Oil vs water objectives
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Quite so. That is why water lenses have NA less than 1.33 -more like 1.1-1.2. Cheers On 5/10/2012, at 3:35 PM, James Pawley <[hidden email]> wrote: >> > > > Very clear. > > But what I would like to add is that, even before you reach the critical angle for TOTAL internal reflection, the interface will still reflect some fraction of the incident light. This fraction increases as you get closer to the critical angle. As a result, it doesn't make much sense to make an NA 1.33 water lens. Not only would the correction collar be very finicky, but the rays between 1.20 and 1.33 would be increasingly attenuated by "non-total" reflections taking place at all (3?) of the flat, horizontal glass-water surfaces. > > Cheers, > > Jim P. > > >> -----Original Message----- >> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel >> Sent: Friday, 5 October 2012 10:29 PM >> To: [hidden email] >> Subject: Re: Oil vs water objectives >> >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> On 04.10.2012 18:20, Cammer, Michael wrote: >>> Also, while people have generated various test samples of >> collagen and latex and acrylic and fat globules and glass beads and carrageen gum and dissolved Scotch tape glue etc., the only test sample that really answers the question is the sample itself. >> >> Again, I agree, in particular if you look at tissues with more or less unpredictable optical properties. Still, bead preparations allow to demonstrate the general effect very clearly and thus convincingly. They also allow quantification and sometimes hard numbers help to convince people. Some person might argue that they don't care about resolution since s/he is planning to use a 1 µm pixel size anyway. But you still can convince these people if you can show them that they will also loose a lot of intensity. >> I use such a demonstration to argue for the usage of 170 µm coverslips and a good embedding medium. >> >> On 05.10.2012 02:40, Guy Cox wrote: >>> Nobody seems to have mentioned so far that the NA of an oil >> objective will NOT be 1.4 if it is imaging a sample in water. >> The maximum it can be is 1.33 - the refractive index of water. >> ... >>> So the oil objective has little or no advantage in NA >> >> Is that really true? If there is oil between the coverslip and the oil objective, but water immersion for the water objective, this should make a noticeable difference in effective acceptance angle, should it not? >> (Assuming the object is rather close to the coverslip). >> >> Steffen >> >> >> -- >> ------------------------------------------------------------ >> Steffen Dietzel, PD Dr. rer. nat >> Ludwig-Maximilians-Universität München >> Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy >> >> Mail room: >> Marchioninistr. 15, D-81377 München >> >> Building location: >> Marchioninistr. 27, München-Großhadern > > > -- > James and Christine Pawley, PO Box 2348, 5446 Burley Place (PO Box 2348), Sechelt, BC, Canada, V0N3A0, 604-885-0840 NEW! Cell (when I remember to turn it on!) 1-765-637-1917, <[hidden email]> Mark B. Cannell Ph.D. FRSNZ Professor of Cardiac Cell Biology School of Physiology& Pharmacology Medical Sciences Building University of Bristol Bristol BS8 1TD UK [hidden email] |
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In reply to this post by Gabriel Lapointe-4
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hallo, If you cannot easily find a point object in your sample, you can optimize the setting of the coverslip adjustment collar by running live view on the confocal and maximizing the signal. Pick a specific feature in the specimen, then turn the collar on the objective a bit and re-adjust the focus. Watch if the feature is getting brighter or dimmer. The signal is the brightest when optimal correction is achieved. I like to use the saturation lookup table to highlight saturated pixels in red (Leica uses a different LUT, but the principle is the same), setting the detector voltage just below saturation for the selected feature in the specimen. It is then easy to see if the signal got stronger after adjusting the collar. The downside of this method is that you can easily photobleach your sample, so do this on a less important area of the specimen. Stan Vitha Microscopy and Imaging Center Texas A&M University On Fri, 5 Oct 2012 07:26:36 +0800, James Pawley <[hidden email]> wrote: >Hi all, > >Looks like you have received a lot of good >advice, but I would like to add one thing. > >Yes, do a test BUT don't forget to be very >careful in adjusting the SA correction collar on >the water lens. )Find a point object: focus up >and down and adjust for symmetry (i.e., for the >same image a little above focus as a little below >focus. DON'T just try to "make it sharper!" The >collar changes the focus plane.) > >Best, > >Jim Pawley, > >Near Harbin, China. > > >> >>On 04.10.2012 15:15, Gabriel Lapointe wrote: >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> Hi, >>> >>> I have a user who insist that using a 1,27NA water immersion objective >>> is brighter and would give better images than using a 1,4NA oil >>> immersion. I understand that deeper into the media that would be true. >>> But, in that particular case, we are talking about imaging GFP at less >>> than 100 micron away with a spinning disk. >>> >>> So, I was wondering at which distance from the coverslips do we start >>> seeing benefits of using a water immersion objective over an oil >>> objective in aqueous media. >>> >>> Thanks for your help. >>> >>> Sincerely >>> *Gabriel Lapointe, M.Sc.* >> > Lab Manager / Microscopy Specialist >>> Concordia University, Biology Department >>> 7141 Sherbrooke St. West SP 534 >>> Montréal QC H4B 1R6 Canada >>> [hidden email] >>> cmac.concordia.ca >>> http://gabriellapointe.ca >>> |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** A similar method (adapted from 4Pi microscopy) which doesn't rely on either point-like objects or sample fluorescence is to image the water-glass interface in reflection mode (4Pi coverslips are typically silvered at the edges to strengthen this reflection - the silvering is probably necessary when using oil or glycerol immersion, but the ri difference for water should give you enough reflection without silvering). If you set the microscope to perform an x-z scan rather than the normal xy, you don't need to refocus (at least for small changes in the collar) as the line representing the interface will just move up and down. I would typically start by maximising intensity, but switch to optimising the depth of focus and symmetry at the end (the maxima are not far apart, and for non 4Pi modes there might not be an advantage in going for the axial half width over intensity). An x-z scan is also my preferred tool when optimising on fluorescent beads, in which case I look for both axial width and symmetry. An additional aspect (other than the evanescent excitation already mentioned) which might make oil objectives considerably brighter than water for a sample close (< ~1 lambda) to the coverslip is that fluorophores near to the interface have a very non-isotropic emission profile with emission being strongly coupled into the glass at the critical angle (see, e.g., Axelrod & Hellen, "Emission of Fluorescence at an Interface", Methods in Cell Biology, Vol 30, Ch 15). As the bulk of this fluorescence is at the critical angle, it will not be captured by a water objective, but will be captured by a higher NA oil objective. cheers, David ________________________________ From: Stanislav Vitha <[hidden email]> To: [hidden email] Sent: Tuesday, 9 October 2012 6:29 AM Subject: Re: Oil vs water objectives ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hallo, If you cannot easily find a point object in your sample, you can optimize the setting of the coverslip adjustment collar by running live view on the confocal and maximizing the signal. Pick a specific feature in the specimen, then turn the collar on the objective a bit and re-adjust the focus. Watch if the feature is getting brighter or dimmer. The signal is the brightest when optimal correction is achieved. I like to use the saturation lookup table to highlight saturated pixels in red (Leica uses a different LUT, but the principle is the same), setting the detector voltage just below saturation for the selected feature in the specimen. It is then easy to see if the signal got stronger after adjusting the collar. The downside of this method is that you can easily photobleach your sample, so do this on a less important area of the specimen. Stan Vitha Microscopy and Imaging Center Texas A&M University On Fri, 5 Oct 2012 07:26:36 +0800, James Pawley <[hidden email]> wrote: >Hi all, > >Looks like you have received a lot of good >advice, but I would like to add one thing. > >Yes, do a test BUT don't forget to be very >careful in adjusting the SA correction collar on >the water lens. )Find a point object: focus up >and down and adjust for symmetry (i.e., for the >same image a little above focus as a little below >focus. DON'T just try to "make it sharper!" The >collar changes the focus plane.) > >Best, > >Jim Pawley, > >Near Harbin, China. > > >> >>On 04.10.2012 15:15, Gabriel Lapointe wrote: >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> Hi, >>> >>> I have a user who insist that using a 1,27NA water immersion objective >>> is brighter and would give better images than using a 1,4NA oil >>> immersion. I understand that deeper into the media that would be true. >>> But, in that particular case, we are talking about imaging GFP at less >>> than 100 micron away with a spinning disk. >>> >>> So, I was wondering at which distance from the coverslips do we start >>> seeing benefits of using a water immersion objective over an oil >>> objective in aqueous media. >>> >>> Thanks for your help. >>> >>> Sincerely >>> *Gabriel Lapointe, M.Sc.* >> > Lab Manager / Microscopy Specialist >>> Concordia University, Biology Department >>> 7141 Sherbrooke St. West SP 534 >>> Montréal QC H4B 1R6 Canada >>> [hidden email] >>> cmac.concordia.ca >>> http://gabriellapointe.ca >>> |
 
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Anje Sporbert |
Position in Microscopy Core Facility of Max Delbrueck Center Berlin Germany
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In reply to this post by Alison J. North
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear all, the MAX-DELBRUECK-CENTER FOR MOLECULAR MEDICINE in Berlin-Buch is offering one POSITION (SCIENTIST) to support the the excellently equipped MICROSCOPY CORE FACILITY of the MDC hosting a broad range of light microscope techniques such as confocal microscopy, 2-photon/intravital microscopy, TIRF microscopy, laser micro-dissection, light-sheet microscopy and a number of image analysis workstations. Job Description: We are looking for a highly motivated person who will work together with scientists in the field of cardiovascular, cancer, neuro and developmental biology. Duties include maintenance of the microscope systems and facility infrastructure, user training and support, assistance of users with data analysis and processing, instrument performance tests and trouble shooting, contribution to the teaching activities of the facility (i.e., tutorials, seminars). Project-based method development in collaboration with MDC research groups is possible. Experience: MSc, PhD or similar in Physics, Biology, Life science, Bioinformatics or related fields. Extensive hands-on experience with fluorescence and confocal microscopy, image analysis, good organisational, problem-solving and communication skills within the team and with collaborators and users are required. Experience with TIRF microscopy, 2-photon microscopy, live cell imaging, laser micro-dissection, superresolution microscopy or advanced image analysis would be an asset, skills in optical instrumentation or programming are highly appreciated. The full position is available immediately, initially limited to 2 years with the possibility of extension. Salary is dependent on qualification. Applications (Ref No: 10342/2012) should be sent by 15. November 2012 including a detailed CV, short description of the relevant experience and motivation as well as contact information for 2 references to: [hidden email] or Personnel Department Max-Delbrueck-Center for Molecular Medicine (MDC) Berlin-Buch, Robert-Rössle-Str. 10, 13092 Berlin-Buch, Germany. Further information on http://www.mdc-berlin.de/en/research/core_facilities/index.html or Dr. Anje Sporbert ([hidden email]). ------------------------------------------------- Dr. Anje Sporbert 2-Photon and Confocal Microscopy Core Facility Max-Delbrück-Center for Molecular Medicine Berlin 13125 Berlin Germany -- ______________________ Dr. Anje Sporbert Max-Delbrück-Center for Molecular Medicine Robert-Rössle-Str. 10 13125 Berlin/Germany |
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