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PRELIMINARY OBSERVATIONS
Alveolar Morphometrics
3: Preliminary
Results with In Vivo Videomicroscopy*
Benedict D.T. Daly, M.D., and John C . Norman, M . D.,F.C.C.P.
An in vivo videomicroscopy system for the study of dynamic alveolar morphology
is described. Slow motion analyses of video recording permits quantification of
changes in alveolar size during respiration. Preliminary results are presented.
e have previously utilized in uiuo photomicroscopy with incident light, dark-field illumination to quantitate changes in the morphology of
alveolar sacs with positive pressure respiration.'s2
The addition of a videotape system to the microscope unit has permitted extended observations of
dynamic lung function to be recorded for subsequent analysis, and an improved technique has been
developed for maintaining the lung in contact with
our microscopy stage without reducing motion at
the lung surface. The purpose of this report is to
describe these adjuncts, with preliminary results.
Sprague-Dawley rats weighing approximately 250 gm
were anesthetized and tracheostomy performed. Ventilation
was maintained with a Harvard rodent respirator adjusted to
produce a peak inspiratory pressure of 15-20 cm of H 2 0 at a
respiratory rate of 50 ( respiratory rate of the animals at rest ) .
A combined anterior and posterior thoracotomy was performed through the sixth intercostal space and the fourth
through seventh ribs were removed. The left upper lobe was
freed from its pleural attachments and its anterior-inferior
margin stabilized by attaching pipe cleaners (length 2.5 cm)
to the lung surface using medical adhesive.
The pipe cleaners provide sufficient traction to maintain
the enclosed lung margin in contact with the coverslip without inhibiting normal lung expansion and contraction, ie in
expiration, the lung relaxes toward the coverslip. The microscope system, previously described in detail,Z consists of a
Wild-Heerbrug inverted metallurgic microscope, with epiobjective lenses and a side arm telescope. Light at 6000°K is
provided by a Xenon arc universal lamp. The unit focuses on
'From the Cardiovascular Surgical Research Laboratories,
Texas Heart Institute, St. Lukes Episcopal and Texas Children's Hospitals, Houston.
Reprint requests: Dr. Daly, PO Box 20269, Texas Heart Institute, Houston 77025
the stabilized lung at depths up to 1.5 mm below the pleural
surface. With the 30 X objective lens three quarters of one
turn with the fine adjustment ( 7 5 p vertical movement of the
lens objective) brings single alveolar sacs completely in and
out of focus. Further movement of the objective lens brings
more superficial or deeper units into focus.
A black and white television camera with 2: 1 interlace
(increased resolution provided by synchronized electron
beam scanning) for greater stability and automatic or manual
light control is attached to the side arm telescope. This is
connected in series with a black and white image enhancer to
increase the sharpness and clarity of video information.
Signals are passed from the image enhancer to a time-lapse
videotape recorder with stepped slow motion and stop action.
Recordings are simultaneously displayed on a fully transistorized 1 6 video television monitor.
Microscopic lung units consisting of several alveolar sacs
are brought into sharp focus on the television monitor and
recorded. The tapes are then played back with slow motion
in steps up to 1/61st of normal speed, or the video frames are
advanced individr~ally. Magnification on the video monitor
( 1,000X) is determined by viewing a calibrated grid through
the microscope and measuring the grid lines ( 4 8 p ) on the
television monitor. Dynamic wall motion can be quantitated
by slow motion analysis.
The topographic anatomy of alveolar sacs visualized in this manner is shown in Figure 1.
Quantitative measurements of alveolar sac dimensions are easily made by stopping the tape at any
point in the respiratory cycle. Although alveolar sac
walls may move slightly out of focus at the extremes
of the respiratory cycle in a single plane, varying the
focus over one full turn of the fine adjustment ( 100
r ) permits sharp outlines of alveolar sacs to be
obtained at any point in respiration.
Forty-two sacs in seven rats measured at peak
CHEST, VOL. 65, NO. 1, JANUARY, 1974
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DALY, NORMAN
FIGURE
I. Wall of alveolar sac at top (open arrow). A's in
alveolar sac at left represent alveoli. Solid arrow points to
shallow fold.
inspiration and end expiration following videotap3p and 33.4
ing had mean diameters of 59.1
1.8p, respectively. Alveolar volumes (AV) were calculated on the assumption they were perfect
spheres, using the formula AV-'/GrD3. Mean alveolar sac volumes were 108,304a3 and 19,527$
at peak inspiration and end expiration, respectively.
A comparison of alveolar measurements obtained
by various methods utilizing different techniques is
*
*
impractical because of the number of possible variables influencing alveolar size. In addition to fixation, the inktion pressure, age of the animal and
area of the lung studied influence recorded dimens i o n ~We
. ~ have standardized the current method so
that measurements of alveolar size are reproducible.
Initially, windows and suction devices were used
to stabilize the lung but these techniques variably
reduced inspiratory and expiratory lung movement
and consequently, normal alveolar dynamics. Lung
contact with the coverslip can be maintained with
the current technique. Some lateral motion is
present but the field recorded on tape usually contains several alveolar sacs, one or more of which
stays within the confines of the monitor screen
throughout a respiratory cycle. Changing tidal volumes or airway pressures does not disturb the effectiveness of this method. It can be used to determine
the effects of the former on alveolar dimensions and,
indirectly, gas exchange.
1 Moreci AP, Norman JC: Quantitative and qualitative
changes in morphology of alveolar sacs with positive-pressure respiration. Proc Soc Exper Biol Med 141:318-321,
1972
2 Moreci AP, Norman JC: Measurements of alveolar sac
diameters by incident light photomicrography 11: Effects of
positive pressure respiration. Ann Thorac Surg 15: 179-186,
1973
3 Glazier JB, Hughes JMB, Maloney JE, et al: Vertical
gradient of alveolar size in lungs of dogs frozen intact. J
Applied Physiology 23 :694-705, 1967
CHEST, VOL. 65, NO. 1, JANUARY, 1974
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