bluelyme said...
maybe toots can explain flourencence microscopy a bit
Typical vertical fluorescent microscope setup:
VIEW IMAGEI'm not an expert on fluoro microscopy, and I've done very little of any type lately on account of being busier with life. "Jack Stravinsky" is more of an authority on it than I am, and he is doing some awesome work with Borrelia using Acridine Orange and a fluorescent microscope. He is "Lymedin2010" on Lymenet.org. Unfortunately, his Photobucket account needs updated to view his pictures on Lymenet, but his videos are awesome. Notice our discussion and the details about
a fluorescent microscope hack using color-specific LED lights.
flash.lymenet.org/ubb/ultimatebb.php/topic/1/120458/19Fluorescent microscopy is used for inspecting various specimens dyed with stains that contain specific fluorochromes. It's the fluorochromes in the stain that cause different parts of the specimen to glow when illuminated under the correct wavelength of light. The correct wavelength (color of light) used is different depending on the type of (fluoro) stain one is using with the specimen, and, depending on what item in the specimen one is trying to illuminate.
Fluoro microscopy can be very helpful for those of us looking at our own blood because certain fluoro stains can illuminate the infectious organisms. For our purposes, acridine orange is the easiest one to use and obtain, and is probably the cheapest. Acridine Orange (AO) stains DNA and RNA, thus making it very easy to visualize bacteria and protozoans even at 400x. Since red blood cells only stain dull green and bacteria and protozoans stain bright orange, it is very easy to pick out microorganisms, especially if one sees the correct morphology (proper shape, size, and
location).
The main reason I wanted to do my own fluorescent microscopy is because I do my own Giemsa smears and it can be nearly impossible sometimes to differentiate a Babesia parasite, or an Anaplasma morulae, from a platelet. Sometimes these pathogens take on variations of morphology that make it very difficult to say with certainty what I am seeing....especially since platelets stain the same color in Giemsa as do pathogens, and platelets typically are the same size as pyriforms and morulas. But, in Acridine Orange, platelets stain a pale, whitish, light green color. So, it's very easy to tell the difference. FWIW, spirochetes
(IN ANY FORM) stain green or red.
VIEW IMAGEHere are some examples of my own work. Notice the color of the red blood cells, the platelets, the white blood cell nuclei (which contain DNA/RNA), and the pathogens:
A Neutrophil chasing a rod bacteria between red blood cells:
VIEW IMAGEA rod bacteria on or in a red blood cell. Notice the pale, whitish, light green platelets:
VIEW IMAGETypical Bartonella (orange dot) on the peripheral of a red blood cell:
VIEW IMAGEProbable Babesia parasite in a red blood cell:
VIEW IMAGEThat last picture is very likely showing a (pyriform) Babesia parasite. Notice in this next picture how much the Babesia (ringform) resembles the platelets to the right of it. This illustrates how much easier it is to discern between platelet and Babesia in a fluorescent stain versus a Giemsa stain.
VIEW IMAGEFor those interested, to get the Acridine Orange (stain) fluorochromes to cause DNA and RNA to glow red and green, the color wavelength of blue must be used to illuminate the specimen (anywhere from 435nm to 500nm is considered in the blue spectrum). In a typical vertical fluorescent microscope, you must have the correct excitation "cube" (longpass filter) to properly use Acridine Orange stain. Incidentally, most fluorescent scopes are equipped with that one. It's always best to check before buying one though!
An illustration of the wavelength spectrum of the color blue (needed for using Acridine Orange):
VIEW IMAGENotice the excitation wavelength specs for Acridine Orange for the illumination of DNA and RNA (in Table 2 on this page):
/www.microscopyu.com/techniques/fluorescence/nikon-fluorescence-filter-sets/blue-excitation-filter-setsAn expensive vertical fluorescent microscope is not necessary to do fluorescent microscopy, though. As long as you have a regular brightfield microscope and some Acridine Orange, you can do fluoro microscopy (with a little improvising). A blue LED light such as this...............:
/www.amazon.com/dp/B010NQMSKA/ref=sxts_bia_sr_1?pf_rd_m=ATVPDKIKX0DER&pf_rd_p=3182441022&pd_rd_wg=mtia4&pf_rd_r=2A229T6NPV74GB11VTR7&pf_rd_s=desktop-sx-top-slot&pf_rd_t=301&pd_rd_i=B010NQMSKA&pd_rd_w=CWTm3&pf_rd_i=rgb+led+bulb+60w&pd_rd_r=8F4VT17YHR0YZHTEGGS1&ie=UTF8&qid=1505418532&sr=1 or this (the higher the wattage the better!):
/www.amazon.com/Ustellar-Changing-Waterproof-Dimmable-Security/dp/B06XJ5QH9T/ref=sr_1_9?ie=UTF8&qid=1505418532&sr=8-9&keywords=rgb+led+bulb+60w .........pointed down on the specimen should excite the fluorochromes in the Acridine Orange and light up the typical stuff. Of course, you would only use the blue LEDs for the Acridine Orange.
Here are some examples of various degrees of hacks:
www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artjun09/ld-fluoro.htmlwbg.wormbook.org/2009/12/01/using-leds-as-a-low-cost-source-to-detect-gfp-and-dsred-2//asymptoticdesign.wordpress.com/2012/03/10/diy-scope/journals.plos.org/plosone/article?id=10.1371/journal.pone.0011890Post Edited (TOOTY) : 9/14/2017 2:17:46 PM (GMT-6)