Person:

Young, Geoffrey

Conventional histopathology with hematoxylin & eosin (H&E) has been the gold standard for histopathological diagnosis of a wide range of diseases. However, it is not performed in vivo and requires thin tissue sections obtained after tissue biopsy, which carries risk, particularly in the central nervous system. Here we describe the development of an alternative, multicolored way to visualize tissue in real-time through the use of coherent Raman imaging (CRI), without the use of dyes. CRI relies on intrinsic chemical contrast based on vibrational properties of molecules and intrinsic optical sectioning by nonlinear excitation. We demonstrate that multicolor images originating from (CH_2) and (CH_3) vibrations of lipids and protein, as well as two-photon absorption of hemoglobin, can be obtained with subcellular resolution from fresh tissue. These stain-free histopathological images show resolutions similar to those obtained by conventional techniques, but do not require tissue fixation, sectioning or staining of the tissue analyzed.

Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

The development and marketing of new volumetric computed tomography (CT) scanners in 1990 made it possible to perform three-dimensional (3-D) imaging of the abdomen without respiratory artifacts and with clarity similar to that achieved in the musculoskeletal and central nervous systems by conventional scanners.6 Before 1990, all CT scanners had x-ray tubes that were connected to the machine's gantry by electrical cables. This limited the excursion of the tube in any one direction because continuous rotation would wind the cables into a knot. Thus, conventional CT scanning is performed by executing a series of individual CT scan slices during which patients are instructed to hold their breath. Between scans the table is moved forward a certain distance and the process is repeated. If the breath holds are not identical, the imaged organ or region contains gaps or skip areas. This limitation is termed “respiratory misregistration.” The technical advantage that made volumetric CT possible was the development of a continuously rotating x-ray tube with slip rings. Slip rings are a pair of matched rings on the tube and gantry that can rotate past one another without limit. This allows for continuous rotation of the tube and the ability to perform a continuous x-ray exposure as the patient is moved forward through the CT gantry. The resulting exposure forms a path that looks like a spiral or helix; hence, today volumetric CT scanning often is referred to as “spiral” or “helical” CT. Spiral CT acquires data in a region of interest using a single continuous x-ray exposure that is fast enough to be executed during a single breath hold, as the patient moves through the gantry, so that respiratory misregistration is eliminated. An entire body region is imaged and a continuous volume of CT data obtained. The resulting volumetric CT data set can be used to create axial images at a desired slice thickness and at a desired increment. Standard axial images from spiral CT of the urinary tract have been shown to be helpful in the diagnosis and staging of renal masses and are now routinely used for evaluation of renal lesions.15,16 However, the same spiral CT data can be reformated in multiple planes or in three dimensions.

Although newer 3-D reconstruction techniques retain more data than did older reformatted display methods, 3-D images cannot contain more information than appropriately filmed axial images from the same source data set. Thus, with the possible exception of CT angiography and future developments in virtual endoscopy, 3-D images, by themselves, are of limited diagnostic utility. However, 3-D imaging of the urinary tract is useful in surgical planning, because it allows surgeons to visualize 3-D anatomic relationships clearly. 3-D images created from the same spiral CT data sets for diagnosis and staging of renal masses have been used to create color-coded 3-D surface renderings of renal masses and surrounding structures for surgical planning of partial nephrectomy.1 A preliminary investigation and one published case report9 have shown that this technique can help the urologist locate small renal masses and can help delineate the relationship between a mass and the urinary collecting system during operative planning. Currently, in addition to standard spiral CT, conventional preoperative assessment of a patient before a partial nephrectomy may include aortography and selective renal angiography to determine the number, location, and pattern of branching of renal vessels. Intravenous pyelography also may be indicated to evaluate the anatomic relationship of any renal masses to the intrarenal collecting system and proximal ureter. Someday, 3-D rendering of the same spiral CT data set used to diagnose the tumor may be able to display the relationship between the tumor and the renal parenchyma, vasculature, and collecting system, thus obviating the need for these additional tests.