Fwd: pgFocus

[Karl Bellve]

> Dear Karl Bellvé,
>
> I found your pgFocus design online and intend to duplicate this
> system. I do have some questions and was wondering if you could share
> your thoughts.
>
> - I believe you use the pgFocus exclusively for TIRF imaging. I plan
> on using it for regular epifluorescence work (without immersion) on an
> inverted microscope. Do you see any issues with using the pgFocus in
> such a use-case? Of course the reflection will be from the (bottom)
> air-cover glass interface and not the (top) cover glas-air interface;
> but I see no harm in that.

It depends on the NA of your objective. The beam can be reflected
because TIRF objectives have a high NA, which allows the beam to come in
at a steep angle and return at a steep angle. I think a NA 1.4 and above
should be fine, but not sure about anything below.

Of course, you don't have to do a reflected beam at a steep angle, but a
straight beam which is how Definite Focus works.

>
> - How did you choose the laser wavelength of 808 nm? Does it require
> an objective that is specified to work in the IR?

808nm was just out of the range of what we normally image. Any
wavelength would work.

>
> - The TSL1401 sensor that is being used has a fairly large pixel pitch
> of 63.5 um. Is there a benefit of going to smaller pixel sizes?

Yes. You can go smaller, and move the sensor closer to the microscope.
The reflected beam is basically like a lever. The longer the beam path,
the bigger the translation at the sensor. If you use a smaller sensor,
then you can use a shorter beam path.

>
> - What is the use of the ADC on the pgFocus? I think it samples an
> external voltage set-point that can be added to the DAC signal output.
> Wouldn't it be possible to use an opamp to sum the DAC and set-point
> voltages?

That is what I do.

The external voltage, is sampled by an ADC, but that external voltage is
also passed all the way through, staying in the analog domain. That
external voltage is modified by a DAC, via an Op Amp to correct focus,
based on a given focus set point. That focus set point can changed based
on the external voltage, which modifies the DAC control response..




--

Karl Bellvé
Biomedical Imaging Group
Molecular Medicine
University of Massachusetts Medical School