 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
December 2019 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)
January
2020
 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)
Autofluorescense
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).
January
2020
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 |
gmail.com