Dr. Teresa,

The most likely issue is the thickness of the mountant.  We place our beads on a glass slide, then overlay with mountant and a 1.5 coverslip (160 to 190 microns thick).  If you have a high mag objective with short working distance (WD) and no correction collar, you indeed may not be able to focus deeply enough to see them.  There is a wonderful tutorial on this by Dr. Davidson at http://www.microscopyu.com/tutorials/java/aberrations/correctioncollar/

One quick check is to image them at lower magnification - if you can see them, the mountant thickness and short WD are the most likely culprits.  (There can also be some variation to coverslip thickness.) Many of the newer objectives have improved WDs at high magnification and numerical aperture and come with correction collars (at a price though).  

We are instituting QC and manufacturing controls to prevent our contribution to this issue going forward.  You may want to consider buying and mounting the desired stand alone beads directly onto a coverslip for the immediate future (cheaper than a new objective!).  We will of course refund your money for the prepared slides if we are unable to resolve the issue with your current equipment.

Kind Regards,

Mike Ignatius

Product Development Manager
Molecular Probes/LifeTechnologies
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541 335-0414


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of MICROSCOPIA CONFOCAL y CCD
Sent: Wednesday, March 24, 2010 9:06 AM
To: [log in to unmask]
Subject: FocalCheck test slide

Does anybody have experience with the FocalCheck 
Fluorescence Microscope Test Slides from Molecular Probes.?
I bought Test Slide #1 and I'd focus problems 
especially with oil immersion objectives in 
different microscopes. They have sent me another 
one and I'm having the same trouble focusing.I do 
not understand what is happening. ¿Someone has had problems with this kit?

Thanks in advance

M. Teresa

SERVICIO de MICROSCOPIA CONFOCAL y CCD
Mª Teresa Seisdedos Domínguez
Oscar Hidalgo Blanco
Amadeo Cazaña Soto
[log in to unmask]
Centro Investigaciones Biológicas (CIB) CSIC
C/Ramiro de Maeztu, 9
28040 Madrid
Phone: + 34-91 8373112 ext.4401
Fax: + 34-91 536 04 32