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In vivo near ultraviolet transillumination with the quartz rod technique; application in electronic quantitation of cellular light absorption and nuclear fluorescence.

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1 N VIVO NEAR ULTRAVIOLET TRANSILLUMINATION WITH THE QUARTZ ROD TECHNIQUE;
APPLICATION IN ELECTRONIC QUANTITATION O F CELLULAR LIGHT ABSORPTION AND NUCLEAR
FLUORESCENCE
CHARLES N. LOESER
Western Reserve Fniversity, School of Hedicine, Clcceland, Ohio
SEVEN FIGURES
INTRODUCTION
This paper deals with methodology in the following two
ways. First, it will discuss quartz rod transillumination
using other than visible light, more specifically dealing with
the near ultraviolet (3200 to 4000 Angstrom Enits). Second,
it will deal with electronic methods of quantitating light that
comes to the eye through the microscope from quartz rod
or other illuminating devices.
The paper has to do with microcirculatory phenomena in
that blood vessels and their contents can conveniently be
irradiated by the quartz rod with chosen wave lengths of
light, and the resultant phenomena (fluorescence, absorption,
photodynamism, etc.) can be studied. Also the electronic
methods mentioned permit quantitative measurement of light
from microscopic portions of blood vessels and their contents,
while these structures are alive and moving. Two of the
methods permit display in ordinarily invisible ultraviolet.
I
Quartz rod transillumination depends upon the use of a
rod of fused quartz, down which a beam of light is passed.
337
338
CHARLES N. LOESEB
The end of the rod is placed under the organ to be studied,
the microscope placed above. For further discussion of the
use of this device we refer to the paper by Knisely ('36) on
the fused quartz rod technique for the microscopic study of
living tissue. The rod being made of quartz, it is ideal for
the transmission of near and true ultraviolet light. During
a search for a method of observing the walls of blood vessels, we came upon the work of Schlegel ('49) who injected
vessels with thioflavine S, and by irradiating with 3650A,
produced endothelial fluorescence. I t was felt that the quartz
rod could be used in this method, and thus the modifications
to h c ~discussed came about.
To change the quartz rod method for short wave length
observation, provision must be made for a good visible light
source by which a field can be found and focussed. At the
suggestion of Dr. E. H. Rloch, the modification shown in
figure 1 was used. The end of the quartz rod which receives
the light is thickened, as at D, and the ultraviolet source, A,
is directly focussed at D. From the side, another rod, F,
directs a visible light source, G, to a portion of the thickened
end, D. When not desired, the second rod can be elevated
o r lowered out of the way. Thus the observer may use the
visible source for focussing, and also for watching irradiation
effects of the ultraviolet source, even while they occur. A
spot source of light from an ordinary microscope illuminator
can be used to replace the extra rod.
I n the utilization of short wave lengths with the quartz
rod, the worker must be aware that the shorter the wave length
he uses, the more readily is the transmitted light absorbed
by the tissues. Thus when working with fluorescence excited
by near ultraviolet, or with tissue reactions due to ultraviolet
light presented by the rod, it may be only the first layer of
cells which actually is affected. Bluntly, to produce desired
effects, light from the rod must reach the cells. I n certain
cases, therefore, it becomes desirable to reverse the rod from
a transillumination device, and have it shine the light directly
on the tissue from above, observing with the microscope at
QLARTZ ROD E L E C T R O S I O QUANTITATIOlr'
339
an appropriate angle. Thus, for example, fluorescence may
be achieved on the surface, at least, of liver when transillumination would produce no visible phenomenon. The quartz
rod works quite well as a n irradiation source directly or
indirectly. Vodifications in the shape of the rod may be
desired to effect irradiation at certain angles.
The light source used depends, of course, upon the experiment. A tungsten bulb gives a continuous spectrum down to
3200A. For many studies, including those dealing with fluorescence maxima in the near ultraviolet, the mercury vapor
lamp produces a strong emission at 3650A and produces
strong band emission in the true ultraviolet. The 100 watt
type is most commonly used, but other stronger forms are
available.
To produce the desired band purity, either filters or a
monochromator may be used.
As examples of the sort of work that can be done, we shall
illustrate with two types of fluorescence microscopy used in
the author's laboratory.
1. Blood vessel wall fiuorescemce a d photodyizamism. Thioflavine S, injected intravenously in frogs and rats, or into
the heart of brine shrimp, has been used to demonstrate
fluorescence of blood vessel walls, and photodynamic constriction of those sensitized walls. The fluorescence and
constriction, both, were produced by bringing the quartz rod
carrying the activating wave length (3650 A) into proximity
with vessels of exposed mesentery, lung, kidney, bladder, o r
liver. Surrounding areas were not affected, nor were control,
non-injected vessels affected by exposure to the same wave
length for the same and longer periods of time. Figure 2
is an example of fluorescence from the injected blood vascular system of the brine shrimp. Photodynamism is discussed
with relation to thioflavine S by Algire and Schlegel ( '50).
2. Nuclear fiorescevzce. Acriflavine hydrochloride injected
intravenously in rats, or into the dorsal lymph sac of frogs,
has been used to stain nuclei in these living animals. By
340
CHARLES N. LOESER
bringing the quartz rod under exposed vessels of injected
animals, endothelial nuclei can be seen, as well as fluorescent
leucocytic nuclei: and in frogs, fluorescent red cell nuclei.
These animals survive doses of dye necessary to produce
these phenomena. Details of such staining techniques have
been reported elsewhere by DeBruyn, Robertson and Farr
( '50) and Loeser ( '53).
These examples serve only a s an indicator of what can be
done with the quartz rod used with near ultraviolet light.
By utilizing the transillumination technique as described, one
can do a great number of experiments involving the following :
( a ) fluorescence microscopy, both autofluorescence and induced fluorescence with addcd fluorochrome in chosen sites,
(b) absorption microscopy where wave length purity added
to the quartz rod permits better definition of desired objects
absorbing at the chosen wave length, and the study of spectra
is inadc possible, (c) the study of effects of localized irradiation with control areas available in the same animal. I n
this same manner, more effects of local irradiation can be
observed such as changes in blood flow characteristics, etc.,
and also photodynamic phenomena may be observed.
Figure 3 conveniently leads us into part I1 of this discussion, for it was the problem presented by just such a picture
that led this laboratory into the work to be described. In
vivo fluorescence emitted by nuclei is at best, weak, and in
living blood vessels, nucleated cells fly by so rapidly that the
eye can detect but little. I n frogs, the difference between
nucleated red and while cells can easily be detected, but no
real estimate can be made by the eye of exact luminous intensities. Figure 3 is a photograph on movie film of fluorescent nuclei of frog white blood cells in a large vein. The
quartz rod is visible at A, the vein at B, and the tiny white
dots seen in successive frames are the leucocytes fluorescing.
At the time this work was going on, reports came out of the
importance of the detection of differential amounts of light
from fluorescent nuclei (Friedman, '50; Mellors and Silver,
'51). Cancer cells were reported to fluoresce more brightly,
QUARTZ ROD ELECTROSIC QUANTITATION
341
and it seemed desirable to attack the problem +n vivo. Photographic methods of differential cell detection are insensitive
(witness fig. 3) and slow, but from the reports of Mellors
and Silver ( 'SO), Zworykin and Flory ( '52), and Young and
Roberts ('52), it seemed that electronic methods might prove
to be more sensitive in the quantitation of light from living,
moving objects.
I1
Four methods will be discussed here for the quantitation
of light from a microscopic field. All of them depend in one
way o r another on the principle of scanning. Scanning as
here used, means that a moving spot travels across a field
of view, and the light energies the spot passes are transformed into an electronic beam. The direction of travel of
the electronic beam will be altered by changes in the light
intensity in the field of view.
Method A. One way t o examine a field of living blood, for
example, is to use the quartz rod for transillumination, ordinary optics for magnification, and a television camera to display the images. The television camera actually scans the
field electronically, transforming the view into a traveling
electron beam which excites fluorescence on a screen to represent proportional light and dark areas encountered. The
scanning of a television camera starts at the top, travels
across the screen, then jumps down a space, travels across
again, etc., until it reaches the bottom of the picture whence
it starts over again. The repetition is rapid, easily delineating
moving objects.
The essential feature to the quantitation of a microscopic
image by television methods is the selection of a single line
from the screen, and the display of that line on an oscillograph in terms of the light and dark areas encountered. The
oscillograph used in this case was a DuMont Type 280. Figure
4 shows the microscope with television camera over it. The
display screen with image is at the right. The white line
through the television screen is the line selected for analysis
342
CHARLES N. LOESEB
by the Type 280 oscillograph (left extreme). The oscillograph displays the light and dark areas of the television
screen picture as pulses, the height of which represents the
degree of lightness o r darkness, and the width of which represents the area of the object being scanned. Figure 6 is a
photograph of the television screen displaying a squamous
cell of a vaginal smear. Over nucleus of the ccll the white
indicator beam can be seen. Below the photo of the screen is
a picture of the oscillographic representation of the light and
dark portions on that part of the television screen shown by
the white line, and it can be seen that the deflections represent
light and dark by hills and valleys. The nucleus, for example,
the darkest spot, has the deepest downward deflection. Such
studies on an Image Orthicon Camera chain can be done in
transmitted light (study of light absorbed by the object) or
in emitted light (fluorescence), see figure 7. I n in vivo work
the use of emitted light proved very difficult due to low intensity, but transmitted light proved much better and more
sensitive. Figure 6 does not show actual rcsolution.
Pick-up tubes are sensitive in the ultraviolet and thus can
be used to display objects otherwise invisible at the shorter
wave lengths. The scanning rate of the television camera is
sufficiently rapid to detect moving blood cells and to study
their characteristics as they go by.
Method B. This method is essentially the same (television
scanning) but depends upon a different camera tube (the
Vidicon) with somewhat different electronic characteristics.
It is not quite as sensitive as the Image Orthicon, and it is
not as linear in its registration of increasing screen brightness per unit increase in light. However, with calibration,
the tube can be used for most absorption studies. Figure 5
shows the small camera being used with the quartz rod.
Method C. The above television methods can be replaced
by a scanning disk and multiplier phototube. The image
from a microscope is sent to a multiplier phototube mounted
over the ocular, hut bctmen the phototube and the ocular
there rotates an opaque disk with a hole at one side near
QUARTZ ROD ELECTRONIC QUANTITATION
343
the periphery. I n rotating, the hole in the disk passes over
a portion of the microscopic field, letting light through to the
phototube, which in turn registers the differential intensities
encountered in the field on a n oscilloscope as pulses. The
method is described in detail by Mellors and Silver ('51).
X e t h o d D. If a traveling spot of light from the fluorescent
beam of a cathode ray tube is permitted to play into a microscope, that traveling beam will pass through (scan) the object,
and then upon entering a phototube mounted over the ocular,
will be registered by another cathode ray tube as pulses
representing, again, the differential amounts of light encountered in the field. This method is described thoroughly
by Young and Roberts ('52), and is reported in the above
application by Loeser and Berkley ( '54).
For the quantitation of light from microscopic fields, any
of the above mentioned methods may be used. They are quite
ideal for the study of blood vessels, blood flow, and blood
cells (as well as a host of other tissues). They are applicable
in the following ways:
1. For the study of cellular light absorption (stained cells
or unstained). Microscopic portions of cells can be quantitated as far as light absorption characteristics, whether those
cells arc moving or still, at almost any chosen wave length.
2. For the study of cellular morphology in single units
or groups, moving or still. (Pulse widths represent area
scanned. )
3. For the gathering of data on large numbers of cells,
by the utilization of known counting and computing methods
linked to the scanning device to record as the objects are
studied.
In closing, it may be emphasized that one of the great
lacks in microscopy, not only in the study of microcirculatory
phenomena, but also in many other fields, has been in the
accumulation of quantitative data beyond the limits of microcinematography. The methods herein described offer an
approach to such objective quantitation, and should aid in
344
CHARLES N. LOESER
taking the subjective element out of the study of microscopic
objects.
SUMMARY
An altered quartz rod transillumination method has been
discussed permitting fluorescence microscopy, absorption
microscopy, and the study of local irradiation effects such
8s photodynamic action.
I n addition, electronic methods of quantitation of light
from microscopic fields have been discussed. These methods
permit study of in viiio, moving objects, as well as still microscopy, and offer opportunity to gather light absorption data
(and in certain cases, fluorescence data) on microscopic fields
for analysis of changing or stationary physico-chemical states
of cells.
LLTERATURK CITED
ALGIRE, G. H., AND J. U. SCHLEGEL1950 Circulatory reactions in photodynamic
action. J. Cell. and Comp. Physiol., 35: 95-110.
DE RRUYN, P. P. IT., R. C. ROBERTSON
AND R. S. FARR 1950 In aiwo affinity of
diaminoacridines for nuclei. hnat. Ree., 108 : 279-308.
fi’RIEDJfAN, H. P., JR. 1950 The use of ultraviolet light and fluorescent dyes
in the detection of uterine cancer by 1-aginal smeai. Am. J. Obstr.
and Gyn., 59: 852.
I<NIBIT~Y,
hf. H. 1936 A method of illuminating living structurcs for micro
scopio study. Anat. Rec., 6 4 : 499-524.
LOESER,C. N. 1953 Studies with the quartz rod trcliuique of transillumination
using near ultrariolrt light. Anat. Ree., 116: 327-344.
LOESER,C. N., AND C. BERKLEYElectronic quantitation of light absorption
and nuclear fluorescence in living cells. Science, 119 : 410-411.
MELLORS, R. c., AND R. SILVER1951 A microfluorometric scanner for the
differential detection of cells: application in exfoliative c y t o l o g ~ .
Science, 1 1 4 : 356-360.
WHLLGEL,
J. U. 1949 Demonstration of blood ressels and lymphatics with a
fluorescent dye i n ultraiiolet light. Anat. Rec., 105: 433.
YOVSG,
J. Z., AND F. ROBERTS3952 The flying spot microscope. Proc. Inst.
Eke. Eng., 9*9: 747.
ZWORYKIN, V. K., AND L. E. FLORY1952 Television in medicine and biolog?.
Elect. Eng., $1: 40.
..
PLATE
crl quartz rod apparatus for use with ultra\-iolet light.
Geiier:il Electric Mcrcury Vapor Lamp, A-H1 Typc.
Arnericau Opticnl Coni~~any
lmup IIousing no. 370.
(londc iixing Icns.
Thickened bulb cmd of quartz rod.
l--ilter.
End piece transmitting visible light.
Visilrle light source.
Hollow tip of rod for ~dlninistrationof fluid.
Ihipper tips.
Rod tip, cornino~~
for both t r p e s of light.
2 Dorsal aspcet of brine shrimp, Ydaemonetes. Arrow points to arterial tree
i i ~ ~ j r c t c\rit,li
d
0.5% tbiofidne 8. Jlluminntion with 3650 A.U. from quartz
rot1 :it l c t t rxiises injectcd VPHSCl to f l ~ ~ o ~ * o s1c1
'1
e1.s X film. Corniug 110. 5874
filter. Exposure two minutes. X 4.
3
Movic strip of fluoreseait white cell nuclei in vein of frog. Vein (B) lies
orer quartz rod ( A ) . h-uelei are risible as white dots at (U). Specially sensitized niorie film. 0.5% (0.5 a n R ) acrilsvinc! hydroehloridc injected int.o dorsal
1ginl)li s:iv of frog. Exposure one franic per R O C O I ~ ~ . Corning filter 5113 hctwc.cn 1,)ulh :ind rod, iiltcn. 3486 b e t m e n ocular and camern. X 100.
4
Left to right. Type 280-A oscjllograph indicator.
orthicon camera orer microscope. Display screen.
5
( 1)
(3)
(3j
(4)
(5 )
{6)
6
TJppcr part of p h o t o is reproducti.on of tclcvi #ion
11 eliowing a normal
squaiiious vaginal ccll, Papsnieolaou stain. White line of oscillograph Type
280 indientor h a m is seen through nucleus. Rdow the screen is oseillograph
line as seen on eathodc ray tulle, reproducing in pulse dcfleetions tlic light
and dark :mas on screcn. Note deep middle downward defloction o f nuclcus,
tlic? darkest a i m sc:iniicd. PllotJogr:lpllic yesolution of scrren i a poor.
7
Television Rcrccn photo n.t top is of fluorescent onion skin nuclei. Upward
pulse deflections are analysis of briglitness of screen a t the two parallcl nuclei.
Stain is acriflavine hpdrochloride.
Monitor screen. Image
Vidicon television c!aniurn.
l\licroscope.
Quartz rod.
Visible light source.
Cltraviolct light SOIITCQ (partiallp cut from photo).
Side quartz rod carrying visible light. Compare figure 1 (F).
347
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