resolution to scan a microscope slide. Not to
magnify 100X.

You need a microscope with a digital camera attached.

I'd certainly get a better picture this way - I do not have a
photographic adapter though for my microscope (an 20y old Olympus CH2,
goes up to 600x)

But IF the scanner actuall gives the above mentioned 4800x9600
resolution, when looking at the picture at 72 dpi screen resolution I
should get a magnification of 67 to 134, ie roughly 100x. Unless the
picture is not as sharp as given by the scanner's specs. And the scanner
does scan in the transmissive mode.

Thank you both

Marc
[/QUOTE]
Finding the focus is not difficult. You need a
flat scale and something of know thickness to set
it on. Best bet is a machinists scale, like the
flat bar from a machinists square (or use an
architects scale). Set one end on the glass and
place a support of known thickness, e.g., a coin,
at a specific distance and scan and check distance
for best focus/ For example, 10mm high at 200mm
length (or 1mm high per 20mm linear).

 

[Marc Heusser]

....

I don't know if getting a new scanner is an option but a film scanner
would considerably improve the result and simplify the workflow.

For example, Nikon scanners have a special holder/adapter for glass
microscope slides. Virtually all Nikon scanners can use this optional
adapter which has to be purchased separately.

I will check - If I can I'd currently avoid the expense - but it
certainly is the better option. Acutally I found a new review comparing
the Epson V750 (newer, better successor of the 4990) compared to the
Nikon 4000 - the dedicated film scanner IS better. And having a
dedicated holder does make it easier. Thank you for the tip.

Finding the focus is not difficult. You need a
flat scale and something of know thickness to set
it on.

Thank you - easy and very useful

I guess I'll make do with my current scanner - at least up to when I'll
get a dedicated Nikonn scanner for my slides. (I do have a few thousand
photo slides too - although a commercial service might be useful there.)

Marc

 

[Don]

I will check - If I can I'd currently avoid the expense - but it
certainly is the better option. Acutally I found a new review comparing
the Epson V750 (newer, better successor of the 4990) compared to the
Nikon 4000 - the dedicated film scanner IS better. And having a
dedicated holder does make it easier. Thank you for the tip.

You're most welcome.

BTW, you don't need an expensive scanner for this. Even an entry model
like the LS-50 also known as Coolscan V will do. I think they can be
had for as little as Euro/$ 600 possibly even less if you shop around.

Don.

 

[Marc Heusser]

Thank you - easy and very useful

I did it now - using a block of Post-It Notes as an easily adjustable
fixed height block.
My Epson 4990 seems to have maximum sharpness (ie focus) at between 1.2
and 1.6 mm above the glass - the effect is quite noticeable at 9600 dpi.
I'll scan at 4800 dpi.

As a quick fix the Post-It Notes will also provide a suiteable ³holder³
for now (scanning some 150 glass microscope slides for my own use).

Thanks again

Marc

 

[Marc Heusser]

My Epson 4990 seems to have maximum sharpness (ie focus) at between 1.2
and 1.6 mm above the glass - the effect is quite noticeable at 9600 dpi.
I'll scan at 4800 dpi.

With the cut now in the plane of focus, test with both 4800 and 9600 dpi
resolution are clear: 9600 is clearly superior in the Epson 4990,
showing much more detail and less "grain" - so if you need this last bit
of resolution it pays to use 9600 dpi. (It even shows when you compress
with jpg afterwards.) Also this does not change when applying an unsharp
mask filter.

Marc

 

[NRen2k5]

There is no use scanning over 4800 ppi. Anything over 4800 ppi is
interpolated resolution. In other words, it is just "made up" pixel
information based on the software's best guess.

Which for all intents and purposes is like scanning the image at 2400ppi
and resampling ("resizing" in most graphics suites) to 4800ppi.

Many people feel there
isn't any real resolution gained over 2400 ppi.

That is *so* true. I don't feel any need to scan most material at any
higher resolution than 600ppi, because even at that resolution, the
individual ink dots in news and magazine graphics come through very clearly.

I have even found that I can scan covers to audio-CD's and save them as
8-bit PNG's... because at that high resolution (where you can
dinstinguish individual ink dots), you no longer need anywhere near 17
million colours.

 

[NRen2k5]

I did it now - using a block of Post-It Notes as an easily adjustable
fixed height block.
My Epson 4990 seems to have maximum sharpness (ie focus) at between 1.2
and 1.6 mm above the glass - the effect is quite noticeable at 9600 dpi.
I'll scan at 4800 dpi.

As a quick fix the Post-It Notes will also provide a suiteable ³holder³
for now (scanning some 150 glass microscope slides for my own use).

Thanks again

Marc

Now onto the next order of business... getting your news client to use
the proper encoding (or do you actually *mean* to use the exponent 3 as
quotation marks?

 

[Marc Heusser]

Which for all intents and purposes is like scanning the image at 2400ppi
and resampling ("resizing" in most graphics suites) to 4800ppi.

Just to clear that: the Epson 4990 has 4800x9600 dpi *optical*
resolution - and scanning at 9600 gives much more detail.

Nonetheless, this only applies if the original actually has that kind of
resolution - this applies for my microscopic slides, (being real
tissue), they have features down to less than 1um (eg nerves).
But then we are using light of a wavelength eg 600 nanometres, so
practical resolution is probably limited at 2 micrometres. (9600 dpi is
2.6 micrometres). And the cuts are 6 micrometres or more thick.

That is *so* true. I don't feel any need to scan most material at any
higher resolution than 600ppi, because even at that resolution, the
individual ink dots in news and magazine graphics come through very clearly.

I have even found that I can scan covers to audio-CD's and save them as
8-bit PNG's... because at that high resolution (where you can
dinstinguish individual ink dots), you no longer need anywhere near 17
million colours.

Very true for most materials - microscopic slides are exceptional in
having details down to much less than 1 micrometre.

Marc

 

[NRen2k5]

Just to clear that: the Epson 4990 has 4800x9600 dpi *optical*
resolution - and scanning at 9600 gives much more detail.

On the y-axis, anyway. On the x-axis, you're getting 4800dpi
interpolated to 9600 when you scan at 9600.

Nonetheless, this only applies if the original actually has that kind of
resolution - this applies for my microscopic slides, (being real
tissue), they have features down to less than 1um (eg nerves).
But then we are using light of a wavelength eg 600 nanometres, so
practical resolution is probably limited at 2 micrometres. (9600 dpi is
2.6 micrometres). And the cuts are 6 micrometres or more thick.


Very true for most materials - microscopic slides are exceptional in
having details down to much less than 1 micrometre.

Marc

Yes and as already pointed out, scanning microscopic slides is not an
intended use of a flatbed scanner. You will get better results with a
microscope+camera combo.

 

[Bart van der Wolf]

Marc Heusser wrote: SNIP SNIP
Now onto the next order of business... getting your news client to
use the proper encoding (or do you actually *mean* to use the
exponent 3 as quotation marks?

Perhaps you should take your own advice, the double quotation marks on
Marc's original message showed as intended on my newsreader, not on
your response.

;-)

Bart

 

[Bart van der Wolf]

"Marc Heusser" <[email protected]>
wrote in message

Just to clear that: the Epson 4990 has 4800x9600 dpi *optical*
resolution - and scanning at 9600 gives much more detail.

Yes, I've recommended something like that ever since the 2450 model,
which shows an improved resolution in the "slow-scan" direction from
half stepping the scan head. It thus samples twice as fine in that
direction, and interpolates in the other direction to maintain aspect
ratio. Since the interpolation doesn't add resolution but the double
sampling does, one can choose to downsample by a factor of two
afterwards and lose very lttle in one but gain resolution in the other
direction.

It pays to do that if the crop area is smallish, because other issues
may be caused by the huge number of pixels of larger crops. How well
it works depends on the original's detail, and the amount of actual
overlap of the half stepped samples versus the scan lens resolution.

Bart

 

[Don]

Just to clear that: the Epson 4990 has 4800x9600 dpi *optical*
resolution - and scanning at 9600 gives much more detail. ....
But then we are using light of a wavelength eg 600 nanometres, so
practical resolution is probably limited at 2 micrometres. (9600 dpi is
2.6 micrometres). And the cuts are 6 micrometres or more thick.

You have to take this rated, optical resolution with a grain of salt.
It's only theoretical. The real resolution is (sometimes a lot) less.

To find out the actual resolution of your scanner you have to run the
so-called "slanted edge test".

In a nutshell, you take a very sharp object e.g. a razor and scan it
at a known angle. This produces a "pixel staircase" (my wording). From
that you can calculate the actual resolution of the scanner using some
math. There's a commercial program to do that at:
www.imatest.com
The procedure has also been standardized under ISO-16067, I believe,
so with that information you should be able to do the math manually.

Googling for "<insert scanner model here>" and "slanted edge" should
return quite a few hits with actual data for you scanner to give you a
rough idea of what to expect.

Very true for most materials - microscopic slides are exceptional in
having details down to much less than 1 micrometre.

I personally am not that much concerned with the actual resolution
because "it is what it is" and that's sufficient for my requirements
(film, photos).

But since you're dealing with real life rather than film or pics which
have limited effective signal data it may be useful to look into this.

Don.