Photomicroscopy and Macro Photography
(Near Ultraviolet to Near Infrared)
Some light sources, especially ultraviolet ones, can pose a significant hazard. Considerable care should be taken with such equipment (1, 2, ICNIRP) which is freely available on the internet. Children must not have unsupervised access to such light sources which should be unplugged and stored away after use or have their batteries removed.
As with aerial archaeology, I am investigating simple, low-cost approaches to microscopy and macro photography using light outside the visible spectrum and blue light where it can be used as a substitute for the near ultraviolet (NUV). In general, I will be using relatively low intensity light sources. This page is intended as a non-sequential blog on my journey, with bits added/removed along the way. I will specify filters etc when I believe that I have the most cost-effective solutions. I will keep the text brief and introduce the various topics with specific examples, including details of the equipment used. Unless otherwise stated, images have not been stacked.
John with grandson Jack
Professionally, I started using microscopes for biological work in 1972 for observing freshly explanted human tumour cells in vitro and also their chromosomes. Before then, my microscopy was limited to physics and measurement. Soon afterwards, I worked on blood and with hair follicles in vitro. Amongst other things, I was interested in the effects of ionising radiation on skin, hair growth and on micronucleus production in relation to biological radiation dosimetry following accidental non-uniform exposures (eg Chernobyl).
John back in the early 1980s at Berkeley Nuclear Laboratories.
I was intrigued to discover that grey/white human hair can act as an annular light guide (Nature, 338, 23, 2 March 1989, see here for a recent review). The central core of the hair, the medulla, does not transmit light but the medulla can disappear, following hair growth after exposure of the hair follicle to ionising radiation in vivo (Int. J. Rad. Biol, 58, 2, 383, 1990) and probably after exposure to other cytotoxic agents too. Sometime after irradiation and regrowth, the medulla can reappear with the length of the medulla deletion (and any reduction in hair diameter) being dose-dependent.
Transmitted and Reflected Near Infrared Light
Microscopes with LED illumination, like the one below, need a tungsten or halogen substage lamp for transmission work in the near infrared (NIR). My tungsten lamp with NIR pass filter on top (eg 720nm, 850nm etc), when needed, will only just fit between the base and the iris. To visualise an image you need a camera, preferably one converted for 'full spectrum' work, so it can also be used for the near ultraviolet. The tube camera below has an NIR blocking, hot mirror filter at the end of the tube which can be removed, thereby converting it easily into a 'full spectrum' camera.
The tube camera set-up for normal visual spectrum work.
When using a standard camera body for NIR work on a microscope and for macro photography it will need to have its hot mirror filter removed as with the image below:
A Gerbera flower viewed through a 720nm infrared filter on a 50mm lens attached by bellows to a converted Sony Nex-5N. Illumination was provided by a small halogen desk lamp.
(click on image to enlarge)
A copystand (~£60) can be used for both macro photography and experimental microscopy.
The camera rail (~£20) at the lower left is also useful
Transmitted and Reflected Near Ultraviolet Light
I am looking at the lowest cost solutions for using UV light which relies on observation via a converted camera and, ideally, a monitor. Ultraviolet light is potentially damaging to the eyes and skin (especially at wavelengths shorter than 300nm), so I will go into much more detail later. A UV light source should not be observed directly. Ideally, a monocular microscope should be used (or digital microscope) fitted with a camera to avoid the risk of direct observation. I always have a pair of 190-540nm protective goggles to hand. If there is a risk that your hands could be in the proximity of a UV light source, especially the shorter wavelengths, gloves should be worn.
A Gerbera flower illuminated obliquely with a TATTU V2, 3 watt, 395nm torch with a 52 mm threaded ZWB2 filter on the front (held in place with a thin ring of Blu Tack) to remove any residual visible light and a visible light blocking filter on a Sony SEL 16mm, f2.8, pancake lens attached by bellows to a converted Sony NEX-5N. This is not an ideal focal length, but the lens is a conventional one that lets through a significant amount of NUV.
(click on image to enlarge)
More will be added about autofluorescence (2, 3) later. Although I will be using the near ultraviolet (300-400nm) for UV imaging, with fluorescence, I will be considering wavelengths from ~190nm (peak 254nm) upwards.
A Gerbera flower illuminated with a TATTU V2, 3 watt, 395nm torch with a 52 mm threaded ZWB2 filter on the front (held in place with a thin ring of Blu Tack) to remove any residual visible light and imaged using an unmodified Sony NEX-5R with 50mm lens on bellows.
(click on image to enlarge).
Fluorescence using Dyes
I will add more about fluorescence microscopy later. The technique usually involves expensive equipment. Over the decades, low cost approaches have been proposed with more recent ones including a portable blue LED illumination set-up and a simple UV microscope built using off-the-shelf components.
Flourescence Backlight Staining
The fluorescence backlight staining technique (FBST) involves converting a solid piece of tissue into a light source. The tissue is nonspecifically stained with a fluorescent dye and illuminated with ultraviolet or blue light. Superficial cells on the surface can be conventionally stained for observation. This technique can be used on large hand-cut sections as well as whole specimens.
In the example here, I used a Nikon fluorescence microscope. I intend to demonstrate this technique with a conventional microscope, probably using acridine orange or fluorescein.
Fluorescence backlit superficially stained solid tissue
(A plucked hair follicle acting like a light bulb)1988
Grandson Jack with his two little microscopes