RE: [Histonet] Fluorescence-filters
Sure you can, all you need is a bright enough lightsource which emits at the correct wavelength. If the spectrum of the light source is too wide, you dump in a filter to narrow it down to your desired excitation. Flow cytometers nowadays even use LED lasers instead of the huge, bulky and vulnerable gas lasers of the old days. Which is a good thing. Jonathan --- Jonathan Cremer Laboratory Technician TARGID - KU Leuven Van: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] namens Emily Sours [talulahg...@gmail.com] Verzonden: woensdag 6 juni 2012 1:31 Aan: histonet@lists.utsouthwestern.edu Onderwerp: Re: [Histonet] Fluorescence-filters Can you do fluorescence with LED? (That may be a stupid question, as I always thought LED was just for brightfield). Emily You see a peanut, day's off to a good start; you witness some soil it's a jamboree for Vince Noir. --Howard Moon, in Charlie, The Mighty Boosh ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Fluorescence-filters
We recently installed a LED light source on our fluorescence scope that our pathologists use for reading IF on kidneys and skins. They love it. No more having to warm up the bulb, not turning it off too soon, or my nightmare - leaving it on overnight. The bulb is supposed to last a really long time and gives off a nice bright light. Sue Hunter, Supervisor Advanced Diagnostics Beaumont Health System Royal Oak MI 248-898-5146 shun...@beaumont.edu -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Emily Sours Sent: Tuesday, June 05, 2012 7:32 PM To: histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Fluorescence-filters Can you do fluorescence with LED? (That may be a stupid question, as I always thought LED was just for brightfield). Emily You see a peanut, day's off to a good start; you witness some soil it's a jamboree for Vince Noir. --Howard Moon, in Charlie, The Mighty Boosh ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet To report this email as SPAM, please forward it to s...@websense.com. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fluorescence-filters
Hi Gudrun: What you wrote is news for me. Dichroic filters with specific lambda transmission of good quality can be used for years. The filters I used were Ernst Leitz, from the early 1950's and the transmission intensity remained strong. We checked the life duration of the mercury lamp, but not of the filters. If you are getting weaker signals perhaps is another cause. René J. From: Gudrun Lang gu.l...@gmx.at To: histonet@lists.utsouthwestern.edu Sent: Tuesday, June 5, 2012 10:37 AM Subject: [Histonet] Fluorescence-filters Hi! Filters for fluorescencemicroscopy tend to burn out after a certain duration of usage. What duration? We have filters for FITC, TRITC, Dapi and a triplefilter. The working-hours are about 150 per year. What do you think? Is it time to change them. I have often bad feedback about weak signals, and I would not be surprised if the microscope is the culprit and not our protocol. Weak signals refer last times to ALK-FISH on lung biopsies. Well fixed but tumourcells mixed within collagenfibers. - and unfortunately unexperienced doctors on reading of this special probe. Hoping for responses Gudrun Lang ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Fluorescence-filters
Filters are usually very stable. What is burning out are fiber optic cables that are present in some illumination systems such as X-Cite in Zeiss microscopes. These optical fiber cables should be replaced after 2000 hours. Anatoli Gleiberman, PhD Director of Histopathology Cleveland Biolabs, Inc 73 High Street Buffalo, NY 14203 phone:716-849-6810 ext.354 fax:716-849-6817 e-mail: agleiber...@cbiolabs.com -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Gudrun Lang Sent: Tuesday, June 05, 2012 10:38 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Fluorescence-filters Hi! Filters for fluorescencemicroscopy tend to burn out after a certain duration of usage. What duration? We have filters for FITC, TRITC, Dapi and a triplefilter. The working-hours are about 150 per year. What do you think? Is it time to change them. I have often bad feedback about weak signals, and I would not be surprised if the microscope is the culprit and not our protocol. Weak signals refer last times to ALK-FISH on lung biopsies. Well fixed but tumourcells mixed within collagenfibers. - and unfortunately unexperienced doctors on reading of this special probe. Hoping for responses Gudrun Lang ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This communication may contain privileged information. It is intended solely for the use of the addressee. If you are not the intended recipient, you are strictly prohibited from disclosing, copying, distributing or using any of this information. If you received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. This communication may contain nonpublic information about individuals and businesses subject to the restrictions of the Gramm-Leach-Bliley Act. You may not directly or indirectly reuse or redisclose such information for any purpose other than to provide the services for which you are receiving the information. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fluorescence-filters
Hi Gudrun, i read about 15 ALK cases a week. If you are seeing a lot of collagen fibers around the tumor cells, I'd try increasing the digestion time of your pepsin (especially if they were fixed for longer than usual). Before altering the pretreatment though, you would want to make sure that these slides were not exposed to light for any period of time. The discrete signals of the probes can quickly fade (much faster than with IF stained slides). I'd also make sure the door is closed in your dark room. If there is light in the corner of your eye the signals can be hard to see. ALK FISH should be scored at high power under oil immersion (60-100x objective). If they are scoring at 40x, it could be a problem. good luck! Mark Tarango On Tuesday, June 5, 2012, Eric Hoy wrote: We do a LOT of fluorescent microscopy in our immunology lab, so I have a bit of experience with fluorescence. The answer to your question depends on what type of filters you have in your microscope, and what type of light source is on the microscope. Older fluorescent systems used absorption filters, which were simply discs of coloured glass. These filters had a fairly wide band-pass, so the fluorescence tended to be less than we see with interference filters. The good news with these filters is that they are nearly indestructible (unless you drop and break them.) Interference filters are produced by vacuum deposition of a thin film of metal vapour on high-quality glass. These filters usually have much sharper band pass characteristics than absorption filters. They are also considerably more expensive. If handled properly, these filters will last for decades, but improper cleaning and handling of the filters can shorten their lifespan. I have also heard that prolonged exposure to solvent vapours (such as we find in a histology lab), can damage the filters, although I have not seen any filters that suffered this type of damage. I have seen interference filters that show delamination of the metal film over time. In my experience these are older filters that were not produced with the current technologies, and filters that have been mishandled. Interference filters made in the past 20 years should last as long as the microscope, if they are properly handled. If you are seeing reduced fluorescence, I would suspect the light source as the most likely problem. Halogen lamps have less intensity than mercury vapour lamps, which are less intense than metal halide lamps, which are less intense than LED sources. We have converted all of our microscopes to LED sources. If you are using an older HBO or halogen lamp, the age of the lamp, the initial wattage of the lamp, and the alignment can all affect the fluorescent output. As you identified, perhaps the most important aspect of immunofluorescence is the skill and experience of the person who reads the slides. Let me know if you have further questions. Eric Hoy === Eric S. Hoy, Ph.D., SI(ASCP) Clinical Associate Professor Department of Medical Laboratory Sciences The University of Texas Southwestern Medical Center Dallas, Texas Email: eric@utsouthwestern.edu === On 6/5/12 9:37 AM, Gudrun Lang gu.l...@gmx.at javascript:; wrote: Hi! Filters for fluorescencemicroscopy tend to burn out after a certain duration of usage. What duration? We have filters for FITC, TRITC, Dapi and a triplefilter. The working-hours are about 150 per year. What do you think? Is it time to change them. I have often bad feedback about weak signals, and I would not be surprised if the microscope is the culprit and not our protocol. Weak signals refer last times to ALK-FISH on lung biopsies. Well fixed but tumourcells mixed within collagenfibers. - and unfortunately unexperienced doctors on reading of this special probe. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu javascript:; http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fluorescence-filters
Wow, that was detailed and interesting!! I didn't know microscopes could even use LED, does that require a different setup, or just a different bulb? Emily You see a peanut, day's off to a good start; you witness some soil it's a jamboree for Vince Noir. --Howard Moon, in Charlie, The Mighty Boosh ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Fluorescence-filters
Can you do fluorescence with LED? (That may be a stupid question, as I always thought LED was just for brightfield). Emily You see a peanut, day's off to a good start; you witness some soil it's a jamboree for Vince Noir. --Howard Moon, in Charlie, The Mighty Boosh ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
