194

IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL.

BME-19,

NO. 3, MAY 1972

Holography and Medicine

ERNEST J. FELEPPA
Abstract-Holography has emerged from the laboratory and is slowly becoming a useful tool in a variety of engineering and scientific areas. The potential exists for holography to serve as such a tool in medical and biological research. Holography can be used to form three-dimensional images. Three dimensionality is a consequence of the recording of phase information in the hologram. The presence of phase information in holographic images makes them extremely versatile and amenable to such a posteriori techniques as interferometry and dark-field imagery. Holography can also be used to analyze images, that is, to enhance image contrast and resolution, or to perform such functions as correlation analyses and pattern recognition. Moreover, the versatility of holography extends beyond the visible portion of the electromagnetic spectrum; holograms made with infrared, ultraviolet, microwave, or ultrasonic illumination, or "synthetic" holograms made using a computer can be used to produce visible images or to analyze nonvisible images. The manner in which holography's potential can be used in biomedical applications is discussed.

LENS
B J
E

-/ PHOTOGRAPHIC PLATE

{~~~~~~TTRNFRENT)

(a)

(b)
Fig. 1. Holographic recording and reconstructing.

INTRODUCTION ]Ej OLOGRAPHY has reached its adolescence. It has matured to the point where it demands attention, and has developed into a potentially useful tool in a variety of scientific realms. One of the realms is medicine. The realization of holography's potential requires that engineers and scientists be made aware of this potential and aware of the characteristics of holography which can make it such a useful tool. Holography has been defined as an interferometric technique for recording the amplitude and phase of a wave [Fig. 1 (a)]. If the wave is one which has been scattered by a specimen, the recording of the wave contains sufficient information to produce a phase-faithful image of the specimen. Thus holography is a method of producing three-dimensional images. Because phase information is retained, the images can be analyzed by

be used to filter waves which are more complicated than a spherical or plane reconstructing wave. The former definition of holography as an interference technique is based upon the methods which were initially used to produce holograms. The latter definition of holography as a filtering technique is based upon holography's applications. The two definitions reflect the progress of holography from a subject of pure research to a subject of applied research. Holography has many applications in medicine and biology. Some applications can be realized in the very near future. Biomedical researchers, with very little guidance from a physicist, can easily construct a simple holographic imaging apparatus. Only slightly more complicated apparatus is required to build a holographic interference, phase-contrast, and dark-field techniques. microscope. The more sophisticated resolution-enhancNow that holography has matured, a more appropriate ing and pattern-recognizing capabilities of holography general definition of holography is that holography is a will be realized in the more distant future. The appamethod of complex spatial filtering. In the familiar image- ratus required to perform these functions is quite forming application, the hologram filters a reconstructing sophisticated, and, if such devices are to be generally wave, and passes components of the wave which will accepted in biomedical laboratories, they must be availform an image [Fig. 1 (b)]. The interference between able as complete units equivalent to current electron an object-scattered wave and a reference wave is photo- microscopes or spectrum analyzers. Thus the challenge graphed to make a hologram. Upon development, the for the engineer exists. photograph diffracts a reconstructing wave to form two IMAGING images of the object. In the geometry depicted in the Theory illustration, using plane reference and reconstruction The ftundamental equations of holography are now waves, one image is virtual, the other is real, and both quite familiar, but warrant a brief review [1]- [5]. have unity magnification. However, the hologram can Superposition of two identically polarized coherent waves results in the addition of their scalar complex Manuscript received May 17, 1971; revised November 15, 1971. field amplitudes. Denoting the complex field ampliThe author is with the Riverside Research Institute, New York, tudes of the waves as U1 and U2, the addition. is simply ;N. Y. 10023.

FELEPPA: HOLOGRAPHY AND MEDICINE

/

195
/

(DI=

DENSITY,i LOG

I outI

0'C
'2

'C,

LOG EXPOSURE (LOG It)

(a)

LOG TRANSMITTANCE (LOG TI LOG IOU!)

II

\S

XC
(b)

(a)

LOG EXPOSURE

(LOGIt)

Fig. 2. Emulsioni response curves.

T
(b)

U1+ U2. In the terminology of image-forming applications, U1 is the reference wave and U2 is the objectscattered wave [Fig. 1 (a)]. A recording made over a period of time greater than the period of the wave contains U1+ U21 2, or, the intensity of the added waves. Expanded, this is

Fig. 3. Uniform lateral magnification. (a) Recording. (b) Reconstructing.

wave, is reconstructed and forms a virtual image. Simultaneously, U2* is produced and forms a real image. Both images have unity magnification. When spherical rather than plane waves are used in either recording or reconstruction, the fundamental processes are the same; sion exposed to an intensity I(x, y) will have an inten- however, the curvature of the reconstructed waves is sity transmittance T1 (x, y) = KI(x, y)-y or equivalently, altered so that one or both images suffer distortion and an amplitude transmittance TA(X, y) =KI (x, y)y/2 are produced with magnification other than unity. A where K is a constant and y is the slope of the linear simple expression for magnification of small regions of portion of the emulsion response curve (Fig. 2). The the image is [6] traditional characteristic curve of a photographic M = + (1 - Z2Z1-1 + Z2Z3-1)-1 medium plots density after development versus exposure as shown in Fig. 2 (a). Somewhat more useful in where Z1, Z2, and Z3 are, respectively, the perpendicular holography is the related curve which plots intensity distances from the reference wave source, the object transmittance after development versus exposure as point, and the reconstructing wave source to the reshown in Fig. 2 (b). The slopes of the approximately cording plane. Positive Z's are associated with sources linear portions of these curves are, respectively, y and on the same side of the hologram as the object. Positive -y. If processing is controlled to give -y = 2, then magnification indicates a real image, and, conversely, TA=KI [1]. However, the requirement that y=2 is negative magnification indicates a virtual image. Disnecessary only in certain filtering applications. In most tortion can be predicted by applying this expression to holographic applications, one simply makes 1o greater several points in the object. than 1, so that the expansion of (0+I,)-y2 can be An interesting property of holographic imaging is the approximated by C1+C218 where cl and c2 are real con- ability to produce an image having constant lateral stants. Thus TA = Kc1 +KC2I1 or K1 +K21. magnification throughout the depth of the image. A During reconstrtuction, the recording imiodulates the lens can be uisedl during recordinig to produce ani image reconstructing wave U3 anid produces a wave U4 = Li TA, of the specinmen; the imiage then serves as tl-he object for so thiat, considering only the signal ternvs, LJ.1 = UJ3 Ul * U2 the hologram. By placing the reference-wave source in + U3U1U2*. If U3- UJ, the first and seconid terms are, the back-focal plaine of this lens [Fig. 3 (a)] arn(l by respectively, the original wave and its complex con- placing the reconstructing-wave source at infinity

Ui1!2 + I U212 + U1*U2 + UlU2* where I is intensity and the asterisk denotes complex conjugate. The two right-hand terms in this expression are the essential signal terms. The two left-hand terms are the bias terms. Let U,1 2+1 U21 21o and U1*U2 +UiU2*'=I, so that 1=1o+1s. A photographic emulI=

jugate, times the constant I U1l 2. Thus Li2, the original

IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, MAY

1972

[Fig. 3 (b)], the magnification of the hologram's real image is Zl/fL, where fL is the focal length of the lens used during recording. Notice that none of the object or image coordinates are included in this expression. Thus magnification does not vary through the entire depth of the image; however, longitudinal magnification remains the square of the lateral magnification, so that for magnification other than unity the image will be elongated. Nonetheless, uniform lateral magnification greatly facilitates botlh visualization and measurement of the image.
Applications The discussion so far has considered holograplhy as an interference technique during recording and an imageforming technique during reconstruction. Image formation includes a large and significant area of applications including several biomedical applications of holograplhy. The unique and useful characteristics of holograplhy in biomedical applications have been amply described; they include three-dimensional imagery [6], [7], interferometry [6], [7], the analysis of vibratory motion [8], the detection of motion in systems containing small, indistinguishable, or slowly moving particles [6], [9], the production of visible images from a recording made in nonvisible illumination [IO], [1I], and the display of nonvisible or hypothetical objects by computer-generated holograms [ 1 2 ]. Summarizing, three-dimensional imagery provides a reconstruction of the entire depth of the specimen. A changing specimen is recorded at a single point in time and can be leisurely examined at a later time. By using a lens during recording, an image lhaving unifornm magnification can be produced during reconstruction. A single, conventional bright-field image can be altered by dark-field, phase-contrast, or interference techlniques to provide additional informationi [6], [7] (Fig. 4). In Fig. 4 (a), the image of the egg and a plane background wave interfere giving a circular contour pattern. In Fig. 4 (b), a small angular shift between exposures produces finite-fringe differential interference. Notice the shearing of the central fringes; this arises from the formation of the mitotic spindle during cell division. In Fig. 4 (c), a long time interval between exposures produces infinitefringe differential interference. The cell has begun to divide, and the pattern in the center arises from cytoplasmic refractive-index variations in the region of furrow formation. (The diameter of the egg is about 100 ,m.) However, holographic interferometry includes the dimension of time. Subtle changes which occur during some time interval can be detected and measured [6]. This is a unique characteristic of holographic interferometry. The analysis of vibratory motion is also an interference technique [8]. Interference is effectivelv obtained from the displacement maxima of the vibrating object. Because specimen motioni reduces the contrast of the fringes recorded by the hologram, the bolo-

(a)

jc) Fig. 4. Holographic iinterferenice demionistrated in doubleexposure interferograms of a sea urchin egg.

FELEPPA: HOLOGRAPHY AND MEDICINE

197

OPAOUE- STOP

(b)

(a)

PHASE- RETARDING STOP

(c)

Fig. 6. Image-altering methods. (a) Bright-field method. (b) Dark-field method. (c) Phase-contrast method.

Mbi
Fig. 5. Holographic motion-induced contrast demonstrated in images of a slime mold.

gram best records only those fringes produced by the object when the velocity of the object is zero, that is, when the object is in its maximal displacement conIfigurations. Similarly, wlveni motion is not vibratory, those regions of the specimen whiclh move during recording do not contribute to the recorded fringe pattern [9]. That is, the fringes arising from the moving parts of the specimen are recorded witlh lower contrast than those arising from the stable parts of the specimen (assuming specimeni absorption is uniform). Thus the reconstruction of the specimen is darker in moving regions than in stable regions (Fig. 5). Fig. 5 (a) is a conventional image made in coherent light. This image is equivalent to the image which would be obtained in noncoherent light except for some apparent contrast enhancement and granularity. Because of the granularity of the specimen and its surroundings, the specimen cannot be distinguished from the background. Fig. 5 (b) is a holographic image. The moving cytoplasm in the specimen is darker in the image than the static background. 'Moreover, darkening is greater in areas of higher motion so that primary flow channels are distinguislhed from eddies and regions of moderate flow. (Magnification is approximately 150.)

These useful properties of holographic imaging can be of value in many areas of medicine. Naturally, a specific discussion of all the potential applications would be monumental; however, the important general areas can be considered. MIicroscopy is the broadest area. Holographic microscopy can improve the observational versatility of microscopic examination of botlh fixed and living specimens. Holographic methods can be especially valuable in the study of living specimens because the entire depth of the specimen is recorded at a single point in time. A series of holograms that records the specimen over a period of time can provide both spatial and temporal information. Data in four dimensions are thus obtained, the three spatial dimensions and time. All planes in the specimen are recorded in each single hologram. In conventional microscopy, the microscope must be refocused for each exposure, thereby limiting the number of recorded planes to the number of exposures. In a dynamic living specimen, the time required to make each exposure effectively reduces information content to one plane for each exposure. Another important advantage of holographic microscopy is the removal of the restriction to a single-image type for each exposure. In conventional microscopy, the microscopist must elect bright-field, dark-field, phase-contrast, or interference imagery, and an individual photomicrographic exposure can contain only one image type. However, a single, bright-field hologram can provide all these image types a posteriori. Again, while this property is useful in studying fixed specimens, it is even more useful in studying dynamic specimens. During reconstruction, the image can be observed as a bright-field image to exam ine absorbing structuires; extremely small structures can be observed in this inmage mode [Fig. 6 (a)]. Thle simple insertioni of a mask permiits examination of

198

IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, MAY

1972

a dark-field image [Fig. 6 (b) ]. A bright-field holographic image can be altered very easily to produce a dark-field or a phase-contrast image. In Fig. 6 (a), illumination of the hologram (H) produces the bright-field image (go) which can be viewed by eye or photographed with a camera (C); 41 is the bright-field image formed on the retina or on the film. In Fig. 6 (b), the addition of an opaque stop at the image of the background wave produces a dark-field image (g2). In Fig. 6 (c), the insertion of a plate, which retards the background wave more (or less) than the image wave, produces a phase-contrast inmage (r3). Even more minute structures can be located in this mode although, strictly speaking, resolutioIi is poorer than in the bright-field mode. Alternately, the equally simple insertion of a phase-shifting plate provides a phase-contrast image [Fig. 6 (c)]. In this mode, transparent structures, which differ from their surroundings only in their refractive index, can be observed. Interference is the most versatile method but requires a somewhat more elaborate setup. Conventional interferometry is an extremely useful technique in biomedical research [21]. The ease and flexibility of holographic interferometry greatly enhances the value of this already useful technique. Interferometry is used in biology to produce amplitude images from phase objects. The amplitude image can be qualitative by producing visible contrast based on phase variations in the object, or the amplitude image can be quiantitative by determining path lengths. (Interference nmethods are superior to phase contrast for producing amplitude images because interference methods provide better resolution.) Optical path length is the product of refractive index and geometrical path length. If one factor is known, the other is readily calculated. While geometrical path length is often an important parameter, refractive index may be of greater general interest. The,significance of refractive index lies in the relationship between the measured index and the concentration of dissolved solute. For a solution, n=n8+ac, where n is the refractive index of the solution, nS is the refractive index of the solvent, a is the specific refraction increment, and c is the concentration of the solute expressed in grams per 100 ml of solution. Each solute has a clharacteristic increment; for example, for proteins it is about 0.18 ml/g.1 Thus the concentration of protein in a differential volume element Ida is c_ (n-n8)/0.18 where / is the geometrical path length through the specimen and do- is the differential surface element. The solvent in biological materials is water, with a refractive index of 1.33, so that for proteins c_(n-1.33)/0.18. Integration over the volume of the specimen gives the total dry mass m of the specimen. However, dry mass can be determined without the knowledge of I; it can be determined from the knowledge of 4, the plhase shift introdluced b1w the specmllen. For a transillulminated
See [21] for Comlpolunnds.
I

specimen, the phase shift is related to the difference between the refractive indices of the specimen and the surrounding medium by the relationship
2w 4) = -l(n--no)

where 4 is the phase shift in radians and no is the index of the surroundinig medium. The difference (v-no) is the quantity which is directly determined by interference methods; however, the surround may not be distilled water. If no=n,+ then 4 becomes

4)

2ir l(n

fs-

For most normiial saline nmedia used witlh living cells, a is sufficiently small to be ignored. Then, substituting n -no= Ca,
+-Ica
--Ca

2w

where C = Ic is the surface concentration. Tlhe total dry mass is then

m =

fCdo-

= (X/27ra) f)dc.
For protein this is

m = (X/0.367r) Ofdr.
Often 4 may be difficult to determine. The most convenient way is to count fringes in infinite-fringe duplication interference (to be discussed next). Counting from the edge of the specimen, each bright fringe corresponds to a phase increment of 2wx radians (Fig. 7). This, however, is not a very accurate method. The most accurate methods utilize differential or duplication methods with finitely spaced background fringes and with the specimen constrained to assume a known shape. Various interference images can be formed a priori

complete

interference patterns obtained with a spherical specimen. In Fig. 7 (a) the image wave is superimposed upon a plane interfering wave which is parallel to the background portion of the image wave. The result is infinitefringe duplication interference. The exaggerated profiles of the interfering waves are shown beneath the image. In Fig. 7 (b), tlle image wave is sutperimposed upon a plaine interfering wave w1iich is ti'lted witlh respect to the background portion of the image wave. Finite-fringe duplication interferenice is obtailned. In list (of inicrem-lenits for specific biological Fig. 7 (c), the inmage wave is superimposed upon itself,

or a posteriori using holography. Fig. 7 illustrates

FELEPPA: HOLOGRAPHY AND

MEDICINE1
ZERO - ORDER BEAM
BEAM- SPLITTER R<

199
HOLOGRAM ,
:/

T)]/

IMAGE - BEARING
t.
(a)

IMA

IbI

Z-4

;P
Id)
d

RECONSTRUCTING
MIRROR-2

ATTENUATOR

BEAM- SPLITTER-2 (R T)

(a)

ZERO-ORDER BEAM
HOLOGRAM
/

/t

BEAM -SPLITTER-1I ( R =T) O~~~~~MRRO-1I

t
{c)

IMAGE- PLANE

Fig. 7. Interference images. (a) Infinite-fringe duplicatimn. (b) Finite-fringe duiplication. (c) InfIinite-frinlge differential. (d) Finite-fringe differential.

RECONSTRUICTING
MIRROR-2

--------

BEAM- SPLITTER-2 (R.T)

tb)

but with a slight axial and lateral shift. The effect is to shadow the image of a phase object using infinite-fringe differential interference. In Fig. 7 (d), the image wave is again superimposed upon itself, but this time with a slight lateral and angular shift. (The lateral shift d is grossly exaggerated here; it is usually made equal to the resolution of the system.) In this way, finite-fringe differential interference is produced. A variety of interferometric arrangements might be (c) employed, such as the Michelson or Mach-Zender sysFig. 8. A posteriori interference. (a) Duplication interference. tems (Fig. 8). By interfering a plane wave with the (b) Differential interference. (c) Two-hologram interference. image wave, duplication interference is obtained [Fig. 8 (a)]. If the interfering waves are parallel, infinitefringe (background-fringe spacing) interference is ob- parallel lines are formed in the background. Where tained which provides a contour map of the specimen these lines pass through the specimen's image they are [Fig. 7 (a)]. The fringes formed in the specimen are deviated. The deviation is a measure of the path length isopath-length contours. Adjacent fringes of the same through the specimen. For a spherical specimen, rephase (e.g., adjacent dark fringes) indicate an optical fractive index is calculated from the displacement of path-length difference of one wavelength. If the the fringes at the equator using the equation geometry of the specimen is known, then refractive |n no = XD/ [2SoV/p2 -X2]. index (the remaining unknown in the pathlength) can be calculated. For a spherical specimen the difference Here D is the fringe displacement measured at the between the refractive index of the specimen and the equator; So is the background-fringe spacing; and x is surrounding medium is the distance along the equator from the center of the specimen to the fringe. In this case, as in the infiniten - nol = Xr/[2iV/p2 - r2] fringe case, the sign of (n-no) cannot be determined where n is the refractive index of the specimen; nO is the from this equation. Of course, for biological materials, refractive index of the surround; X is the wavelength of n>no. In general, however, by knowing the relative the illuminating light; r is the distance from the center positions of the image background and interfering of the sphere to the contour line or fringe; S is the waves, and by changing the angle appropriately, the fringe spacing at r; and p is the radius of the specimen. direction of fringe movement indicates the sign of The sign of (n-no) cannot be determined from this (nf-fno) [see Fig. 7 (b)]. The image wave can be superimposed upon another equation. However, as discussed earlier, biological main general have a refractive index greater than image wave [Fig. 7 (c)]. This type of interference is terials called differential interference. It can be used to enhance that of water. An alternative method of duplication interference em- the visualization of surface or path-length variations, or ploys a plane interfering wave which is tilted with re- to measure shape or refractive index. Holography makes spect to the image wave [Fig. 7 (b)]. Thus straight possible the measurement of changes in these parameters
-

200

IEEE

TRANSACTIONS ON BIOMEDICAL ENGINEERING,

MAY 1972

over a period of time. (This will be discussed more fully later.) The interfering waves can be derived from a single hologram [Fig. 8 (b)]. This is done by using a beam splitter to form two image waves, and using mirrors to superimpose the two image waves. If they are superimposed with only a slight lateral and axial shift, destructive interference appears in some regions of the image and constructive interference appears in others. The visual effect is one of shadowing; the specimen appears to be obliquely illuminated. If an angular shift is imposed, as well as a slight lateral shift, fringes appear in the background as in duplication interference [Fig. 7 (d) ]. Again, fringes appear in the specimen, and, if the geometry of the specimen is known, refractive index can be calculated. For a

sphere

(n-no) = XD[2So(Vp2 -(x-d)2- v\p2- (x+d)2)]-I

where d is the lateral displacement between the images. In this case, however, the sign of (n-no) can be determined; it depends on the sign of D. If the fringes in the specimen are displaced witlh respect to the background fringes away from the center of the specimen, then D > 0 and n > no [Figs. 4 (b) and 7 (d) ]. A third method of differential interference is used to detect or measure temporal changes [Fig. 8 (c)]. In this case, no angular or linear displacements are effected. Fringes then appear only where path-length differences exist in the image waves [Fig. 4 (c) ]. Obviously, such interference is not obtained from a single singly exposed hologram; it is obtained from a single doubly exposed hologram, from two singly exposed holograms [Fig. 8 (c) ], or from a hologram and the specimen itself. Using these methods, differences of a fraction of a wavelength can be analyzed (>X/100). All of these interference methods can be utilized by doubly exposing a single hologram while effecting the appropriate displacements (if any) between exposures. However, doubly exposed holograms can provide only fixed interference patterns; the nature, spacing, and orientation of the fringes cannot be varied. A posteriori interference, using one or two holograms, provides far more flexibility, but it requires a more elaborate reconstructing apparatus. In many cases, the added complexity is well justified (Fig. 8). Microscopy using hiolograms can fruitfully utilize the motion-analyzing potential of holography. A laser of adequate power permits control of exposure time and, therefore, of image darkening arising from localized specimen motion. Thus darkening duie to motion can be distinguished from darkening dtue to absorption or scattering. While a quantitative determination of velocity is difficult, velocity profiles can readily be ascertained. Moreover, the detection and localization of low and relative flow rates are biological parameters of great importance. For example, the cytoplasmic streaming exhibited by many simple organisms (e.g., amoel a

and slime molds) has been explained by two models. In one, cytoplasmic flow is caused by sweeping or pumpinlg action along the borders of the flow channel, and in the other, flow is caused by distal pressure. In the former case, the flow velocity would be greatest near the borders of the channel; in the latter, it would be greatest in the center. For a number of reasons, the latter is more reasonable and the holographic images of the slime mold physarum connatum support this conclusion [Fig. 5 (b) ]. In addition to its use in analyzing motion, motioninduced darkening can be useful simply as a means of enhancing contrast. Thus holography can provide detail in microscopy by utilizing phase contrast, dark-field interference, and motion-induced darkening, all from a single bright-field hologram. Microscopy has been discussed in some detail, but it is representative of a number of biomedical imaging methods. Many other observational techniques might be improved by utilizing holography. One outstanding example is ophthalmoscopy. Current ophthalmoscopic methods permit examination of only very limited regions in a single observation. Analysis of shape is difficult but important in many diagnostic procedures. Moreover, a number of pathologies are manifested by the existence of transparent or semitransparent structures, for example, bands in the vitreous humor, or a stratification of the vitreous. A holographic recording could provide an easily visualized high-quality image which, because it contains absorption, phase, and motion information, can be extremely valuable in ophthalmology. Changes of shape as well as absolute shape can be determined by interference; transparent structures can be observed by phase contrast or interference; minute particles can be analyzed by dark field; regions of very high or very low blood flow can be detected by motion-induced darkening. Many systems cannot be adequately observed using visible light. Nonvisible illumination, such as ultraviolet or ultrasound, must be employed. Often, however, a visible image must eventually be produced; it may be produced photographically, with a fluorescent screen, or electronically. Moreover, optical elements must be made from special materials (such as quartz lenses for ultraviolet) or have inherent optical limitations (such as mirror lenses for ultraviolet). Holography greatly simplifies the procedure of image formation while simultaneously providing visible images which contain far more information than is contained in conventional images made using nonvisible illumination. The problems encountered when using nonvisible illumination in holography are essentially problems in recording. Ultraviolet can be recorded on a photographic emulsion, but scattering within the emulsion becomes severe at ultraviolet wavelengths. Ultrasonic waves can also be recorded by pressure-sensitive emulsions, but alternative methods exist such as photography of surface-standing waves [10], [11], or electronic recording, uising piezoelectric vidicons [19].

FELEPPA: HOLOGRAPHY AND MEDICINE

201

where F denotes Fourier transformation. Alternatively, U3 = U2Uo where U3, U2, and Uo are, respectively, the Fourier transforms of V3, V2, and Vo. U2 is the modulation transfer function or MTF. (If V3 exists in one focal plane of a lens, U3 will be produced in the other focal plane; thus a lens can be used to effect the transformation.) By obtaining Ur-1, Uo can be obtained from U3 because U2-'U3 = U2-'U2Uo = UO. Thus by filtering U3 to get Uo, the resolution of the image can be significantly improved. The problem reduces to that of obtaining U271, but U2-1= U2* U21-2. As shown earlier, U2* is obtained from a hologram, and is the transmittance of a photograph of U2 when the photograph is developed to y=2. Alternatively, a synthetic or computer-generated filter [15] might be made by calculating U2'- from the spread function or from the MTF. In order to produce U2-1 optically, a point source is imaged by the system which is to be corrected and this image is recorded on transparency film. If the final element of the system is a photographic emulsion, then the normal emulsion of the system can be used to record the point-source image. In this case, the U2-1 filter will include the MTF of the film. The product of the gammas of the photographic steps outside the system which is to be corrected must equal 2 so that the amplitude transmittance of the final transparency is a linear function of the system-transmitted intensity. The final transparency is placed in the focal plane of a lens and illuminated by a plane, coherent wave [Fig. 9 (a)]. This lens transforms the wave V2 transmitted by the transparency, and forms U2 in its other focal plane. A photograph made in the second focal plane records 2. By controlling the development of the photograph so that -2 A ,y = 2, the transmittance of the photograph is second phofograph made in the focal plane, but with a plane reference wave U, superimposed upon U2, can record Ul1 2+I U212+ U,* U2+ U, U2* [Fig. 9 (b) ]. This is the familiar intensity distribution recorded by a conventional hologram, and, as discussed earlier, U2* is FILTERING one component of the wave transmitted by a hologram. Resolution-Enhancement Theory By sandwiching these two photographs, in proper regisResolution enhancement using holographic spatial ter, the deconvolution filter U2*1 U21-2 is obtained. filters is essentially a deconvolution operation [13], [14]. Subsequently, a recording of the image for which resoluIn any image-forming system, the complex amplitude tion enhancement is required is placed in the focal plane (listributioni in the image V13 is eqLual to the convolution of the trainsforminig lens and illuminated with a plane of the geometrical or ideal image distribution Vo and coherent wave [Fig. 9 (c)]. This lens transforms the

Ultraviolet and ultrasonic holography can be useful in a number of biomedical areas. Ultraviolet microscopy, for example, can be used to determine the distribution of nucleic acids in cells. The distribution is indicative of the functional state of the cell. In abnormal cells the distribution differs from that in normal cells and it can be used to diagnose abnormal states, for example, cancer. Conventional ultrasonic techniques are becoming quite common in medicine for visualizing softtissue structures. Ultrasonic waves, generated by an external transducer, are propagated through body tissues and are reflected at boundaries between tissues. Current techniques employ pulses of ultrasound and time the echo return in order to ascertain depth. Each pulse may be reflected at several boundaries, giving rise to several echoes. When visualization is not necessary, depth is determined by an oscilloscope trace displaying intensity along the y axis and time along the x axis. Alternatively, an image can be produced by displaying echo intensity as oscilloscope intensity and time along an axis determined by the position of the transducer. By sweeping the transducer, a single plane in the specimen is displayed. The grey scale of such a display is extremely limited which, in turn, limits the versatility of the technique. Despite these limitations, the technique has considerable potential value in medicine. Naturally, the production of a visible three-dimensional image from ultrasonic waves would increase this value manyfold. Holographic ultrasonography would be invaluable in ophthalmology, obstetrics, gynecology, mammography, and the detection of soft-tissue tumors in general. Synthetic holograms also might be extremely useful in medicine. While the interference arising from the superposition of U1 and U2 is conveniently recorded by a photographic emulsion it also can be calculated by a computer [12]. This calculation is simplest when the object can be considered to be a finite collection of points or line elements. The data from a variety of scanning techniques used in medical diagnosis could be converted directly into a synthetic hologram for subsequent visual display. Because these data are collected as a set of coordinates and intensities, they are ideally suited to such treatment and analysis. For example, ultrasonic scans made in several parallel planes would provide data in four dimensions, x, y, z, and intensity. By knowing the modulation transfer function (MTF) of the system, significant resolution enhancement might also be achieved using this method. (The MTF shall be discussed in the next section.)

the spread function of the system V2, that is,

V3

V2 0) VO.

The Fourier transform of this equation is

F[V3] = F[V2 (D Vol

=

F[V2]F[Vo]

I U21-2

U21 I U21

20)2

IEEE TRANSACTIONS ON BIOMEDICAL

ENGINEERING, MAY 1972

--0-

V2

-ffi

_
If

/
f

U2V.El

(a)

U3

U4~

L2
V4

Fig. 9. Resolution-enhancing filter. (a) Recording U2 1-2. (b) Recording U2. (c) Filtering U3.

Fig. 10. Convolution-analyzing filter. (a) Recording U2. (b) Filtering U3.

transmitted wave V3 and forms U3 in its second focal plane. The U2-1 filter, placed in the second focal plane, passes U3 U2- = UO. Subsequently, U0 can be transformed by a second lens, forming Vo in its focal plane. Thus the geometrical-optical image is obtained. Resolution-Enhancement Applications An obvious and direct application of these principles is radiology [20]. Conventional radiograms suffer from image degradation due to penumbra effects. The cause is the relatively large size of the X-ray source, 1 to 3 mm. In order to make a resolution-enhancing filter, a radiogram is made of a point object, that is, a pinhole aperture. This radiogram is then used to make the U2* and filter components. Subsequently, a diagnostic radiogram is illuminated with coherent light and filtered using the U1-2 sandwich. An interesting aspect of the enhanced image is that not only is resolution enhanced, but contrast is enhanced as well. Moreover, a single radiogram can be deblurred in specific planes thereby providing three-dimensional information from a single two-dimensional photograph. Because this resolution-enhancing technique eases the requirement for a small source of X-rays (i.e., X-ray tube focal spot), a larger source or several sources can be used. This permiiits higher anode currents, higher X-ray intensities, and, tlherefore, shorter exposures. By reducing exposure time, the irreversible resolution degradation due to random patient motion is reduced. Similarly, resolution-enhancement methods can be applied to scanning systems. Such methods can permit the use of mutltiple scanning detectors, thereby increasing thie sensitivity and/or rate of the scan. However, in scanning systems, stclh as isotope-activity scanners,

U21-2

U*1

computer calculation of the inverse of U2-1 might be more feasible than the optical formation of U2-1. A scan is made using a point or line source of radiation. This scan provides the spread function for a given distance from the source to the scanning plane of the detector. The computer-calculated Fourier transform of the spread function provides the MTF. By making scans for various distances from the source to the scanning plane, a number of different MTFs are obtained. The inverse of each MTF enhances the resolution of a distinct plane in the patient while blurring other planes so that a three-dimensional evaluation of isotope distributions can be made from a single scan of the patient. Thus deconvolution can be effected by spatial filters using purely optical methods, or the inverse of the MTF can be calculated, retained in a computer, and applied directly to the data. Alternatively, an optical filter can be made using a computer to calculate the inverse of the MTF [15]. The inverse of the MTF is displayed and photographically recorded. This photograph then serves as an optical filter. These techniques can fruitfully be applied to any system which can be described by an MTF and in which the resolution is not limited solely by random or noise factors. Some obvious examples are fluoroscopy, cystoscopy, electron microscopy, radioautography, isotope-activity scanners, and gamma-ray cameras.

Pattern-Recognition Theory While resolution enhancement is a filtering function which can be considered deconvolution, holographic filters can be used to measure convolu-tion in pattern recognition or optical computing applications [17 ], [18 ]. As in deconvolution, convolution analysis occurs in the Fourier or spatial-frequency domain, that is, the analy-

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sis is made on Ui and U3 which are the Fourier transforms of V1 and V3 [Fig. 10 (a) ]. By using a lens to transform V, and V2, and then superimposing the transforms U1 and U2, a recording of the superposition contains the familiar expression I= U112 + U2 12 + U1*U2 + UlU2*. The two right-hand terms are I -F[V1*]F[V2] + F[V1]F[V2*]. In this case, V2 is derived from a point source at the focal plane of the transforming lens; thus U2 is a plane wave. Upon reconstruction using U3 [Fig. 10 (b)], the transform of V3, the wave transmitted by the recording is -U4 = U3UU2 + U3UlU2Again, only the signal terms are considered. In Fourier notation tlhis is

(a)

U4= F[V3]F[V1*]F[V2]
+

+ F[V3]F[Vu]F[V2*1 F[V3 0 V1]F[V2]

F[V3 0 V1]F[V2*]
(b) Fig. 11. Pattern recognition by complex optical spatial filtering applied to the recognition of cells. (a) Recording cell-recognition filters. (b) Specimen analysis.

where 0 denotes correlation and (D denotes convolution. By transforming the transmitted wave with a second lens, we obtain V4 (V3 0 V)V2 + (V3 0 V2)V2*. Thus one diffracted wave consists of V2 and this wave forms an image of a point source whose amplitude is V, determined by V3 08 the correlation of V3 and V. The other diffracted wave consists of V2*, and this wave forms an image of a point source whose amplitude is determined by V3 0 Vi, the convolution of V3 and Vi. Like resolution-retrieval filters, correlation-analysis filters are conveniently made by purely optical methods. Alternatively, a computer could synthesize a filter based upon a hypothetical object. Moreover, U1* U2 + U1 U2* might be utilized entirely within the computer during data analysis. Correlation can be analyzed simultaneously for a number of patterns in a tested wave. That is, if a correlation filter were made from a specific reference pattern, for example, the letter E, that filter could analyze the correlation of the reference pattern E with a number of test patterns, for example, the letters on a page. A number of point images would be formed in the output plane, the brightest points corresponding to the letters E on the page. Less bright points would be formed for the letters F, L, P, I, and so on.

thousands of cells, is spread on a microscope slide. The slide is then examined, cell by cell, and malignant cells are identified, often against an overwhelming background of benign cells. Using this optical technique, filters would be made for a large number of benign and malignant cells. The filters might most simply be recorded on movie film, each frame containing a separate filter [Fig. 11 (a) ]. The entire specimen would be filtered, one frame at a time [Fig. 11 (b)]. By integrating the output of all the frames, the tremendous variations in cell configurations would be averaged and the final output would be statistically reliable. In the scheme, variations in cells are taken into account by using many filters and averaging the output electronically. This hybrid method, using optical filters to calculate correlation or convolution and computers to average the output, may be simpler and cheaper than a complete computer analysis. In addition to reducing the time required to diagnose a specimen, this method of analysis should enhance the accuracy of diagnosis by eliminating the tedium of examining each cell on a microscope slide. Instead, the technician or pathologist only needs to Pattern-Recognition Applications examine those cells which the correlation analyzer indiThis technique might be profitably applied to cyto- cates are suspicious. Diagnostic cytology is one useful application of these logical or histological diagnosis [161. In cytological diagnosis, the specimen, consisting of hundreds or methods, an application which can be considered a

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screening application. The same principles can be ap plied to more complicated specimens such as lhistological specimens. Again, the problem currently is to single out diagnostically relevant cells against a background of irrelevant cells. A useful simple application is hematology. Current methods require tedious visual inspection of blood smears and time-consuming manual counting of blood cell types. Holographic methods might significantly improve the speed and accuracy of blood counts. In fact, ultimately such counts might be done in real time. Counting might be performed in a continuous monitoring of the patient's blood, for example, after surgery or to evaluate chemotherapeutic treatments. Computer-synthesized filters also might be useful in recognizing less-complicated patterns. These filters might be applied to pattern recognition in electron micrographs or diffraction patterns in crystallography. While humans are excellent in recognizing patterns, we are not so adept in detecting periodicities, especially if periodic patterns are masked by nonperiodic details. Thus correlation filters might be especially useful in detecting periodicities or repeated patterns in complicated images. Not only can correlation filters be used to detect and analyze patterns, they can also be used in strictly computational applications. For example, a symptomanalyzing optical computer might be based on holographic correlation filters. Because such filters are complex-amplitude filters, they can analyze data consisting of both intensity and phase information. Filters might be made from two- or three-dimensional arrays of points where the coordinates identify the symptom and the number of points or the scattering intensity indicates symptom intensity. A two-dimensional array could be made on a single transparent sheet, or a threedimensional array could be made from sandwiched transparent sheets. The scattering points could be made by placing light-absorbing and/or phase-retarding material at the appropriate coordinates on the sheets. As in the suggested method for cytological diagnosis, the filters might be recorded on movie film, an individual frame containing a single filter. (Using a three-dimensional array, a single filter on a single frame of movie film could contain all the data from a sizable stack of original data sheets.) The entire movie film would then contain an enormous amount of data. The diagnostician could make a symptom array for a given patient and analyze it using the movie-film filters. The output of the analyzer would be the relative correlation or convolution of the patient's symptoms and the various disease symptoms contained on the movie film.
CONCLUSION Holography is now being recognized as a versatile concept. Often it is a specific tool, as in recording and reconstructing waves. However, it also can function as

a basis for more general techniques such as optical conmputing. In medicine, holograplhy can be useful in forlning images in microscopy, interferometry, vibratorymotion analysis, continuous-motion analysis, ultrasonic diagnosis, and the visual display of nonvisible or hypothetical objects. It also can be useful in analyzing images or treating data in radiography, autoradiography, isotope-distribution analysis, cytology, pathology, ophthalmology, cystoscopy, ultrasonic diagnosis, electron microscopy, X-ray diffraction, and opticalmathematical correlation or convolution analysis. Many of the specific medical applications which have been suggested or discussed are currently purely speculative; some are the subjects of present research. The underlying theory for each of these applications has been experimentally verified, but most of these techniques require considerable development in their specific areas of application. Engineers, scientists, and physicians also must communicate their needs and interests in this area. If technological progress continues and avenues of communication are created and utilized, holography's potential value in biology and medicine can be realized.
REFERENCES
[1] D. Gabor, "A new microscopic principle," Nature, vol. 161, pp. 771-778, 1948. [2] - -, "Microscopy by reconstructed wavefronts," Proc. Roy. Soc., Ser. A., vol. 197, pp. 454-487, 1949. [3] E. N. Leith and J. Upatnieks, 'Reconstructed wavefronts and communication theory, " J. Opt. Soc. Am., vol. 52, pp. 1123-1130, 1962. [4] -, "Wavefront reconstruction with continuous-tune objects," J. Opt. Soc. Am., vol. 53, pp. 1377-1381, 1963. , "Wavefront reconstruction with diffused illumination and [5] three-dimensional objects," J. Opt. Soc. Am., vol. 54, pp. 12951301, 1964. [6] E. Feleppa, "Holography and its application to biological research," Ph.D. dissertation, Columbia University, New York, N. Y., 1968, pp. 1-103. [7] G. Ellis, "Holomicrography: Transformation of image during reconstruction a posteriori," Science, vol. 9, pp. 1195-1197, 1966. [8] R. Powell and K. Stetson, "Interferometric vibration analysis of three-dimensional objects by wavefront reconstruction," J. Opt. Soc. Am., vol. 55, p. 612, 1965. [9] E. Feleppa, "Holographic, motion-induced-contrast images," J. Cell. Biol., vol. 40, pp. 838-842. 1969. [10] A. Metherell, E. El-Sum, J. Dreher, and L. Larmore, "Image reconstruction from sampled acoustical holograms," A ppl. Phys. Lett., vol. 10, pp. 277-279, 1967. [11] E. Feleppa, "Holography applied to biomedical research," in Advances in Biomedical Engineering and Medical Physics, vol. 4, S. Levine, Ed. New York: Wiley, 1971, pp. 1-45. [12] A. Lohman and D. Paris, "Binary Fraunhofer holograms generated by computers," Appl. Opt., vol. 6, pp. 1739-1748, 1967. [13] G. Stroke and R. Zech, "A posteriori image-correcting 'deconvolution' by holographic Fourier-transform division," Phys. Lett., vol. 25, pp. 89-90, 1967. [141 A. Lohman, "Holographic production of spatial filter for code translation and image restoration," Phys. Lett., vol. 25, pp. 570-571, 1967. [15] A. Lohnian and D. Paris, "Computer generated spatial filters for coherent optical data processing," Appl. Opt., vol. 7, pp. 651656, 1968. [16] E. Feleppa, "Biomedical applications of holography," Phys. Today, vol. 22, pp. 25-32, July 1969. [17] D. Gabor, G. Stroke, R. Restrick, A. Funkhouser, and D. Brumm, "Optical image synthesis (complex amplitude addition and subtraction by holographic Fourier transformation)," Phys. Lett., vol. 18, pp. 116-118, 1965. [18] G. Stroke, R. Restrick, A. Funkhouser, and D. Brumm, "Reso-

lution-retrievinF compensation of source effects by correlative reconstruction in high-resolution holography," Phys. Lett., vol.

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18, pp. 274-275, 1965. [19] E. Marom, D. Fritzler, and R. Mueller, "Ultrasonic holography

by electronic scanning of a piezoelectric crystal," Appl. Phys. Lett., vol. 12, pp. 26-28, Jan. 1968. [20] G. Krusos, S. Hilal, W. Seaman, and G. Myers, "Reduction of

penumbra in X-ray images by optical spatial filtering," Appl. Phys. Lett., vol. 16, pp. 37-40, Jan. 1970. [21] R. Barer, "Phase contrast and interference microscopy in cytology," in Physical Techniques in Biological Research, vol. 3-A, A. Pollister, Ed. New York: Academic Press, 1966, pp. 1-56.

Mass-Balance Model of Pulmonary Oxygen Transport

GERALD M. SAIDEL, THOMAS C. MILITANO,
Abstract-A dynamic lumped-parameter model for pulmonary gas transport has been developed to characterize the lung and predict the effect of various parameter changes. The gas side of the lung is modeled as a series and parallel arrangement of five perfectly mixed, variable-volume compartments that correspond roughly to airway and alveolar regions. The blood side of the lung is modeled as a series of perfectly mixed, constant-volume compartments that represent the pulmonary capillary bed. From nonsteady mass balances, equations are derived which yield the time course of concentration for each compartment. Model simulations indicate that the oxygen-hemoglobin reaction does not reach equilibrium in the pulmonary capillaries, an assumption commnonly made in analyses of pulmonary oxygen transport. Simulations also show the extent to which breathing amplitude and rate can affect the oxygen level in the blood leaving the lung. A comparison of simulations for a normal state and chronic obstructive lung disease (COLD) with identical input conditions demonstrates that the oxygen level in the blood leaving the lung is much lower in COLD. Also, the simulations are compared with experimental findings.
AND

EDWARD H. CHESTER, MEMBER, IEEE

INTRODUCTION

O M THE airway opening to the gas-exchange region, the human lung consists of dichotomously branching airways that lead to microsacs called alveoli. Associated with these alveoli are capillaries through which blood flows. Gas exchange occurs between gas in the alveoli, which are ventilated with environmental air, and blood in the capillaries. Blood leaving the lung has increased oxygen content and lower carbon dioxide content than the blood that enters.
Manuscript received April 9, 1971, revised September 14, 1971. This work was supported in part by grants from the National Tuberculosis and Respiratory Disease Association, and by the National Institutes of Health under Grant GM-12302. This paper was presented in part at the 1971 Rocky Mountain Bioengineering Symposium, Fort

Collins, Colo. The authors are with the Departments of Biomedical Engineering, Medicine, and Biometry, Case Western Reserve University, Cleveland, Ohio, and the Veterans Administration Hospital, Cleveland, Ohio 44106.

For convenience, we divide the transport of oxygen into several distinct processes: 1) gas-phase transport in the airways and the alveoli; 2) interphase transport between alveolar gas and capillary blood; and 3) liquidphase transport in the capillary blood. Gas-phase transport, which occurs by convection and molecular diffusion, results in the mixing of the alveolar air with fresh air from the environment. The effectiveness of this mixing depends on the distribution of inspired air to the alveolar region. Properties such as airway resistance to flow and tissue distensibility play an important role in the transport and mixing of gases. Transport across the alveolar-capillary interface may be thought of as diffusion across a membrane separating ventilated alveoli and perfused capillaries. Alveolar-capillary transport depends on the effective surface area of this membrane, which in turn is a function of the relative distributions of ventilation and perfusion. In addition, the interphase transport across the membrane depends on the membrane permeability to the particular chemical species. Oxygen transport through the blood involves convection and diffusion in plasma, diffusion across red cell membranes, and chemical reactions of oxygen and hemoglobin. To characterize the lung with respect to gas transport, various experiments have been devised with gases of different physical and chemical properties which can be measured easily at the mouth. Interpretation of these data is discussed by Forster [1] in his review, where he points out the difficulties. Ayers et al. [2] note that these difficulties are increased in the presence of a chronic obstructive lung disease (COLD) such as emphysema and/or chronic bronchitis. To separate the effects of transport processes within the airways and alveoli from the transport processes