the significant features or preferred embodiments of the invention , illustrated schematically in fig1 that distinguishes itself from other types of blood flow monitors include : a ) the delivery of a long - wavelength visible light emanating from a laser or semiconductor laser at a specific yet adjustable incidence angle , b ) the collection optics which magnify that area of the tissue illuminated not only by the initially inserted laser light but also by laser light back scattered to the surface of the tissue as the initially inserted laser light propagates beneath the tissue &# 39 ; s surface , c ) the partitioning and contiguous sampling of photons emanating from the surface of the tissue within this area but along the expected trajectory of the propagating laser light by a translating optical fiber or an array of optical fibers , and d ) the user or computer selectable algorithm ( s ) developed for the data analyses and presentation of the resulting measurements which determine blood flow characteristics at precise depths within the tissue , the thickness and the elasticity of the tissues above the region ( s ) where blood flow first occurs and / or 2 - dimensional representation of the structure of the blood flow region ( s ) in terms of blood perfusion characteristics , or any combination thereof . we now describe in detail each significant aspect of the invention whose simplified schematic is diagrammed in fig1 . a vertically polarized laser beam emitted by a laser source 1 is reflected by a mirror 2 and passes through a lens 3 . at this stage of the process , it is necessary that the diameter of the laser light focal point need be small , in the range of ˜ 40 μm to ˜ 100 μm , and the laser polarization be normal in orientation with respect to the back scattered light collection optics . as shown in fig2 the focal point of the lens 3 is positioned on the surface an object 4 which is on a stage resting at the focal plane of the collection lens 5 . that is , the focal point of the laser beam exiting the lens 3 , the sample surface 4 and the focal point of the collection lens 5 are coincident on the same plane . fig2 also illustrates the overall laser light incidence and back scattered laser light collection geometry of the system , as well as the positioning of the initially inserted laser beam within the field of view of the collection optics 5 . the collection optics 5 may be commercially available compound microscope objectives whose magnification can range from 4 × to 32 × or greater if necessary , provided that the field of view be sufficently large to enable triangulation onto the required depth of tissues . all scattered light collected within this field of view is then collimated into a microscope body 6 . the back scattered collumated light within the boby of the microscope 6 is passed through a polarizer 7 whose orientation can be adjusted to allow for polarized (∥) or depolarized (□) scattered light sampling . a portion or most of the collumated light is reflected by a partial reflector 8 into the side port which contains a lens coupled to a reticle 9 that focuses the collected image 18 , shown in fig2 and fig3 onto a ccd or televison camera 10 . the image obtained by the camera 10 is forwarded to a monitor 11 which would then enable the user to properly align the focused laser beam and the collection optics 5 onto the object resting on a stage 4 . once optical alignment is satisfacory , the partial reflector 8 may be rotated out of position to allow for all back scattered collumated light to pass towards either a translating optical fiber probe or an arrray of optical fiber probes 12 . fig3 displays an image of both the laser light initially focused on the surface propagating into the deeper regions of a rats &# 39 ; foot skin , and the subsequent sampling of the back scattered laser light collected within the field of view of the microscope at contiguous locations on the surface of the object by either a translating optical fiber probe or an arrray of optical fiber probes 12 . as also depicted in fig3 only a portion ( a partitioned region ) or small area of the back scattered laser light collected within the field of view of the microscope is sampled by either a translating optical fiber probe or each individual optical &# 34 ; pick - up &# 34 ; fiber within the array 12 , at any given time . the back scattered light sampled within a specific area is guided to an individual avalanche photo diode ( apd ) or photo multiplier tube ( pmt ), or a bank of apd &# 39 ; s or pmt &# 39 ; s 13 through each respective optical fiber . the photon flux sampled from within each area on the surface of the object is amplified and converted to an electrical signal that is delivered to a signal processor unit 14 and then treated in accordance with another aspect of the invention which provides user selectable algorithms . the algorithm ( s ) developed for the invention are based on both knowledge of the back scattered light characteristics obtained by the collection optics 5 , 6 , 7 and 12 as a focused beam of laser light propagates through the skin of a rats &# 39 ; foot under - side whose cross - section is illustrated in fig4 as well as the measurement type desired . irrespective of measurement type desired or the cost determinant version of the invention utilized , the starting point of the algorithm ( s ) is the measurement of the static intensity properties of the ∥ and / or □ □ polarized back scattered laser light emitted at the surface of the skin . fig5 displays the typical results of the focal diameter of the laser light on the skin surface as shown here is ˜ 80 μm , and the field of view diameter is ˜ 350 μm . the focal diameter of the polarized laser light expands from ˜ 50 μm to ˜ 80 μm due to the multiple scattering of photons within the skin . in addition to beam expansion , multiple scattering also effects changes in the polarization of the incident laser light . regardless of multiple scattering effects , laser penetration of the tissue is evidenced also by photons emitted at the surface along the expected trajectory of the laser light propagation . the observed image is an electronic reproduction of a photograph taken at 40 × magnification . the white circles , later inserted by a computer , represent the different locations from which photons emitted from the surface of the skin are selectively monitored by the translating optical pick - up fiber . at each location , the optical pick - up fiber , covering an area of ˜ 100 μm 2 , monitors both the static and dynamic properties of the back scattered laser light . the translated distance between different locations is approximately 0 . 0125 mm . that is , while maintaining the focal positions of both the incident laser and the microscope objective on the surface the skin , we translated the &# 34 ; pick - up &# 34 ; optical fiber in approximately 0 . 0125 mm intervals along the microscope objective focal plane following the expected trajectory of the laser beam as it propagates through the sample . the observed behavior of the static and dynamic properties of back scattered light can then be classified into regions , dependent solely upon the location from which photons emitted from the surface of the skin were collected . with reference now to fig4 there is displayed the static intensity properties of the back scattered photons emitted at the surface of the tissue as a function of translation position and collection polarization . all data were collected using a vertically polarized laser light with an incidence angle . o slashed . i = 60 °, and a 4 × microscope objective that collects the back scattered light at a scattering angle . o slashed . s = 120 °. as evidenced , the behavior of the static intensity properties of the back scattered light can be classified into five perceptible regions that are dependent solely on the optical pick - up fiber position and independent of the collection polarization . differences in the static intensities of polarized and depolarized back scattered light within specific regions are , however , also readily evident due to the effects of multiple scattering of photons . multiple scattering effects on the back scattered laser light are least observed at the focal point of the incident laser light . that is , the further the laser light propagates through the dense regions of the skin , the more the polarization of the back scattered light is altered due to multiple scattering . hence , the static behavior of the intensity of back scattered light , by region , can be described as follows : region 1 . near the focal point of the incident laser light , but lies in the opposite direction of laser light propagation . for photons to be observed at the surface of this region , they must undergo numerous &# 34 ; collision &# 34 ; events or multiple scattering . region 2 . the focal point of the incident laser light on the stratum corneum of the skin . region 3 . the magnitude of polarized and depolarized back scattered laser light intensities observed emanating from the surface to this region are difficult to predict . the initial polarized laser light is expected to reach this region due to forward scattering , which is expected to maintain polarization . the intensity of the initial laser light also should not only diminish exponentially with respect to the depth of propagation , but also due to multiple scattering . changes in the scattering cross - section of the malphigian layer had , however , increased the primary contributions to both the polarized and depolarized back scattered light intensities . the magnitude of difference between polarized and depolarized intensities had also diminished , because photons scattered from this region undergo more &# 34 ; collision &# 34 ; events prior to reaching the surface of the tissue . region 4 . the epidermal / dermal boundary region exhibits a further increase in both the polarized and depolarized back scattered intensities , contrary to the expected steady exponential diminishment of laser light intensity as it propagates further into the tissue . the increase in both the polarized and depolarized back scattered intensities is due not only to changes in the scattering cross - section of the epidermal / dermal boundary , but also due to scattering form the presence of red blood cells . this will become more evident upon comparison with the dynamic properties of the back scattered laser light . once again , the magnitude of difference between polarized and depolarized intensities had also diminished , because photons scattered from this region undergo further &# 34 ; collision &# 34 ; events prior to reaching the surface of the tissue . region 5 . farthest from the initial focal point but along expected trajectory of propagating laser light , propagation . for photons to be observed at the surface of this region , they must undergo numerous &# 34 ; collision &# 34 ; events , as in the case of region 1 . moreover , the scattering cross - section of the dermis is also weak upon comparison with red blood cells . thus both the polarized and depolarized back scattered intensities observed at the surface of the tissue diminish somewhat exponentially as the laser light propagates further within this region , as expected . there is also no apparent difference between polarized and depolarized static back scattered light intensities . as shown , the diameter of the focused laser light on the skin surface is ˜ 80 μm , and the field of view diameter is ˜ 350 μm . clearly visible are the three areas of the skin representing the stratum corneum , malphigian layer and the dermis . also to be noted is the highly non - uniform boundary between the epidermal and dermal layers where capillary blood flow is known to occur . as evidenced , the data can be classified into five perceptible regions , dependent solely on the optical position independent of collection polarization . the frequency component of the data representing the average motion of rbc &# 39 ; s is noticeably broader than the polarized back scattered light data obtained from the same optical fiber positions . moreover , the amplitudes of the power spectra obtained from optical fibers positioned in region 2 are noticeably diminished . the frequency component of the data represent the average motion of rbc &# 39 ; s over the time course of several heart beats , where rbc &# 39 ; s motions are rapid when blood flow is occurring ( high frequency ) and slow at other times ( low frequency ). regardless , the notable features of the averaged spectra are the readily evident differences in the amplitudes and spectral widths of the data with respect to the specific positions of the optical fibers . observations of the temporal fluctuations in the scattered light signal yields blood flow and volume characteristics within a specific region of interest . implicit in all analyses of the temporal fluctuations of scattered light signal is the assumption that the characteristics of blood flow and volume are directly correlated with red blood cell motion and concentration . that is , it is generally considered that the singular difference between the static and dynamic properties of the scattered light signal measured in all studies relating to blood flow is that light scattered by all cells within a specific area contribute to the former , whereas only the motions of red blood cells contribute to changes in the latter . the relative rate of red blood cell ( rbc ) motions are thus determined by the &# 34 ; frequency &# 34 ; or the &# 34 ; ac &# 34 ; component of the scattered light intensity fluctuations , and their relative concentrations are determined by the magnitude of these fluctuations as well as changes in the static intensity . several well - known means are available for analysis of the temporal fluctuations in scattered light signal . for the case of monitoring the &# 34 ; real &# 34 ; time characteristics of blood flow , the most frequently used algorithm calculates the changes in the power spectral density , in time . in this particular study , however , we had used two alternate means available for determining the averaged properties of rbc motions , over a period of several heart beats , in order to develop a more thorough understanding of the spectral characteristics of intensity fluctuations within the five perceptible regions of the skin . based on the results of these studies , we will then describe appropriate algorithms for determining blood flow characteristics , in real time . fig5 displays the dynamics of the vertically polarized back scattered photons emitted at the surface of the tissue form the five different regions . all data were collected using a vertically polarized laser light with an incidence angle . o slashed . i = 60 °, and a 4 × microscope objective that collects the back scattered light at a scattering angle . o slashed . s = 120 °. a simple polarizer in the &# 34 ; vertical &# 34 ; configuration was placed in front of a microscope objective to collect back scattered light of the same polarization as the incident laser light . the polarized back scattered laser thus collected within a small area within each region at the surface of the skin was then &# 34 ; piped &# 34 ; to a photo multiplier tube ( see fig2 ) and converted to an electrical current . this current is next amplified by a factor of ˜ 10 5 and forwarded to a high - pass filter with a cut - off frequency at 110 hz . the photon flux thus represented as voltage is next transferred to a spectrum analyzer which converts temporal fluctuations into a power spectrum in the frequency range of 0 - 2500 hz . that is , the spectrum analyzer first digitizes the temporal fluctuations in the back scattered light intensities within a short period of time . it next performs a fast - fourier transform of the temporal data thereby converting it into the frequency domain . the frequency range of the data corresponds directly to the time period of data collection . the amplitude of the power spectrum is the sum of the squared real and imaginary components of fourier transform and represents the magnitude of temporal fluctuations . in the current configuration , this process is repeated over a period of 1 to 2 minutes and averaged in order to obtain spectra with good signal - to - noise ratios . hence the spectra represent the averaged motions of rbc &# 39 ; s over the time course of several heart beats , where rbc &# 39 ; s motions are rapid when blood flow is occurring and slow at other times . more specifically , the frequency &# 34 ; widths &# 34 ; of the power spectra correspond to the averaged motions of the rbc &# 39 ; s . upon completion of spectrum averages , the data were then transferred to a computer for further analyses . the method ( s ) developed for the invention are based on both the knowledge of the back scattered light characteristics obtained by the collection optics and electronics 5 , 6 , 7 , 12 , 13 , and 14 as a focused beam of laser light propagates through the skin of a rats &# 39 ; foot under - side , whose cross - section is illustrated in fig4 as well as the measurement type desired . irrespective of measurement type desired or the cost determinant version of the invention utilized , the starting point of the method ( s ) is the measurement of the ∥ and / or □□ polarized static or time averaged intensity properties of the back scattered laser light emitted at the surface of the skin . fig5 displays the typical results of time averaged sampling 15 of both ∥ and □□ polarized orientations of the intensity of back scattered laser light collected within the field of view of the microscope at each contiguous location on the surface of the skin of a rats &# 39 ; foot under - side . the data were obtained by means of focusing a vertically polarized laser light on the surface of the skin at an incidence angle i = 60u , and a 4 × magnification microscope objective 4 for collecting the back scattered light from the surface of the skin within the field of view of the microscope at a scattering angle s = 120u . the intial focal diameter of the laser light was ˜ 50 μm , which had expanded to ˜ 70 μm on the surface of the skin due to the multiple scattering of photons within the skin . the diameter of the filed of view of the microscope is ˜ 350 μm . the partitioned area within the field of view of the microscope sampled by optical pick - up fiber ( s ) 12 after passing through the polarizer 7 is ˜ 100 μm 2 , with the center - center distance between different partitions ( areas being sampled by an optical pick - up fiber along the expected trajectory of the laser beam beneath the surface of the tissue ) being 0 . 0125 mm . the back scattered light intensty signals thus collected were converted to electrical voltage 13 , processed through signal processor which obtains a time average 14 , then sent to the voltage read - out 15 for retreival by a computer that displays the data in useful form by the algorithm step s1 illsutrated in schematic form in fig6 . the observed behavior of the static intensity properties of back scattered light within each partition / sampling area on the surface of the skin is dependent solely upon the location of the partition or area from where photons were collected , but independent of the or ∥ or □ collection polarization . the observed static intensities for both polarizations are next identified into five perceptible regions in the algorithm step s2 . the significant characteristics of the observed static intensity for both ∥ or □ polarized back scattered light from each region are simply defined as follows : all of the data presented not only in fig5 but also in fig6 , 8 & amp ; 9 were obtained at essentially the same locality on the rats &# 39 ; foot .
a non - invasive , non - traumatic technology for measuring continuously the blood flow and volume at precise depths within tissues is presented . the new methodology makes use of 1 ) a long - wavelength visible laser or semiconductor laser light delivered at a variable incidence angle for triangulating onto a specific region under investigation , 2 ) a lens system which magnifies the area illuminated by the laser light as it propagates within the tissue , 3 ) a translating optical fiber or a linear array of optical fibers that collect the back scattered photons emanating from the surface of the tissue along the expected trajectory of the laser light propagating within the tissue , 4 ) a photo diode or a photo multiplier , or a bank of photo diodes or photo multipliers that amplify and convert the back scattered photons into electrical signals , 5 ) a system for collectively or selectively monitoring both the static and dynamic properties of the back scattered photons , and 6 ) an algorithm or algorithms which determine not only the location , volume and velocities of blood flow at precise depths within the tissue , but may also determine the thickness as well as the elasticity of the tissues above the region where blood flow first occurs . furthermore , this newly developed technology can also provide a 2 - dimensional map of the blood flow regions within tissues in terms of depth and blood flow characteristics .