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Zeiss LSM700 has a new stage insert

We got a new stage insert for Zeiss LSM700. The old insert was already partially broken and it ground the objective and cover glasses so that there was always small pieces of glass under the metal plates. So we decided to get a new one.

On the new insert a sample is lower than on previous insert and there is a danger to scratch other objectives than what you are using. We have tried to minimize the risk but you have to be more careful than previously when you use the microscope. Especially when you use 5x objective you have to be very careful not to scratch big objective beside it.

Dipping objectives should not be attached to turret unless you use them and with them you also have to be extra careful. Write a Session Request if you need a dish adapter because the adapter may not be changed by users.

We have limited the movement of the stage more than previously to prevent most of the accidents. To change a slide you might want to lower the stage more than Load Position -button gives you to avoid scratching an objective.

stage - 10xb_annotatedb

Green arrow shows movable side clamps which holds the slide stable. The back knobs of the clamps (shown by red arrows) are dangerous for big objectives when using 5x or dipping objectives. Do not use automatic return to focus level with these objectives.

– Mika

Status of LMU microscopes December 2014

Leica DM 6000:

New wide field microscope which had first trainings during week 51. Together with IT department we got computer working with special ‘Start Microscope’ -button. Everyone who has been at trainings can reserve and use the microscope. Normal trainings by LMU staff will start on January.

 

Leica SP5:

The problem is that occasionally scanning field does not center properly. You see this if an image has some black corners with zoom 1. Also if zoom in -area does not go to correct position, the reason is that centering of the whole scanning field has been wrong either before or after zoom in. If you have this problem try changing zoom or moving scanning area by arrow buttons during Live-scan. Problem does not affect resolution or laser powers.

 

Leica SP5II HCS A

Z-Stage has unhealthy noice sometime (about 7kHz). Calibration of the stage did not help. Field size in bright field is smaller with 2.5x and 5x objectives as it should be. Hybrid Detector 4 is very sensitive with extra light and might shut down itself. If it shut down, it might be impossible to get it back on without long wait after shutting down the whole system. DIC Prism with ScanDIC mode at Transmitted light PMT goes away when you activate Hybrid Detector on fluorescence detection. You have to click ScanDIC mode setting again. Slide insert has lost its left nut so that it can’t get attached into the stage as firmly as it should. We try to find a new one. When you change inserts, be aware of the small parts (screws, nuts, small white rubber ring on plate insert) and don’t loose them! They are difficult to replace.

 

Leica SP5MP

Argon laser power fluctuates within 10-20 minutes. Leica maintenance is trying to find a solution. SPAD detectors alignment is still bad so that they are not usable. Support from Germany is required.  Multiphoton laser is not fully aligned and requires realignment.

 

3I Marianas

FLIM alignment is going on and it is not usable at the moment. Definite focus requires that objective zero level is within working distance of the focus (for example 250-300μm with 63x water immersion objective) and it needs to be started before the microscope. Zero level is the level where the objectives are when microscope starts up. If you need to get zero level to focus: 1) focus your sample 2) Shut down microscope by Power Supply 232 (White box and Definite focus 3) Turn on Definite focus 4) Turn on microscope.

 

Zeiss LSM 700

Working as designed

 

CellInsight

System needs restart after every plate. Erroneous images with wrong excitation or filters starts to appear more frequently in time. We have not got helpful support from the company.

 

Cell-IQs

Working as designed

 

– Mika

Christmas 2014 and New Year 2015

Support is limited at 20.12.-6.1.2015.

During this thime LMU persons at work are:

Mika: 21.12.
Harri: 29.12.-31.12.

Kimmo and Marko are at holiday until 7.1.2015.

 

Merry Christmas and Happy New Year!

 

– Mika

Square Pinhole

A Very good article from Leica why they use square pinhole instead of some other geometry. Pinhole Geometry: Four Corners are Perfect.

– Mika

Labtek II chambers

There is a message from Confocal list for a solution to use the Labtek II chambers in microscopes where the tab at the end causes problems.

 

– Mika

————————————————————————————

Date: Fri, 15 Mar 2013 22:32:48 +0000
From: “Cammer, Michael” <Michael.Cammer@MED.NYU.EDU>
Reply-To: Confocal Microscopy List <CONFOCALMICROSCOPY@LISTS.UMN.EDU>
To: CONFOCALMICROSCOPY@LISTS.UMN.EDU
Subject: Re: Nunc Lab-Tek problem :  A Solution

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

After all the angst about the new chambers with tabs at the end, we came up with a
very simple cheap solution.  We just clip the ends off.  It’s low tech but works.

Thank you for the discussion without which we would not have come upon this simple
solution.

Anyone interested can see

fixed with razor scraping

followed by

How To Use Box Plots in ImarisVantage

Box Plots in Imaris Vantage to get more out of your data!

www.bitplane.com/learning/box-plots-in-imarisvantage-tutorial

 

– Mika

Step by Step Guide to Hybrid Detection and Photon Counting

Interactive tutorial for phonton counting by Leica

http://www.leica-microsystems.com/science-lab/step-by-step-guide-to-hybrid-detection-and-photon-counting/

– Mika

LMU imaging project competition

Dear all,

I would like to remind you about the LMU imaging project competition.
Submission deadline is at the end of January 2014:

Win free imaging time! (Success stories from LMU)

During the past years we have had the honor of seeing many brilliant
imaging projects come to fruition. Undoubtedly, many of you have planned
and executed great projects without any input from us.  Now we would like
to hear from you. Unfortunately, many of our users still don’t know
everything they could try to do. Imaging, as relevant as it can be, is not
widely discussed and the results people share tend to focus on the
biological findings more than the means that were used to achieve them.
>From our point of view, this is a waste as other people from different
fields could potentially benefit from the ideas behind the methods used
elsewhere. We are planning to establish a method wiki, detailing what can
be done with each of our machines. And for that, we need your help.

To facilitate this, we are opening a competition for the best imaging projects.
We ask the participants to write a 1½ page paper with relevant pictures about
your project. What have you done and why? How have you done it? How were the
results?

The papers we receive will form the basis of the LMU-wiki as described
above. We hope that by sharing the ideas we can benefit whole of the
scientific community in Helsinki. The authors of the three best papers, as
determined by us, will be asked to give a brief talk about their project
at the third annual LMU seminar (on 4th of March 2014). These three
will be also rewarded 10 hours of free imaging time to be used as they
wish (within the limitations of normal booking rules).  During the
seminar, the LMU-staff will also reward the best presentation with an
additional more personal prize.

Best regards,

LMU staff

Bounding box statistical variable in Imaris

If you need to know how long some structures are but their orientation vary, Imaris has a nice variables called Bounding boxes for surface objects.

Bitplane has made a nice short tutorial for them: Bounding box tutorial.

– Mika

Stack overlays to tiff-series by Fiji

Quick and dirty method to make a overlay tiff-series from stacks by Fiji.

1) Open a stack to Fiji by Hyperstack

2) Change the stack to Image5D stack

  • Plugins/Image5D/Stack to Image5D
    Default Colorscheme for channels is RGB, non-default is typically  grayscale for every channel
  • Choose Overlay from dropdown list beside the image
  • Click Color
  • Change color for the channels if needed
  • If channel intensity is weak, adjust LUT (not for quick and dirty method)

4) Change Image5D Overlay to RGB

  • Plugins/Image5D/Image 5D Stack to RGB

5) Save as Image Sequence choosing TIFF as a Format

 

– Mika