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I would like to hear some criticism on the following concepts
relative to focus drift.
This is more of a philosophical approach than what I have seen in the
past but this is how I see and approach it.
I think it is nearly unreasonable to expect the variant factors in
the world that affect focus drift to remain constant or to be
controlled enough to prevent the problem.
Therefore, I think it is reasonable to approach the problem in
acceptance and anticipation of the variants.
So far all of the attempts to maintain constant focus that I have
seen adjust the space between the objective and the specimen to
compensate for the following:
thermal expansion
mechanical settling
perfusion induced focus shift
evaporation when the gravitational force on a coverslip is reduced
when the fluid level changes.
The problem is that you are always out of focus while you are trying
to find focus.
You are not in focus again until you arrive at the right point.
During that time an automated image capture could occur or you may be
unnecessarily photo-bleaching your specimen while finding focus.
If you use the coverslip as a reference point and the specimen moves
relative to the coverslip you are still at a disadvantage.
So, you might ask. What else can be done?
Here is where I am requesting criticism of a proposed system.
I have prepared a model of it located at:
http://66.39.20.117/autonomous/autonomous.gif
The system achieves focus by splitting the image emerging from the
objective lens into two pathways, (for you photon hungry types a 100%/
100% mirror can be used) a focus profiling pathway, diverted path,
and an observation pathway, straight path. Each pathway contains an
electronically indexed lens module. The focus profiling pathway lens
module is continuously varied providing Z slices to the focus
camera. The numeric value of the lens position in the focus
profiling pathway is correlated and evaluated with the image in the
focus profiling camera by computer. The indexed value for the
optimal image correction, obtained from the test path, is then
compared to the index value of the observation path lens module where
immediate correction is applied if and when necessary. Therefore,
time and three-dimensional information which would otherwise be lost
with conventional focusing methods is acquired, processed, and
utilized without negative effects to the observation path. In this
system all focusing is done in the back focal plane of the
objective. The distance between the specimen and the objective
remains fixed. Variations in focus can be made over a limited but
useful range + and - with respect to the objectives principle working
distance with simultaneous compensation for spherical aberration.
Advantages:
Ambient temperature regulation for the scope will not be necessary.
Dynamic compensation is independent of real world variants. (listed
above)
Specimens can be tracked in 3D independent of the coverslip surface.
The quantitative or observation path always knows where focus is and
can go directly to it without "delay"
Focus profiling can occur in the IR where there is no photo damage
and the numerical value of the focus for the observation path can be
offset for visual wavelengths.
Focus corrections in the observation path are only applied precisely
and only when necessary so there are no unnecessary interruptions in
the observation path.
A further extension of this technology can be applied to serpentine
scan applications where optical information profiled in the
peripheral region of an image can be combined with the intended
vector of travel so that the system can predetermine focus correction
before the specimen is brought into position. This saves time in
applications where repetitive focusing is required along a
predetermined path.
There is far more I could write about this system but don't want to
be more wordy than I already have.
I would appreciate if you would look at it and send me your comments
off line.
I submitted a grant to continue development of this but the reviewers
commented that there were already enough commercially available focus
correcting systems and it was not necessary. What do you think?
Does this have merit or am I barking up the wrong tree?
Dan
On Nov 17, 2006, at 1:15 AM, Stephen Cody wrote:
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G'day Olaf,
I did some rough experiments to get an idea of this using a Nikon 60x
Oil lens, 1.4na.
These experiments were conducted as a teaching exercise and I show
the results at Jim Pawley's 3D Microscopy of Living Cells course www.
3dcourse.ubc.ca/index.htm (Free plug Jim :) ).
In a nutshell I found that with a temperature fluctuation of about
1.5Deg C the focus drift was approximately 1.5um.
This was a simple "hand wavy" experiment, rather than designed to be
empirically correct.
This experiment has been discussed on the list before....see subject:
"time-lapse trouble on SP2"
For those who may be interested I've posted details of this
experiment on my web site www.ludwig.edu.au/confocal/drift .
I recommend anyone setting up live cell imaging for the first time
use this technique to monitor temperature fluctuations and z-drift.
Cheers
Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute For Cancer Research
PO Box 2008 Royal Melbourne Hospital
Parkville Victoria 3050
Australia
Tel: 61 3 9341 3155 Fax: 61 3 9341 3104
email: [log in to unmask]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Dr. Olaf Selchow
Sent: Wednesday, 15 November 2006 7:12 AM
To: [log in to unmask]
Subject: Thermal Focus Drift - Olympus BX 51 WI -
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Hello List,
anybody out there who has an idea about how much focus shift
(approximately) I can expect from a 5 (10) deg celsius temperature
change in the lab on an Olympus BX 51 WI?
With aproximately I mean: is it 1, 5 or 10 µm?? Or even more?
I know I should keep the temperature constant (the goal in our case is
plus minus 1 deg C) but we need to know how much the focus shifts to
have good arguments to ask for better air conditioning the lab... A
measurement would take time - therefore it would help a lot if someone
has done the test already and can help out with his experience.
Thanks very much!
Olaf
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Cancer Research.
Dan Focht
Bioptechs Inc.
V (724)282-7145
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