Deep tissue fluorescence imaging with time-reversed light

Advances in optical techniques have enabled many breakthroughs in biology and medicine. However, light scattering by biological tissues remains a great obstacle, restricting the use of optical methods to thin ex vivo sections or superficial layers in vivo. In this thesis, we present two related methods that overcome the optical depth limit—digital time reversal of ultrasound encoded light (digital TRUE) and time reversal of variance-encoded light (TROVE). These two techniques share the same principle of using acousto-optic beacons for time reversal optical focusing within highly scattering media, like biological tissues. Ultrasound, unlike light, is not significantly scattered in soft biological tissues, allowing for ultrasound focusing. In addition, a fraction of the scattered optical wavefront that passes through the ultrasound focus gets frequency-shifted via the acousto-optic effect, essentially creating a virtual source of frequency-shifted light within the tissue. The scattered ultrasound-tagged wavefront can be selectively measured outside the tissue and time-reversed to converge at the location of the ultrasound focus, enabling optical focusing within deep tissues. In digital TRUE, we time reverse ultrasound-tagged light with an optoelectronic time reversal device (the digital optical phase conjugate mirror, DOPC). The use of the DOPC enables high optical gain, allowing for high intensity optical focusing and focal fluorescence imaging in thick tissues at a lateral resolution of 36 µm by 52 µm. The resolution of the TRUE approach is fundamentally limited to that of the wavelength of ultrasound. The ultrasound focus (~ tens of microns wide) usually contains hundreds to thousands of optical modes, such that the scattered wavefront measured is a linear combination of the contributions of all these optical modes. In TROVE, we make use of our ability to digitally record, analyze and manipulate the scattered wavefront to demix the contributions of these spatial modes using variance encoding. In essence, we encode each spatial mode inside the scattering sample with a unique variance, allowing us to computationally derive the time reversal wavefront that corresponds to a single optical mode. In doing so, we uncouple the system resolution from the size of the ultrasound focus, demonstrating optical focusing and imaging between highly diffusing samples at an unprecedented, speckle-scale lateral resolution of ~ 5 µm. Our methods open up the possibility of fully exploiting the prowess and versatility of biomedical optics in deep tissues.

Doppler echocardiography in athletes from different sports

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo Doppler-ecocardiográfico de pacientes com talassemia major em regime de hipertransfusão sanguínea

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Utilzzo dei parametri ecocardiografici tradizionali ed a Doppler tissutale nella valutazione della disfunzione atrio-ventricolare in cani affetti da malattia mitralica cronica

In questo lavoro ci siamo posti come obiettivo lo studio della disfunzione atrio-ventricolare mediante tecniche ecocardiografiche avanzate (come il Tissue Doppler Imaging - TDI) in cani affetti da malattia mitralica cronica (MVD). Una prima parte è volta alla valutazione della funzionalità diastolica del ventricolo destro. Ci siamo proposti di analizzare la funzione del ventricolo destro in cani affetti da malattia del cuore sinistro per comprendere se quest’ultima possa condizionare direttamente la performance del settore cardiaco controlaterale. I risultati più importanti che abbiamo riscontrato sono: l’assenza di differenze significative nella disfunzione sisto-diastolica del ventricolo destro in cani con MVD a diverso stadio; la diretta correlazione tra le variabili TDI di funzionalità del ventricolo destro con il grado di disfunzione del ventricolo sinistro, come indicatori di interdipendenza ventricolare; ed infine il riscontro di una maggior tendenza ad alterazioni diastoliche del ventricolo sinistro in cani con ipertensione polmonare. A quest’ultimo proposito, per quanto riguarda le variabili TDI, il rapporto E/e’ dell’anulus mitralico laterale e settale è risultato avere una differenza significativa tra i cani con ipertensione polmonare e quelli privi di ipertensione polmonare (P<0,01). Nel secondo studio abbiamo applicato il TDI per l’analisi della funzione sisto-diastolica dell’atrio sinistro. Il lavoro è stato articolato in una parte di validazione della metodica su cani normali ed una su animali affetti da MDV. I risultati ottenuti mostrano che la valutazione ecocardiografica delle proprietà di deformazione dell’atrio sinistro basata sul TDI è attuabile e riproducibile nel cane. Abbiamo fornito dei valori di normalità per questa specie e confrontato questi dati con quelli ricavati in cani portatori di MVD. Le differenza tra le varie classi di malattia, nei diversi gradi di dilatazione atriale, sono risultate limitate, ma abbiamo individuato delle correlazioni tra i parametri TDI ed alcune variabili di funzionalità atriale.

Application of a new laser Doppler imaging system in planning and monitoring of surgical flaps

There is a demand for technologies able to assess the perfusion of surgical flaps quantitatively and reliably to avoid ischemic complications. The aim of this study is to test a new high-speed high-definition laser Doppler imaging (LDI) system (FluxEXPLORER, Microvascular Imaging, Lausanne, Switzerland) in terms of preoperative mapping of the vascular supply (perforator vessels) and postoperative flow monitoring. The FluxEXPLORER performs perfusion mapping of an area 9 x 9 cm with a resolution of 256 x 256 pixels within 6 s in high-definition imaging mode. The sensitivity and predictability to localize perforators is expressed by the coincidence of preoperatively assessed LDI high flow spots with intraoperatively verified perforators in nine patients. 18 free flaps are monitored before, during, and after total ischemia. 63% of all verified perforators correspond to a high flow spot, and 38% of all high flow spots correspond to a verified perforator (positive predictive value). All perfused flaps reveal a value of above 221 perfusion units (PUs), and all values obtained in the ischemic flaps are beneath 187 PU. In summary, we conclude that the present LDI system can serve as a reliable, fast, and easy-to-handle tool to detect ischemia in free flaps, whereas perforator vessels cannot be detected appropriately.

Pulsed-wave tissue Doppler echocardiography for the analysis of fetal cardiac arrhythmias

Rhythm analysis of the fetal heart is hampered by the inability to routinely obtain electrocardiographic recordings of the fetus. Doppler studies of fetal cardiac tissue movements, assessing cardiac movements both qualitatively and quantitatively, have recently been described. We used a conventional high-resolution ultrasound system to obtain rhythm data from pulsed-wave tissue Doppler signals of the fetal heart in normal cardiac rhythm and in a variety of fetal cardiac arrhythmias.

Assessment of melanocytic skin lesions with a high-definition laser Doppler imaging system

Early detection is a major goal in the management of malignant melanoma. Besides clinical assessment many noninvasive technologies such as dermoscopy, digital dermoscopy and in vivo laser scanner microscopy are used as additional methods. Herein we tested a system to assess lesional perfusion as a tool for early melanoma detection.

Laser Doppler imaging for intraoperative human brain mapping

The identification and accurate location of centers of brain activity are vital both in neuro-surgery and brain research. This study aimed to provide a non-invasive, non-contact, accurate, rapid and user-friendly means of producing functional images intraoperatively. To this end a full field Laser Doppler imager was developed and integrated within the surgical microscope and perfusion images of the cortical surface were acquired during awake surgery whilst the patient performed a predetermined task. The regions of brain activity showed a clear signal (10-20% with respect to the baseline) related to the stimulation protocol which lead to intraoperative functional brain maps of strong statistical significance and which correlate well with the preoperative fMRI and intraoperative cortical electro-stimulation. These initial results achieved with a prototype device and wavelet based regressor analysis (the hemodynamic response function being derived from MRI applications) demonstrate the feasibility of LDI as an appropriate technique for intraoperative functional brain imaging.

Comparison between pulsed-wave Doppler- and tissue Doppler-derived Tei indices in fetuses with and without congenital heart disease

OBJECTIVES The aim of this study was to compare the right (RV) and left (LV) ventricular Tei indices obtained by pulsed-wave Doppler (PD) and tissue Doppler (TD) methods in fetuses with structurally normal and abnormal hearts. METHODS This was a retrospective cross-sectional study of 147 fetuses that had a fetal echocardiogram and Tei index measured during a 2-year period. The RV and LV Tei indices were measured using both PD and TD methods. The difference between the two methods of Tei index measurement was tested using paired sample t-test, Pearson correlation coefficient was used to examine their relationship, and the agreement between the methods was tested using Bland-Altman analysis. RESULTS A total of 87 fetuses had normal hearts and 60 had a congenital heart defect. Both PD and TD Tei indices were measured successfully from at least one ventricle in 123 cases and from both ventricles in 110 cases. The mean TD Tei index was significantly higher than the mean PD Tei index for both ventricles (P < 0.0001). There was a weak but statistically significant correlation between the PD and TD Tei indices of the right ventricle (r = 0.20, P = 0.029), whereas the PD and TD Tei indices of the left ventricle did not correlate significantly (r = 0.04, P = 0.684). When pairs of Tei indices measured by two different methods (123 pairs for the right ventricle and 111 for the left ventricle) were tested with Bland-Altman analysis, the bias and precision were 0.147 and 0.254, respectively, for the right ventricle, and 0.299 and 0.276, respectively, for the left ventricle. CONCLUSIONS Correlation between Tei indices measured by PD and TD methods is weak and the agreement between individual measurements is poor. Therefore, they should not be used interchangeably in the assessment of fetal cardiac function.

Relative indexes of cutaneous blood perfusion measured by real-time laser Doppler imaging (LDI) in healthy volunteers.

We used real-time LDI to study regional variations in microcirculatory perfusion in healthy candidates to establish a new methodology for global perfusion body mapping that is based on intra-individual perfusion index ratios. Our study included 74 (37 female) healthy volunteers aged between 22 and 30 years (mean 24.49). Imaging was performed using a recent microcirculation-imaging camera (EasyLDI) for different body regions of each volunteer. The perfusion values were reported in Arbitrary Perfusion Units (APU). The relative perfusion indexes for each candidate's body region were then obtained by normalization with the perfusion value of the forehead. Basic parameters such as weight, height, and blood pressure were also measured and analyzed. The highest mean perfusion value was reported in the forehead area (259.21APU). Mean perfusion in the measured parts of the body correlated positively with mean forehead value, while there was no significant correlation between forehead blood perfusion values and room temperature, BMI, systolic blood pressure and diastolic blood pressure (p=0.420, 0.623, 0.488, 0.099, respectively). Analysis of the data showed that perfusion indexes were not significantly different between male and female volunteers except for the ventral upper arm area (p=.001). LDI is a non-invasive, fast technique that opens several avenues for clinical applications. The mean perfusion indexes are useful in clinical practice for monitoring patients before and after surgical interventions. Perfusion values can be predicted for different body parts for patients only by taking the forehead perfusion value and using the perfusion index ratios to obtain expected normative perfusion values.